Method and device for preparing tubular biological structures

Abstract

An apparatus (105) for manufacturing a tubular biological structure (165), comprising a tubular member (150) configured to be rotatable wherein the tubular member (150) comprises a tubular wall extending from a first end (151) and a second end (152) wherein the tubular member (150) is configured to receive a biological material (160) on an inner surface (154) thereof, and wherein the tubular member (150) is configured to be rotatable, and wherein the tubular member (150) is configured to be rotatable. The tubular member (150) comprises at least one opening (156) through the tubular wall, the at least one opening being configured to, in use, allow expulsion of excess biological material (160).

Description

Technical Field

[0001] The present invention relates to an apparatus and method for preparing a tubular structure. In particular, but not exclusively, the present invention relates to an apparatus and method for preparing a tubular multilayer biological structure. Background Art

[0002] Layered tissues are ubiquitous throughout the human anatomy, with notable examples found in cardiac, skin, intestinal, and vascular tissues. This layering features a range of different cell types and extracellular matrices, resulting in a highly specialized composite structure that imparts a variety of properties and functions to tissues. Tissue engineering, which aims to create anatomically and functionally precise tissues, typically uses large numbers of cells suspended in liquid hydrogels, which can be deposited and subsequently gelled. When using such methods to form layered tissues, researchers have few technical options. Extrusion-based 3D bioprinting technologies, the mainstay of the industry, are not suitable for assembling hydrogels into planar layered structures. In particular, they cannot achieve the deposition resolution and repeatability required to assemble the multiple thin, continuous layers observed in natural tissue architectures. In addition, these technologies impose restrictions on the range of compatible hydrogels, requiring the printing liquid to gel rapidly to assume the desired 3D spatial morphology.

[0003] Recognizing the limitations of 3D bioprinters in manipulating hydrogels to microscale layers, some researchers have attempted to develop improved biofabrication techniques. One strategy is to sequentially dip, spray, or extrude cell-laden hydrogels onto the outer surface of a cylinder to build layer by layer. The advantage of this method is the formation of tubular macrostructures, which are observed in many natural layered tissues such as blood vessels, intestines, trachea, and bile duct tissues. In addition, the fabrication of tissues by cutting layered tubular structures allows for easy conversion to planar tissues. The limitation of rod-based methods is that they require the cell solution to be dipped or sprayed onto the outer surface. This requires a large volume of cell-laden hydrogel that is ultimately wasted and inhibits the achievable cell density. Removing the fabricated tissue may also be challenging due to the shrinkage of the hydrogel on the cylindrical mold. Rotating the cylinder at high speed under motor control can achieve a level of control over the layer thickness, which is otherwise determined only by the viscosity of the liquid hydrogel and the surface properties of the basal layer. However, this approach limits the cell density that can be achieved in the resulting layer. This means that they are further away from the structure of natural tissues, reducing their clinical relevance.

[0004] For example, US10688694B2 (Acevedo et al) discloses a method for manufacturing multi-layer hollow tubes using different biomaterials using a layer-by-layer rod immersion method.

[0005] An alternative approach is centrifugal casting, in which the material is applied to the interior of a closed cylinder, which is then rotated. Centrifugal forces push the material, such as a hydrogel, onto the inner surface, producing a tube with a layer thickness of typically about 1 mm, determined by the volume added. However, this technique has received limited attention since the seminal work of Mironov et al.

[0006] Mironov et al ("Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in an in situ crosslinkablehyaluronan-gelatin hydrogel"; Biomaterials 26 (2005) 7628-7635) disclosed the use of centrifugal casting to produce tubular biostructures made of a crosslinkable hyaluronan-gelatin hydrogel (sECM).

[0007] US6969480 (Dalton et al) discloses hollow structures made by rotary spinning. In this document, phase separation of a soluble solution or emulsion is induced when a filled mold is rotated about one of its axes, and the density difference between the phases causes precipitation to occur in the mold cavity under the action of centrifugal force.

[0008] EP2755599B1 discloses engineered tissues and organs comprising one or more layers of muscle, the engineered tissue or organ consisting primarily of cellular material, provided that the engineered tissue or organ is implantable in a vertebrate subject and is not a blood vessel.

[0009] However, centrifugal casting methods are generally not suitable for preparing multilayer biostructures because adding different volumes of hydrogel will form inconsistent thickness. In addition, biomaterials such as collagen have generally proven difficult to manipulate using existing biofabrication techniques due to a combination of low viscosity and extended thermal gelation time, which requires a high level of self-support and temperature control in the transition from liquid to gel.

[0010] It is an object of the present invention to solve and / or alleviate one or more problems associated with the prior art.

[0011] It is an object of the present invention to provide an improved method for manufacturing a tubular biological structure, such as a multilayer tubular biological structure. Summary of the invention

[0012] According to a first aspect, there is provided a device for manufacturing a tubular biological structure, the device comprising:

[0013] a tubular member configured to be rotatable,

[0014] wherein the tubular member comprises a tubular wall extending between a first or proximal end and a second or distal end,

[0015] wherein the tubular member is configured to receive biological material on an inner surface thereof, and

[0016] Wherein the tubular member comprises at least one opening through the tubular wall, the at least one opening being configured to allow the escape of excess biological material in use.

[0017] The device may comprise a rotatable support configured to be rotated.The tubular member may be configured to be mounted on or attached to the rotatable support.

[0018] The tubular member, such as a tubular wall, may define an outer surface and an inner surface.

[0019] Typically, the tubular member may define an open tube. The first end of the tubular member may define a first open end. The second end of the tubular member may define a second open end. The first end may be disposed opposite the second end. The first end may be a proximal end or may define a proximal end. The second end may be a distal end or may define a distal end.

[0020] Advantageously, the tubular member may have a substantially constant diameter between the first end and the second end. The tubular member may have or may define a substantially constant or continuous circular cross-section between the first end and the second end. With this arrangement, a uniform thickness of biomaterial on the inner surface of the tubular member may be achieved when rotating.

[0021] The device may include a motor capable of actuating rotation of the tubular member.

[0022] The support may be connected to a motor or may be actuated by a motor. The support may be rotated by actuating the motor. Thus, when the tubular member is mounted to the support or attached to the support, the tubular member may be rotated by actuating the rotatable support via a motor.

[0023] The support may be configured to engage a tubular member (eg, a tubular wall).

[0024] The support and the tubular member may have complementary features.

[0025] The support may comprise a tubular or cylindrical portion.

[0026] The cylindrical portion of the support can be configured to fit within an end of the tubular member, such as the first end or the proximal end. The outer surface of the cylindrical portion of the support can be configured to engage with an inner surface of an end of the tubular member, such as the first end or the proximal end.

[0027] The tubular portion of the support may be configured to mate with an outer surface of the tubular member.The inner surface of the tubular portion of the support may be configured to engage with an outer surface of an end of the tubular member, such as a first end.

[0028] The support and tubular member may be configured to engage with the tubular member (eg, a first end or a proximal end thereof) to form a watertight seal. One or more seals may be provided to provide a sealed engagement between the support and the tubular member.

[0029] In use, the support may extend substantially horizontally. The support may be configured to rotate the tubular member on a substantially horizontal axis. This may help achieve a consistent layer thickness of the biomaterial.

[0030] The device may comprise a barrier element.The barrier element may be configured to prevent, in use, the discharge of biological material at the second or distal end.

[0031] The blocking element may typically be disposed at an end of the tubular member, such as the second or distal end.

[0032] The blocking element may be separate and / or distinct from the tubular member.

[0033] The barrier element may be configured to provide a sealing engagement with the tubular member, for example at the second or distal end thereof.

[0034] The barrier element may be configured to engage with the inner surface and / or outer surface of the tubular member. Typically, the barrier element may be configured to engage with the inner surface of the tubular member.

[0035] The device may be configured to allow transport of biomaterial inside the tubular member, such as on the inner surface of the tubular member, such as on the tubular wall. In an embodiment, the barrier element may be configured to allow transport of biomaterial inside the tubular member, such as on the inner surface of the tubular member, such as on the tubular wall.

[0036] The barrier element may include an opening therebetween. The barrier element may include an opening near its central region. With this arrangement, the opening may allow for the delivery of biomaterials without compromising the barrier element's ability to prevent the discharge of biomaterials when the tubular member is rotated.

[0037] The blocking element may comprise or may be a plug.

[0038] The stopper may be made of an elastic or compressible material, such as foam or rubber. By this arrangement, the opening may not affect the ability of the stopper to prevent the discharge of the biomaterial, while allowing an object, such as a delivery device such as a syringe, to be inserted through the opening to allow the delivery of the biomaterial.

[0039] The barrier element may comprise a tubular barrier element or may be a tubular barrier element. The tubular barrier element may be rigid. The tubular barrier element may be made of a rigid material such as plastic, metal or glass.

[0040] The tubular barrier element may be configured to engage an end of the tubular member, such as the second end or the distal end.

[0041] The tubular barrier element may extend partially into the tubular member from the second or distal end.

[0042] The blocking element or parts of the blocking element may be integrally formed with the tubular member.

[0043] The blocking element may include or may define a ridge or shoulder extending radially inwardly, typically at or near an end of the tubular member, such as the second or distal end.

[0044] The tubular member, such as its second end or distal end, may include or define a ridge or shoulder that generally extends radially inward. Thus, the barrier element may define an opening having a size (e.g., inner diameter) that is smaller than the size (e.g., inner diameter) of the tubular member. With this configuration, when in use, the barrier element, such as the ridge or shoulder, may allow the delivery of biomaterial within the tubular member and may prevent the discharge of the biomaterial when the tubular member is rotated.

[0045] The blocking element may include one or more combinations of the above features.

[0046] In an embodiment, the blocking element may include:

[0047] a raised ridge or shoulder, which may typically extend radially inwardly at or near an end of the tubular member, such as the second or distal end; and

[0048] A tubular barrier element may extend partially from the second or distal end into the interior of the tubular member.

[0049] The ridge or shoulder and the tubular barrier element may be integral or one-piece. Alternatively, the tubular barrier element may be separate from the ridge or shoulder. The tubular barrier element may be configured to engage with the ridge or shoulder.

[0050] The tubular member may define an annulus extending from the second or distal end, located between the inner surface of the tubular member and the tubular barrier element. This may help prevent expulsion of biological material when the tubular member is rotated.

[0051] The tubular member comprises at least one opening configured to allow, in use, the escape of excess biological material.The at least one opening may extend through the wall of the tubular member from the inner surface to the outer surface thereof.

[0052] The at least one opening may be defined as or function as at least one leakage hole.

[0053] The tubular member may comprise a plurality of openings configured to allow escape of excess biological material in use.

[0054] Advantageously, providing at least one opening in the tubular member, for example a plurality of openings may allow for precise control of the thickness of each layer of biomaterial.

[0055] It may also be advantageous to provide at least one opening, for example a plurality of openings, in the tubular member to allow for removal of any gelling / cross-linking agent that may need to be added to the biomaterial to induce gelling and / or cross-linking thereof.

[0056] For example, when the biomaterial is a hydrogel, such as a low viscosity hydrogel, the gelling and / or crosslinking agent may be added after a layer of the biomaterial is formed. In use, after the gelling and / or crosslinking agent is added, the tubular member may be rotated to distribute the gelling and / or crosslinking agent, such as substantially evenly, over the biomaterial. The provision of at least one opening on the tubular member provides a path for any excess gelling and / or crosslinking agent to escape, thereby avoiding or reducing the risk of unreacted residual gelling and / or crosslinking agent remaining on the surface of the biomaterial prior to adding a fresh layer of the biomaterial, such as a hydrogel, thereby avoiding undesirable premature crosslinking and / or gelling thereof.

[0057] However, it will be appreciated that, depending on the nature of the biomaterial, gelation and / or cross-linking may not require the addition of a separate gelation and / or cross-linking agent. In this case, providing at least one opening, such as a plurality of openings, in the tubular member may still allow for precise control of the thickness of each layer of the biomaterial. The openings may be circumferentially disposed on the tubular member, such as the tubular wall. The openings may be located in a plane substantially perpendicular to the longitudinal axis of the tubular member. The openings may be located at substantially the same distance from the ends of the tubular member, such as the second end.

[0058] The location of the openings can allow the expulsion of excess biomaterial to be confined to a specific location and collected as it is expelled. This can be particularly useful if the material being used is high value and can be reused, as it can reduce waste of biomaterial. This is especially true compared to other methods where the layer is formed on the outer surface of the rotating rod.

[0059] The openings may be arranged symmetrically around the circumference of the tubular member. This may help to evenly distribute the flow of any excess biomaterial around the circumference of the tubular member, thereby providing a consistent layer thickness on the inner surface of the tubular member.

[0060] The openings may be of substantially the same size. This may further assist in evenly distributing the flow of any excess biomaterial over the circumference of the tubular member, thereby providing a consistent layer thickness on the inner surface of the tubular member.

[0061] N openings may be provided that are circumferentially arranged at about 360° / N relative to each other. For example, 3 openings may be provided that are arranged at about 120° relative to each other. 4 openings may be provided that are arranged at about 90° relative to each other. 5 openings may be provided that are arranged at about 72° relative to each other. 6 openings may be provided that are arranged at about 60° relative to each other.

[0062] A plurality of groups of openings may be provided, each group of openings being symmetrically arranged around the circumference of the tubular member.

[0063] A first set of openings may be provided, symmetrically arranged around the circumference of the tubular member, the first set of openings being located near the second end of the tubular member and at a first distance from the second end of the tubular member. The first set of openings may include N1 openings, which are circumferentially arranged at approximately 360° / N1 relative to each other.

[0064] A second set of openings may be provided, symmetrically arranged around the circumference of the tubular member, the second set of openings being located near the first end of the tubular member and at a second distance from the second end of the tubular member. The second set of openings may include N2 openings, which are arranged circumferentially at approximately 360° / N2 relative to each other.

[0065] Typically, at least one of the openings, such as the first set of openings and / or the second set of openings, may be substantially circular. This may facilitate the drainage of biological material.

[0066] At least one opening, such as the first group of openings and / or the second group of openings, can include a chamfer on the inner surface of the tubular member, such as the tubular wall. This can help or promote the discharge of the biomaterial and can help achieve a consistent thickness of the biomaterial.

[0067] The number of opening groups may depend on the location to which the biological material is desired to be fed or delivered.

[0068] If the biomaterial is intended to be delivered near an end of the tubular member, such as near a first end or a proximal end, a set of openings may be provided. These openings may be provided near a second end or a distal end of the tubular member. With this arrangement, upon rotation, the biomaterial may be coated on at least the inner surface of the tubular member between the delivery location and the set of openings.

[0069] If the biomaterial is expected to be delivered near the opposite end of the tubular member, such as near the second end or the distal end, a set of openings can be provided. The openings can be provided near the first end or the proximal end of the tubular member. With this arrangement, the biomaterial can be coated on the inner surface of the tubular member at least between the delivery position and the set of openings during rotation. This configuration can allow the biomaterial to be delivered or distributed near the second end or the distal end. Advantageously, this can minimize the insertion of a delivery device, such as a syringe or a nozzle, toward the first end or the proximal end of the tubular member. In other words, this can help reduce the overlap between the tubular member and the delivery device, which in turn can help reduce the risk or contact between the tubular member and the delivery device during rotation.

[0070] If the biomaterial is intended to be delivered at a location away from either end of the tubular member, such as near the central region of the tubular member, a set of openings may be provided near the first end or the second end of the tubular member, or multiple sets of openings, such as two sets of openings, may be provided. A first set of openings may be provided near the first end, and a second set of openings may be provided near the second end. With this arrangement, the biomaterial may be coated on the inner surface of the tubular member at least between the first set of openings and the second set of openings during rotation.

[0071] The advantage of the present invention is that when using low viscosity materials such as hydrogels, multilayer biomaterials can be formed. From the perspective of tissue engineering and biological research, these types of hydrogels are useful because they often simulate the characteristics of natural tissues. Collagen is an example of this material. Unfortunately, using the prior art, these materials are often difficult to form biologically relevant structures (such as microscopic scale multilayers) because they cannot maintain their 3D shape in the process of changing from liquid to gel form. The present invention allows the use of low viscosity biomaterials such as hydrogels to form multilayer structures that simulate the macroscopic and microscopic scale architectures of natural tissues.

[0072] The biomaterial may include or may be a hydrogel. The biomaterial may include alginate, collagen, etc. It should be understood that the term "biomaterial" is to be understood herein in the context of its use in tubular structures in biological applications. However, in some cases, the biomaterial may include a synthetic material suitable for biological applications, such as a polyether homopolymer or copolymer such as F-127( F-127).

[0073] The biological material may include cellular material, such as cells. Thus, the present apparatus and methods may allow for the preparation of multilayered cellular structures.

[0074] The device may comprise or may be coupled with a first control unit configured to control the rotation of the tubular member.The control unit may be configured to control the actuation of the rotatable support and / or the motor.

[0075] The first control unit may be configured to control the direction of rotation.

[0076] The first control unit may be configured to control the rotation speed. Typically, the first control unit may be configured to control and / or adjust the rotation speed to within a range of about 1000-15000 rpm, such as about 4000-10000 rpm.

[0077] The device may include or may be coupled to a second unit configured to control the delivery of the biomaterial.

[0078] The first control unit and the second control unit may be the same or different.

[0079] The device, such as the first and / or second control unit, may be manually actuated or operated.

[0080] The means, such as the first and / or second control unit, may be activated or operated automatically, such as by a computer.

[0081] The present invention provides a cost-effective method for biological tissue engineering, especially compared to bioprinting equipment, which is sometimes an economic barrier for researchers trying to biomanufacture tissues in the laboratory. The speed of manufacturing layered tissues using the present method provides researchers with a further advantage, especially compared to techniques such as cell-sheet engineering that require extended maturation times.

[0082] According to a second aspect, there is provided a method for manufacturing a tubular biological structure, the method comprising:

[0083] (i) providing an apparatus comprising a tubular member configured to be rotated, wherein the tubular member comprises a tubular wall extending from a first end and a second end, wherein the tubular member comprises at least one opening through the tubular wall, the at least one opening being configured to allow, in use, the escape of excess biological material;

[0084] (ii) feeding a first amount of biological material onto the inner surface of the tubular member; and

[0085] (iii) rotating the tubular member.

[0086] The tubular member may be the tubular member described in relation to the first aspect.

[0087] The apparatus may be the apparatus described in relation to the first aspect.

[0088] The method may include providing the tubular member on a rotatable support configured to be rotated. The method may include mounting the tubular member on the support.

[0089] The method may include mounting the tubular member on the support such that the tubular member extends substantially horizontally.

[0090] The method may include feeding a predetermined amount of biomaterial. It is understood that the amount of biomaterial fed into the tubular member may depend on the size of the tubular member, the desired thickness of the layer / each layer of the biomaterial, and / or the rotation speed expected to be adopted. Although the thickness of each layer is mainly controlled by the rotation speed and the viscosity of the material, it should be understood that the amount of material fed into the tubular member should be sufficient to generate a complete layer of material on the inner surface of the tubular member. Typically, the amount of material, such as volume, should be sufficient to induce a rimming flow on the inner surface of the tubular member, and any excess material can be discharged via at least one opening, such as a drain hole. Therefore, it is understood that the minimum amount of material required may depend on the size of the tubular member and the desired thickness of each layer. Typically, the method may include feeding about 0.002-4ml, such as about 0.01-1ml of biomaterial.

[0091] The method may comprise feeding the biological material via the opening in the plug at the second end of the tubular member.

[0092] The method may include rotating the rotatable support, thereby rotating the tubular member.

[0093] The method may include forming a layer, such as a first layer of a biomaterial, on an inner surface of a tubular member, such as a tubular wall.

[0094] The method may include actuating a motor coupled to a support.

[0095] The method may comprise rotating the tubular member on a substantially horizontal axis. This may help to achieve a consistent layer thickness of the biomaterial.

[0096] The method may include controlling the rotation speed of the rotatable support and / or tubular member. This may help achieve a desired layer thickness of the biomaterial.

[0097] The method may include rotating the tubular member at a speed of about 1000-15000 rpm, such as about 4000 to 10000 rpm.

[0098] Step (ii) may be performed before step (iii). In other words, the first amount of biomaterial may be fed into the tubular member before the tubular member is rotated. In this case, step (ii) may be performed when the tubular member is stationary and / or not rotating. This may minimize any risk of spillage and / or damage during the feeding step.

[0099] Steps (ii) and (iii) can be performed simultaneously. In this case, the first amount of biomaterial can be fed into the tubular member while the tubular member is rotated. In this case, step (ii) can be performed when the tubular member is already rotating. This can improve the uniformity of the layer thickness in the tubular member.

[0100] The method may comprise maintaining the same rotational speed during step (ii).

[0101] Alternatively, the method may comprise feeding a first amount of a biological at a first rotational speed, and subsequently setting a second rotational speed, such as increasing the rotational speed, in order to form a corresponding layer of material on the inner surface of the tubular member.

[0102] The method may include allowing excess biomaterial to drain out via at least one opening, such as via a plurality of openings, such as weep holes, during the rotating.

[0103] The method may include forming a layer of biomaterial on an inner surface of the tubular member, such as a tubular wall.

[0104] The method may include forming a layer of biomaterial between the first end of the tubular member and at least one opening, such as a first set of openings.

[0105] The method may include delivering a predetermined amount of biomaterial near a first end of the tubular member. In this case, a set of openings may be provided near a second end of the tubular member. The method may include forming a layer of biomaterial between the first end and the set of openings circumferentially located near the second end.

[0106] The method may include delivering a predetermined amount of biomaterial at either end remote from the tubular member, for example near a central region thereof. In this case, a set of openings may be provided near the second end of the tubular member, or multiple sets of openings, for example two sets of openings, may be provided. A first set of openings may be provided near the first end, and a second set of openings may be provided near the second end. The method may include forming a layer of biomaterial between the first end and the set of openings circumferentially located near the second end, or between the first set of openings and the second set of openings.

[0107] The method may further comprise:

[0108] (iv) adding or feeding a first amount of a gelling / crosslinking agent into the interior of the tubular member. With this arrangement, the gelling / crosslinking agent can initiate gelling and / or crosslinking of the first amount or first layer of the biomaterial.

[0109] It is understood that some biomaterials, such as alginate, may require the addition of a cross-linking agent or gelling agent to gel or cross-link. However, other types of biomaterials, such as collagen or agarose, may not require the addition of a cross-linking agent or gelling agent to gel or cross-link, and therefore may not require this additional step after each discrete layer of the biomaterial is formed.

[0110] The method may include feeding a gelling and / or cross-linking agent through an opening in the plug at the second end of the tubular member.

[0111] The method may include:

[0112] (v) rotating the tubular member, for example at a speed of about 1000 to 15000 rpm, for example about 4000 to 10000 rpm.

[0113] Preferably, the method may include adding or feeding a first amount of gelling / crosslinking agent inside the tubular member during rotation of the tubular member. The method may include performing steps (iv) and (v) simultaneously. Advantageously, this may prevent or reduce the risk of local or uneven / inhomogeneous crosslinking when adding the gelling / crosslinking agent.

[0114] In other words, while the biomaterial may be fed before or during rotation of the tubular member, any addition of gelling / cross-linking agents is preferably performed during rotation of the tubular member to increase the uniformity of the cross-linking process.

[0115] The method may include allowing excess gelling and / or cross-linking agent to drain off during the rotation via at least one opening, for example via a plurality of openings, ie, drain holes.

[0116] Advantageously, the method may allow for a substantially uniform distribution of the gelling and / or cross-linking agent on the biomaterial. Also advantageously, the method may allow for flushing or draining of any excess gelling and / or cross-linking material drained from the surface of the first layer of biomaterial. This may avoid or reduce the risk of unreacted residual gelling and / or cross-linking agent on the surface of the first layer of biomaterial prior to adding a fresh layer of biomaterial, such as a hydrogel, thereby avoiding undesirable premature cross-linking and / or gelling thereof.

[0117] Thus, the method may include forming a first layer of gelled or cross-linked biomaterial on the inner surface of the tubular member.

[0118] The method may comprise triggering cross-linking, for example, by applying a stimulus such as radiation (eg UV light) or temperature.

[0119] The method may include forming a second layer of a second biomaterial on the first layer. The second biomaterial may be the same as the first biomaterial, or may be different.

[0120] The method may include feeding a second amount of a second biomaterial onto the interior surface of the first layer; and

[0121] Rotate the tubular member.

[0122] Feeding of the second amount of the second biological material may be performed before rotating the tubular member, or may be performed while rotating the tubular member.

[0123] The method may include adding or feeding a second amount of gelling / crosslinking agent to the interior of the tubular member (preferably while rotating the tubular member). By this arrangement, the gelling / crosslinking agent may initiate gelling and / or crosslinking of the second amount or layer of biomaterial.

[0124] The method can include repeating this process to form multiple layers of the biological structure on the inner surface of the tubular member.

[0125] The method may comprise manually actuating or operating the device, such as a control unit.

[0126] The method may comprise automatically actuating or operating the device, such as a control unit.

[0127] The method may include disconnecting the tubular member from the rotatable support.

[0128] The method may include removing the plug.

[0129] The method can include removing the biological structure from the tubular member.

[0130] The method may comprise cutting the structure, for example longitudinally, in order to obtain a planar biological structure.

[0131] Thus, while an initial structure made using the present apparatus and methods may be in the form of a tubular structure, the final structure may not be tubular, and may be planar, for example.

[0132] According to a third aspect, there is provided a method of manufacturing a tubular multilayer biostructure, the method comprising:

[0133] (i) providing an apparatus comprising a tubular member configured to be rotated, wherein the tubular member comprises a tubular wall extending from a first end and a second end, wherein the tubular member comprises at least one opening through the tubular wall, the at least one opening being configured to allow, in use, the escape of excess biological material;

[0134] (ii) feeding a first amount of biological material onto the inner surface of the tubular member;

[0135] (iii) rotating the tubular member to form a first layer;

[0136] (iv) optionally adding or feeding a first amount of a gelling / crosslinking agent inside the tubular member to cause gelling and / or crosslinking of the first layer; and rotating the tubular member;

[0137] (v) Repeating steps (ii)-(vi) to form one or more additional layers.

[0138] According to a fourth aspect, there is provided a biological structure obtained or obtainable by a method according to the second or third aspect.

[0139] The biological structure may be tubular.

[0140] The biological structure may be planar, for example after cutting a tubular structure.

[0141] Features described in relation to any aspect of the present invention are equally applicable to any other aspect, and for the sake of brevity only, the features are not repeated. For example, features described in relation to an apparatus may be applied to a related method, and vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Embodiments of the present invention have been described with reference to the accompanying drawings, which show:

[0143] Figure 1 -5(b) a device for manufacturing a tubular biological structure according to the first embodiment;

[0144] Figure 6 use Figure 1 -5(b) Multilayer alginate-based hydrogel tubes with different inner diameters made by the device;

[0145] Figure 7 Figure 6 Bright field microscopy image of the tube wall showing discrete alginate layers, scale bar in the lower right is 500 μm;

[0146] Figure 8 (a)-8(e) Bright field microscopy images of tube walls formed using alginate 1% (w / v) at different rotation speeds, and (e) a graph showing the relationship between rotation speed and layer thickness;

[0147] Fig. 9 (a)-9(e) Bright field microscopy images of tube walls formed using alginate compositions at different concentrations (w / v) a) 4%, b) 2%, c) 1% and d) 0.5%, and (e) a graph showing the relationship between concentration and layer thickness;

[0148] Fig.10 (a)-10(e) Combined bright field and fluorescence microscopy images of tube walls formed using HEK cells encapsulated in 1% (w / v) alginate, showing live (green) and dead (red) cells at a) day 1, b) day 4, c) day 7, and d) day 10, and (e) a graph representing cell viability;

[0149] Fig.11 (a)-11(d) Combined bright field and fluorescence microscopy images of the tube wall showing the location of pre-labeled red and green high-density HEK293 cells in adjacent patterned layers formed using 1% w / v alginate.

[0150] Fig.11 (e)-11(f) Combined bright field and fluorescence microscopy images of the tube wall showing the location of the pre-labeled red and green high-density HEK293 cell layers formed using 1% w / v alginate with 5 ( Fig.11 (e)) or 10( Fig.11 (f)) No cell layer.

[0151] Fig.12 (a)-12(i) Studies of tubular structures made using human vascular smooth muscle cells (hSMCs) encapsulated in collagen hydrogels, showing (a) macroscale collagen tubular architecture with opaque appearance; (b) microscale acellular collagen layer formation; (c to f) viability of hSMCs in collagen, with live (green) and dead (red) cells at days 1, 4, 7, and 10 after biofabrication; (g) hSMC orientation 4 days after layering, with yellow arrows indicating circumferential direction; (h and i) confocal stacks showing hSMC localization, with acellular layer spacing of 20 (h) and 10 (i), respectively;

[0152] Fig.13 The distance between the human smooth muscle cell layers is caused by a set number of cell-free layers between the two populations. These correspond to Fig.12 (h) and double vertical lines in 12(i);

[0153] Fig.14 (a)-14(e) Bright field microscopy images of tube walls formed using agarose 1% (w / v) at different rotation speeds, and (e) a graph showing the relationship between rotation speed and layer thickness;

[0154] Fig.15 (a)-15(e) Bright field microscopy images of tube walls formed using agarose compositions at different concentrations (w / v) a) 8%, b) 4%, c) 2%, and d) 1%, and (e) a graph showing the relationship between concentration and layer thickness;

[0155] Fig.16 : a device for manufacturing a tubular biological structure according to another embodiment;

[0156] Fig.17 : Comparison of layer thickness obtained using manual method compared to automated method;

[0157] Fig.18 : Comparison of cell viability measured using manual method compared to automated method. DETAILED DESCRIPTION

[0158] In the present disclosure, some terms are mentioned, and unless the context indicates otherwise, these terms have the meanings provided below. The nomenclature used to define compounds (particularly compounds according to the present invention) herein is generally based on the rules of the IUPAC organization for compounds, specifically the "IUPAC Compendium of Chemical Terminology (Gold Book)". For the avoidance of doubt, if there is a conflict between the rules of IUPAC and the definitions provided herein, the definitions herein shall prevail. In addition, if there is a conflict between the structure of a compound and the name provided for the structure, the structure shall prevail.

[0159] The term "comprise" or variations thereof should be understood herein to mean the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of other elements, integers or steps.

[0160] The term "consisting of" or variations thereof should be understood to mean the inclusion of the stated elements, integers or steps, or groups of elements, integers or steps, and the exclusion of any other elements, integers or steps, or groups of any other elements, integers or steps.

[0161] The term "about" herein, when used to qualify a number or value, refers to a value within ±5% of the specified value. For example, if the specified temperature is about 5 to about 13°C, temperatures of 4.75 to 13.65°C are included.

[0162] References to the physical state of matter, such as liquid or solid, refer to the state of that matter at 25°C and atmospheric pressure, unless the context requires otherwise.

[0163] Figure 1 -5(b) illustrates an apparatus 105 for manufacturing a tubular biological structure according to a first embodiment, and a related method for manufacturing a tubular biological structure.

[0164] like Figure 1 As shown, the device 105 comprises a motor 110 configured to actuate a rotatable support 120 in the form of a cylinder.

[0165] The tubular member 150 defines an open tube having a circular cross-section.

[0166] The tubular member 150 has a first end 151 configured to engage with the support 120. The tubular member 150 has a second end 152 facing opposite to the support 120.

[0167] The tubular member 150 has an outer surface 153 on its outer side and an inner surface 154 on its inner side.

[0168] In the present embodiment, the support member 120 is a cylinder configured to sealingly engage the inner surface 154 of the tubular member 150 .

[0169] The device 105 includes a plug 155 configured to fit within the second end 152 of the tube 150 and configured to sealingly engage the inner surface 154 of the tube to prevent discharge of biological material at the second end 152 during use.

[0170] Plug 155 is configured to allow delivery of biological material 160 within tubular member 150, typically onto interior surface 154 thereof.

[0171] As best shown in Fig. 2(a), the stopper 155 has an opening 157, such as a slot or hole, near its central region. With this arrangement, the opening 157 allows the delivery of the biomaterial 160 via a delivery device 162 (a syringe in this embodiment) without compromising the ability of the stopper 155 to prevent the discharge of the biomaterial 160 when the tubular member 150 is rotated.

[0172] The tubular member 150 includes openings 156, two openings in this embodiment, disposed adjacent the second end 152 of the tube 150 and configured to allow for the escape of excess biomaterial in use, as will be described below.

[0173] FIG. 2 shows a first step of feeding a first amount (typically between 2 μl and 4 ml) of biomaterial 160 onto the inner surface 154 of the tubular member 150. As explained above, the plug 155 has an opening 157, such as a slot or hole, near its central region. With this arrangement, the opening 157 allows the biomaterial 160 to be delivered via a delivery device 162 (a syringe in this embodiment) without compromising the ability of the plug 155 to prevent the discharge of the biomaterial 160 when the tubular member 150 is rotated. As shown in FIG. 2 (b), in this embodiment, a predetermined volume of biomaterial 160 is delivered to the vicinity of the central region of the tubular member 150. However, as mentioned above, in other embodiments, the biomaterial 160 may be delivered to a different region of the tubular member 150, such as near the end of the tube 150 opposite to the plug 155 and / or the opening 156.

[0174] like Figure 1-3 As shown by the rotation arrow in , in this embodiment, the tubular member rotates in a continuous manner. However, as explained above, in other embodiments, the biomaterial 160 can be transported under static conditions, and the tubular member 150 can be rotated after the feeding step.

[0175] The delivery device 162 is then removed.

[0176] like Figure 3 As shown, tubular member 150 is rotated at a predetermined rotational speed. Advantageously, providing openings 156 in tubular member 150 allows excess biomaterial 160 to be drained from tube 150, which allows for precise control of the thickness of the layer of biomaterial 160 being formed on inner surface 154 of tube 150.

[0177] In this embodiment, there are two openings 156, which are arranged diametrically opposite each other. However, it will be appreciated that there may be any number N of openings, preferably arranged circumferentially in a symmetrical manner, for example at an angle of about 360° / N relative to each other.

[0178] In this embodiment, the opening 156 is substantially circular and also has a chamfer on the inner surface 154 of the tubular member 150. This may assist or may promote the drainage of the biomaterial and may help achieve a consistent thickness of the biomaterial.

[0179] Advantageously, the location of the openings 156 allows for the expulsion of excess biomaterial 160 to be restricted to a specific location and collected as it flows out. This can be particularly useful if the material being used is high value and can be reused, as it can reduce waste of biomaterial 160. This is especially true compared to other methods of forming a layer on the outer surface of a rotating rod.

[0180] If the biomaterial 160 requires the addition of a gelling agent or cross-linking agent to gel or cross-link the material between successive layers, for example for a hydrogel such as alginate, the method includes a cross-linking step, as illustrated in FIG4 . In this step, a first amount of a cross-linking agent or gelling agent 170 (in this embodiment, 100 mM CaCl) is fed onto the inner surface of the first layer of the material 160 via a delivery device 172 (in this embodiment, a syringe), which is inserted through the plug 155 via the plug opening 157. As shown in FIG4 (b), in this embodiment, a predetermined volume (usually between 2 μl and 4 ml) of the cross-linking agent 170 is delivered to the vicinity of the central region of the tubular member 150. The tube 150 is rotated so that the cross-linking agent or gelling agent 170 is coated on the surface of the biomaterial (alginate) 160 and causes its cross-linking or gelling. During the rotation of the tubular member 150, the addition of the first amount of gelling / cross-linking agent 170 is performed inside the tubular member 150. Advantageously, this can promote homogeneous cross-linking of the biomaterial 160 .

[0181] Also advantageously, the provision of openings 156 allows for the removal of any excess gelling / cross-linking agent 170, thereby avoiding or reducing the risk of unreacted residual gelling and / or cross-linking agent 170 on the surface of the biomaterial 160 prior to the addition of a fresh layer of biomaterial, thereby avoiding its unwanted premature cross-linking and / or gelling.

[0182] This step thus produces a first layer 165 of cross-linked or gelled biomaterial.

[0183] To build additional layers, the same process is repeated, as shown in FIG. 5 , where a second biomaterial 180 is fed onto the inner surface of the first layer 165 via a delivery device 182 and the tubular member 150 is rotated to produce a second layer.

[0184] It will be appreciated that the second material 180 (and any subsequent materials for additional layers) may be the same as or different from the first material 160, depending on the desired application and the desired biological structure.

[0185] The advantage of the present invention is that when using low viscosity materials such as hydrogels, multilayers of biomaterials can be formed. From the perspective of tissue engineering and biological research, these types of hydrogels are useful because they often simulate the characteristics of natural tissues. Collagen is an example of this material. Unfortunately, using the prior art, these materials are often difficult to form biologically relevant structures (such as microscopic scale multilayers) because they cannot maintain their 3D shape in the gel state form from liquid. The present invention allows the use of low viscosity biomaterials such as hydrogels to form multilayer structures that simulate the macroscopic and microscopic scale architectures of natural tissues.

[0186] Figure 6 Shown using the above Figure 1 -5(b) An example of a multilayer alginate-based hydrogel tube formed by the method described above, using tubes 150 of different diameters, in this case 3 mm, 5 mm, 7 mm and 10 mm, respectively.

[0187] Figure 7 Shows Figure 6 Bright field microscopy image of the wall of one of the tubes showing discrete alginate layers, scale bar at bottom right, 500 μm.

[0188] Fig.16 An apparatus for producing a tubular biological structure according to another embodiment is shown. Fig.15 The device 205 is generally similar to Figure 1 - The device 105 of Figure 5, like parts are indicated by like reference numerals, but with the addition of "100". However, in this embodiment, the device 205 is automatically operated.

[0189] Therefore, in this embodiment, the computer 290 controls:

[0190] - Rotation of the tubular member 250, e.g., actuation, speed, and duration;

[0191] - Operation of the feed delivery device 295, which here comprises a plurality of circulation units (such as pneumatic systems or pumps), which controls the delivery of the biological material 260 stored in the first container 263 via the delivery device 262, and controls the delivery of the cross-linking or gelling agent 270 stored in the second container 273 via the delivery device 272.

[0192] It will be appreciated that a plurality of control units or computers may be provided, each configured to control individual elements of the apparatus, such as actuation of tubular element 250 and operation of feed delivery device 295. A plurality of containers or other receptacles may also be provided for feeding or delivering additional material.

[0193] In addition, Fig.16In an embodiment, the device 205 does not include Figure 1 The compressible plug 155 shown in the drawings, instead, includes a blocking element 255, which is composed of a ridge or shoulder extending radially inward at the second or distal end 252, and a tubular blocking element engaged with the ridge or shoulder and extending partially from the second or distal end 252 into the tubular member 250. Figure 1 Like the plug 155 in FIG. 2 , the blocking element 255 has an opening in its central region that allows the delivery devices 262 , 272 to extend into the tubular member 250 without interfering with the tubular member 250 , even when rotated.

[0194] Example

[0195] Examples 1 and 2: Investigation of the relationship between rotation speed and layer thickness

[0196] Example 1

[0197] according to Figure 1 The multilayer tubes were made using the method described in -5, using 1% (w / v) alginate at different rotation speeds. At each stage, a volume between 30 μl and 100 μl was deposited in the tubes before rotation.

[0198] Layered tubes were produced at speeds of 4500, 6000, 7500 and 9000 rpm. Figure 8 (a)-8(d) show bright field microscope images of the tube wall of each tube, respectively. Figure 8 (e) is a graph showing the relationship between the rotation speed and the layer thickness.

[0199] As shown in the figure, the layer thickness is found to be a function of the rotation speed. Analysis of the layer thickness shows that increasing the rotation speed, the thinner the layer is formed, e.g. Figure 8 (e) as shown.

[0200] Example 2

[0201] Similar experiments were performed using 1% (w / v) agarose at different rotation speeds. However, the preparation of agarose-based structures did not require the addition of cross-linking agents. At each stage, volumes between 30 μl and 100 μl were deposited in the tubes before spinning. Layered tubes were made at speeds of 4500, 6000, 7500 and 9000 rpm. Fig.14 (a)-14(d) show bright field microscope images of the tube wall of each tube, respectively. Fig.14 (e) is a graph showing the relationship between the rotation speed and the layer thickness.

[0202] As shown in the figure, the layer thickness was found to be a function of the rotation speed. Analysis of the layer thickness showed that increasing rotation speed formed thinner layers, e.g. Fig.14 (e) as shown.

[0203] Examples 3 and 4: Investigation of the relationship between concentration and layer thickness

[0204] Example 3

[0205] according to Figure 1 -5, using different concentrations (w / v) of alginate solutions, formed at a rotation speed of 9000 rpm, to make multilayer tubes.

[0206] Layered tubes were made using alginate at concentrations (w / v) of (a) 4%, (b) 2%, (c) 1%, and (d) 0.5%.

[0207] Fig. 9 (a)-9(d) show bright field microscope images of the tube wall of each tube, respectively. Fig. 9 (e) is a graph showing the relationship between alginate concentration and layer thickness.

[0208] As shown in the figure, the layer thickness was found to be a function of the alginate concentration. Analysis of the layer thickness showed that increasing concentrations formed thicker layers, e.g. Fig. 9 (e) as shown.

[0209] Without wishing to be bound by theory, it is believed that higher percentage concentrations of alginate have greater viscosity, establishing a positive correlation between layer thickness and viscosity. It is believed that higher viscosity materials require higher rotational speeds, and therefore greater centrifugal forces, to enable a transition to annular flow, and therefore to achieve a specific thickness.

[0210] When trying to use low viscosity hydrogels, such as low concentrations of alginate or collagen, researchers often find that they are unable to form microscale features using traditional bioprinting methods. There is often a trade-off between degradation properties and printability. The present system is able to form microscale multilayer structures using low viscosity biomaterials (such as alginate) by forming a stable layer of material before adding a gelling agent (if necessary).

[0211] Example 4

[0212] Similar experiments were performed using agarose compositions at a rotation speed of 9000 rpm to form tubes. However, the preparation of agarose-based structures did not require the addition of a cross-linking agent.

[0213] Layered tubes were made using agarose at concentrations (w / v) of (a) 8%, (b) 4%, (c) 2%, and (d) 1%.

[0214] Fig.15 (a)-15(d) show bright field microscope images of the tube wall of each tube, respectively. Fig.15(e) is a graph showing the relationship between agarose concentration and layer thickness.

[0215] As shown above for the alginate compositions, the layer thickness with agarose was found to be a function of agarose concentration. Analysis of the layer thickness showed that increasing concentrations formed thicker layers, e.g. Fig.15 (e) as shown.

[0216] Example 5: Using this method to study the viability of living cells

[0217] according to Figure 1 -5, HEK cells encapsulated in 1% (w / v) alginate were used to form multilayer tubes at a rotation speed of 9000 rpm. In this example, the concentration of HEK cells in the biomaterial was 2.8×10 6 cells / ml.

[0218] Fig.10 (a)-10(d) show combined bright field and fluorescence microscopy images of tube walls formed using this material, showing live (green) cells and dead (red) cells at a) day 1, b) day 4, c) day 7, and d) day 10.

[0219] Cell viability data after layer encapsulation are shown in Fig.10 (e) Error bars are ± SD, n = 3, and white scale bar is 100 μm.

[0220] HEK cells encapsulated in a 1% (w / v) alginate layer were shown to have a day 1 viability of 97.5% ± 0.4. Fig.10 As shown in (e), the proportion of viable cells decreased over the following days, falling to 60.4% ± 1.2 on day 10. Without wishing to be bound by theory, the decrease in viability may be attributed to the formation of a necrotic core as the cells proliferate and transform into larger spheres.

[0221] The data show that trypsinization, precipitation, suspension in alginate and the overall formation scheme of this process lead to higher cell viability, and its percentage is similar to that obtained using extrusion bioprinting. Throughout the process, cells are subjected to the centrifugal force generated by tube rotation and the shear force generated by fluid flow. Simple centrifugal force calculations show that single cells are subjected to a force that is lower than the standard centrifugal force used during precipitation (1.55×10-11N vs 1.22×10-10N). Shear force is also considered to have an adverse effect on cell viability, and Mironov et al. proposes that the shear force generated by cell transfer rather than centrifugal force is the cause of cell death in their closed cylinder system. This viability data shows that shear force is low enough to keep cells alive, and it is possible to show that cell encapsulation with a higher rotation speed than shown in this article may be feasible.

[0222] Example 6: Study of cell layer localization

[0223] according to Figure 1 -5, using HEK293 cells encapsulated in 1% (w / v) alginate, formed at a rotation speed of 9000 rpm to make multilayer tubes. The composition includes a high-density cell population.

[0224] Fig.11 (a)-11(d) show combined bright field and fluorescence microscopy images of the tube wall, showing the location of pre-labeled red and green high-density HEK293 cells in adjacent patterned layers formed using 1% w / v alginate. The cell concentration in the biomaterial used to make these structures was 1.0×10 8 cells / ml.

[0225] Use different patterns to assemble the labeled layers, e.g. Fig.11 (a)-11(d) as shown.

[0226] Fig.11 (e)-11(f) show combined bright field and fluorescence microscopy images of the tube wall, showing the location of pre-labeled red and green high-density HEK293 cell layers formed using 1% w / v alginate with 5 ( Fig.11 (e)) or 10( Fig.11 (f)) Cell-free layer. The cell concentration in the biomaterial used to make these structures was 2.7×10 6 cells / ml.

[0227] The results confirmed that each layer was formed independently and separated from the adjacent layers, which made it possible to form highly diverse composite layered structures. The high-density cell layer was observed to be thicker than the cell-free layer and the low-density layer.

[0228] The results also showed that the low volumes of hydrogels required for assembly enabled high cell density encapsulation. In contrast, conventional tubular biofabrication techniques typically require large volumes for immersion, spraying, or extrusion.

[0229] Layered tissues vary significantly throughout mammalian anatomy, with a wide variety of combinations of cell types, matrices, and thicknesses of microlayer anatomy. Biofabrication methods that can account for this variation by controlled layer deposition that encapsulates high-density cells could play an important role in the future development of highly representative 3D tissues. Precise spatial distances between independent cell populations could also have applications beyond tissue engineering, such as in studies of cell-to-cell signaling.

[0230] Example 7: Formation of vascular collagen-based cell structures

[0231] according to Figure 1 -5, using hydrogel collagen and encapsulated human vascular smooth muscle cells (hSMCs) to make multilayer tubes, formed at a rotation speed of 4500 rpm and carried out at 37° C. A gelation time of 3 minutes was applied to each collagen layer instead of adding an ionic agent of alginate.

[0232] Fig.12 (a)-12(f) show studies of this structure.

[0233] Fig.12 (a) shows a macro-scale collagen tube architecture with an opaque white appearance fabricated by the present method.

[0234] Acellular tubes were also formed. Fig.12 (b) shows the formation of a microscopic acellular collagen layer for the structure. This shows evidence of microscopic layer formation when observed using bright field microscopy. However, due to the opacity of the gelled collagen, observation of the layer is more difficult compared to the alginate tube, with a layer of ≈15 μm visible on the outer edge of the wall ( Fig.12 (b)). This need to allow 3 minutes per layer to gel prolongs the biofabrication time, with approximately 1.5 hours required to make 25 collagen layers.

[0235] Fig.12 (c) to 12(f) show the viability of hSMC cells in collagen using the present process, with live (green) and dead (red) cells at 1, 4, 7, and 10 days after biofabrication. The cell concentration in the biomaterial used to make these structures was 7.4×10 6 These images demonstrate that hSMCs added as a monolayer between cell-free layers were viable up to 10 days after biofabrication.

[0236] Fig.12 (g) shows the hSMC orientation 4 days after delamination, with the yellow arrow indicating the circumferential direction. The cell concentration in the biomaterial used to make this structure was 0.6×10 6 Cells / ml. This observation reveals the network of hSMC arranged in the circumferential direction). This cell orientation in the microscopic scale layer is very similar to the microscopic scale anatomical structure observed in natural vascular tissue. The cell orientation after encapsulation is a challenge faced by tissue engineering strategies. It is not desirable to be bound by theory, but it is believed that the flow of the circumferential direction of the liquid phase collagen caused by the rotation of the molded tube, and the static tension generated by compaction on the inner core shaft, may be the reason causing hSMC to be arranged. Tensile force can be considered as a key factor, because the hSMC encapsulated in the monolayer does not show any visible compaction force on the macroscopic tube in viability and positioning experiments, and does not show any discernible arrangement.

[0237] Fig.12 (h) and Fig.12 (i) shows a confocal stack showing the localization of hSMCs with 20 (h) and 10 (i) cell-free layers in between. Observation of F-actin via phalloidin staining and confocal microscopy showed that hSMCs were spread out in two separate, independent, microscopic scale layers with narrow cell widths. The distance between cell layers increased proportionally in the 10- and 20-layer assemblies, with an average layer thickness of 12.5 ± 1.7 μm. This distance is very close to published measurements of the thickness of the native medial lamellar unit measured at 13.9 ± 1.2 μm (rat) and 13.2 μm (human). The assembly of viable hSMCs into concentric, anatomically accurate layers represents an important advance in vascular biofabrication technology. Bioprinting studies typically involve only the macroscopic layers of the intima, media, or adventitia, oversimplifying the complex microscopic layered architecture of these structures, such as the medial lamellar unit. The present system allows for the encapsulation of vascular cells in anatomically accurate concentric medial lamellae within the native ECM material of collagen.

[0238] Fig.13 The distances between the two populations caused by a set number of cell-free layers are shown. These correspond to Fig.12 (h) (20 layers, hence larger gaps) and Fig.12 Double vertical lines in (i) (10 layers, hence smaller gap).

[0239] Example 8: Automation of research equipment

[0240] With Figure 8 (Example 1) and Fig.10In a similar manner to the experiment of (Example 5), using Fig.16 Layer thickness and cell viability were studied using an automated setup.

[0241] Fig.17 A comparison of the layer thickness obtained using the manual method compared to the automated method is shown. It can be seen that the automated method produces similar results and therefore represents a reliable alternative method.

[0242] Fig.18 A comparison of cell viability measured using the manual method compared to the automated method is shown. It can be seen that the automated method produced similar results and therefore represents a reliable alternative method.

[0243] It should be understood that the present embodiment is provided by way of example only and that various modifications may be made to the present embodiment without departing from the scope of the present invention.

Claims

1. A device for manufacturing a tubular biological structure, the device comprising: a tubular member configured to be rotatable, wherein the tubular member comprises a tubular wall extending between a first or proximal end and a second or distal end, wherein the tubular member is configured to receive biological material on its inner surface, and wherein the tubular member comprises at least one opening through the tubular wall, the at least one opening being configured to allow expulsion of excess biological material in use.

2. The device according to claim 1, wherein: The device comprises a rotatable support configured to be rotated, wherein the tubular member is configured to be mounted on or attached to the rotatable support.

3. The device according to claim 1 or claim 2, wherein: The tubular member defines an open tube having a first open end and a second open end.

4. The device according to any one of claims 1 to 3, wherein: The device comprises a motor capable of actuating a rotation of the tubular member or a rotation of the rotatable support.

5. A device according to any one of the preceding claims, wherein The device comprises a barrier element configured, in use, to prevent discharge of the biological material at the second or distal end.

6. The device according to claim 5, wherein: The blocking element is disposed at the second or distal end of the tubular member.

7. The device according to claim 5 or claim 6, wherein: The barrier element is configured to allow transport of the biological material within the tubular member and onto the inner surface of the tubular member.

8. The device according to any one of claims 5 to 7, wherein: The blocking element includes an opening therein.

9. The device according to any one of the preceding claims, wherein: The at least one opening extends through the wall of the tubular member from the inner surface to the outer surface thereof.

10. The device according to any one of the preceding claims, wherein The tubular member comprises a plurality of openings configured to allow escape of excess biological material in use.

11. The device according to claim 10, wherein: The openings are circumferentially arranged on the tubular member.

12. The device according to claim 10 or claim 11, wherein: The openings are symmetrically arranged around the circumference of the tubular member.

13. The device according to any one of the preceding claims, wherein: The biomaterial comprises a hydrogel.

14. A device according to any one of the preceding claims, wherein The biological material includes cellular material.

15. The device according to any of the preceding claims, further comprising a control unit configured to control the rotation of the tubular member.

16. The device according to claim 15, wherein: The control unit is configured to control a rotation speed of the tubular member and / or the rotatable support.

17. The device according to claim 16, wherein: The control unit is configured to control and / or adjust the rotation speed to a speed within the range of about 4000-10000 rpm.

18. A method for manufacturing a tubular biological structure, the method comprising: Providing an apparatus comprising a tubular member configured to be rotated, wherein the tubular member comprises a tubular wall extending from a first end and a second end, wherein the tubular member comprises at least one opening through the tubular wall, the at least one opening being configured to allow, in use, the evacuation of excess biological material; feeding a first amount of biological material onto the inner surface of the tubular member; and The tubular member is rotated.

19. The method of claim 18, further comprising adding or feeding a first amount of a gelling / cross-linking agent into the tubular member and rotating the tubular member.

20. A method of making a tubular multilayer biostructure, the method comprising: (i) providing an apparatus comprising a tubular member configured to be rotated, wherein the tubular member comprises a tubular wall extending from a first end and a second end, wherein the tubular member comprises at least one opening through the tubular wall, the at least one opening being configured to allow, in use, the escape of excess biological material; (ii) feeding a first amount of biological material onto the inner surface of the tubular member; (iii) rotating the tubular member to form a first layer; (iv) optionally adding or feeding a first amount of a gelling / crosslinking agent inside the tubular member to cause gelling and / or crosslinking of the first layer; and rotating the tubular member; (v) Repeating steps (ii)-(vi) to form one or more additional layers.

21. The method according to any one of claims 18 to 20, further comprising cutting the structure, optionally longitudinally, so as to obtain a planar biological structure.

22. A biological structure obtained or obtainable by a method according to any one of claims 18 to 21.

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