Method and apparatus for preparing annular structures

Abstract

An apparatus (105) for manufacturing a tubular biological structure (165) comprises a tubular member (150) configured to be rotated, 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 internal surface (154) thereof, and wherein the tubular member (150) comprises at least one opening (156) through the tubular wall configured to allow egress of excess biological material (160), in use.

Description

Technical Field

[0001] The present invention relates to an apparatus and a method for preparing a tubular structure. In particular, although not limited thereto, the present invention relates to an apparatus and a method for preparing a tubular multi-layer biological structure.

Background Art

[0002] Layered tissues are common throughout human anatomy, with notable examples found in heart tissue, dermal tissue, intestinal tissue, and vascular tissue. Such layering creates a highly specialized composite structure characterized by a variety of different types of cells and extracellular matrix, conferring multiple properties and functions to the tissue. In tissue engineering aimed at creating anatomically and functionally accurate tissues, methods of suspending cells in a liquid phase hydrogel, depositing it, and then gelating it are frequently used. When forming layered tissues using such an approach, researchers have few technical options. Industry mainstream extrusion-based 3D bioprinting technologies are not suitable for assembling hydrogels into planar layered structures. In particular, they cannot achieve the deposition resolution and reproducibility required to assemble the multiple thin continuous layers observed in native tissue structures. These technologies also impose constraints on the range of compatible hydrogels because the printed liquid requires rapid gelation in order to take on the intended 3D spatial form.

[0003] Recognizing the limitations of 3D bioprinters that manipulate hydrogels into microscale layers, some researchers have attempted to develop improved biofabrication techniques. One such strategy involves sequentially immersing, spraying, or directly extruding cell-containing hydrogels onto the outer surface of a cylinder to construct layers. The advantage of this method lies in its ability to form tubular macrostructures, similar to those observed in many naturally occurring layered tissues such as vascular, intestinal, tracheal, and biliary tissues. Furthermore, the layered, tubular tissues can be easily converted into planar tissues by dissection. The limitation of the rod-based method is the need to immerse or spray the cell solution onto the outer surface. This requires a large amount of cell-containing hydrogel, which is ultimately wasteful and hinders the achievable cell density. Removal of the fabricated tissue can also be difficult due to the shrinkage of the hydrogel on the cylindrical mold. By rotating the cylinder at high speed under motor control, it becomes possible to control the layer thickness, which is determined by the viscosity of the liquid hydrogel and the surface properties of the substrate layer. However, this approach limits the cell density that can be achieved in the resulting layers. This means that the cells move away from the original tissue structure, reducing their clinical validity.

[0004] For example, Patent Document 1 (Acevedo et al.) discloses a method for manufacturing multilayer hollow tubes, in which a rod dipping method is used for each layer using different biological materials.

[0005] Another method is centrifugal casting, in which the material is applied to the inside of a sealed cylinder, which is then rotated. The centrifugal force extrudes the hydrogel-like material, coating the inner surface. However, this technique has received limited attention since the initial pioneering research by Mironov et al.

[0006] Non-patent document 1 by Mironov et al. discloses the use of a centrifugal casting method for producing tubular biological constructs made from crosslinkable hyaluronic acid-based (HA) synthetic extracellular matrix (sECM).

[0007] Patent Document 2 (Dalton et al.) discloses a hollow structure manufactured by a rotary spinning method. In this document, phase separation of a soluble solution or emulsion is induced within the mold as the filled mold rotates around one of its axes, and a precipitate is formed in the lumen of the mold due to centrifugal force caused by the density difference between the phases.

[0008] Patent Document 3 discloses artificial tissues and organs consisting of one or more layers of muscle, wherein the artificial tissues or organs are essentially made of cellular material, but are implantable in vertebrate subjects and are not blood vessels.

[0009] However, centrifugal casting is generally unsuitable for preparing multilayer biostructures because the addition of variable hydrogel volume results in inconsistent thickness. In addition, biomaterials such as collagen have generally proven difficult to handle using existing biofabrication techniques due to their low viscosity and the long thermal gelation time required for high levels of self-supporting and temperature control during the transition from liquid to gel. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent Publication No. 10688694B2 [Patent Document 2] U.S. Patent Publication No. 6969480 [Patent Document 3] Japanese Patent Application Publication No. Hei EP2755599B1 [Non-patent literature]

[0011] [Non-Patent Document 1] “Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in situ crosslinkable hyaluronan-gelatin hydrogel”, Biomaterials 26 (2005), 7628-7635 [Overview of the project] [Problems that the invention aims to solve]

[0012] The object of the present invention is to address and / or mitigate one or more problems related to the prior art.

[0013] The object of the present invention is to provide an improved method for producing tubular biological structures—for example, multilayer tubular biological structures. [Means for solving the problem]

[0014] According to a first embodiment, an apparatus for manufacturing a tubular biological structure is provided. The apparatus includes a tubular member configured to rotate, the tubular member including a tubular wall extending between a first end, i.e., a proximal end, and a second end, i.e., a distal end, the tubular member being configured to receive biological material on its inner surface, and the tubular member including at least one opening penetrating the tubular wall, configured to allow for the discharge of excess biological material during use.

[0015] The apparatus may include a rotatable support configured to rotate. The tubular member may be mounted on the rotatable support or configured to be mounted on the rotatable support.

[0016] A tubular member, such as a tubular wall, can have its outer surface and inner surface defined.

[0017] Typically, the tubular member can define an open tube. The first end of the tubular member can define a first opening. The second end of the tubular member can define a second opening. The first end may be provided on the opposite side of 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.

[0018] Advantageously, the tubular member may have a substantially constant diameter between the first end and the second end. The tubular member can have or define a substantially constant or continuous circular cross-section between the first end and the second end. With such a definition, an even thickness of the biological material on the inner surface of the tubular member can be achieved during rotation.

[0019] The device may include a motor capable of actuating the rotation of the tubular member.

[0020] The support may be connected to the motor or may be actuated by the motor. By actuating the motor, the support can be rotated. Thus, when the tubular member is attached to or mounted on the support, the tubular member can rotate via the rotatable support by actuating the motor.

[0021] The support may be configured to engage with the tubular member, for example, a tubular wall.

[0022] The support and the tubular member may have complementary features.

[0023] The support may include a tubular portion or a cylindrical portion.

[0024] The cylindrical portion of the support may be configured to fit within the end of the tubular member, for example, the first end, i.e., the proximal end. The outer surface of the cylindrical portion of the support may be configured to engage with the inner surface of the end of the tubular member, for example, the first end, i.e., the proximal end.

[0025] The tubular portion of the support may be configured to fit onto the outer surface of the tubular member. The inner surface of the tubular portion of the support may be configured to engage with the outer surface of the end of the tubular member, for example, the first end.

[0026] The support and the tubular member may be configured to engage with the tubular member, for example, its first end or proximal end, to form a waterproof seal. One or more seals may be provided to provide a sealed engagement between the support and the tubular member.

[0027] During use, the support extends substantially horizontally. The support may be configured to rotate a tubular member on a substantially horizontal axis. This allows for a constant layer thickness of the biological material.

[0028] The device may include a blocking section. The blocking section may be configured to block the discharge of biological material at the second end, i.e., the distal end, during use.

[0029] The blocking portion may typically be provided at the end of the tubular member, for example, at the second end or the distal end.

[0030] The blocking portion may be separate from and / or distinct from the tubular member.

[0031] The blocking portion may be configured to provide a sealing engagement to a tubular member, for example, its second end, i.e., its distal end.

[0032] The blocking portion can be configured to engage with the inner and / or outer surfaces of the tubular member. Typically, the blocking portion may be configured to engage with the inner surface of the tubular member.

[0033] The device may be configured to allow the supply of biological material into a tubular member, for example, onto the inner surface of the tubular member, for example, onto the tubular wall. In one embodiment, the blocking portion may be configured to allow the supply of biological material into a tubular member, for example, onto the inner surface of the tubular member, for example, onto the tubular wall.

[0034] The blocking portion may include an opening within it. The blocking portion may have an opening near its central region. Such a provision allows the opening to supply biological material without impairing the blocking portion's ability to prevent the discharge of biological material into the tubular member during rotation.

[0035] The blocking part may include a stopper, or it may be a stopper.

[0036] The stopper can be made of an elastic or compressible material—for example, foam or rubber. In this way, the opening does not impair the stopper's ability to prevent the discharge of biological material, while allowing an object, such as a syringe or other feeding device, to be inserted through the opening to enable the discharge of biological material.

[0037] The blocking portion may include a tubular blocking portion, or it may be a tubular blocking portion alone. The tubular blocking portion may be made of a rigid material. The tubular blocking portion can be made of a rigid material such as plastic, metal, or glass.

[0038] The tubular stopper may be configured to engage with the end of the tubular member, for example, the second end, i.e., the distal end.

[0039] The tubular blocking portion may partially extend into the tubular member from the second end, i.e., the distal end.

[0040] The blocking portion or a part of the blocking portion may be integrated with the tubular member.

[0041] The blocking portion may include, or define, a protrusion or shoulder that typically extends radially inward at or near the end of the tubular member, for example, the second end, i.e., the distal end.

[0042] The tubular member, for example, its second end, i.e., the distal end, may typically constitute or define a protrusion or shoulder extending radially inward. Thus, the blocking portion can define an opening having a dimension smaller than the dimensions of the tubular member, for example, the inner diameter, e.g., the inner diameter. With such a configuration, during use, the blocking portion, for example, the protrusion or shoulder, can enable the supply of biological material within the tubular member and prevent the discharge of biological material when the tubular member is rotated.

[0043] The blocking unit may have a combination of one or more of the above features.

[0044] In one embodiment, the blocking part is At the end of the tubular member, for example, at the second end or distal end, or in its vicinity, typically a protruding portion or shoulder portion extending radially inward, and A tubular blocking portion that partially extends into the tubular member from the second end, i.e., the distal end, It may be provided.

[0045] The raised portion or shoulder portion and the tubular stopper portion may be integral or integral. Alternatively, the tubular stopper portion may be separate from the raised portion or shoulder portion. The tubular stopper portion may be configured to engage with the raised portion or shoulder portion.

[0046] The tubular member may have an annular portion defined between its inner surface and the tubular retaining portion, extending from a second end or a distal end. This may help prevent the discharge of biological material into the tubular member as it rotates.

[0047] The tubular member comprises at least one opening configured to allow the discharge of excess biological material during use. The at least one opening may penetrate the wall of the tubular member and extend from its inner surface to its outer surface.

[0048] At least one opening may define at least one drain hole, or may function as at least one drain hole.

[0049] The tubular member may include a plurality of openings configured to allow for the discharge of excess biological material during use.

[0050] Advantageously, by providing at least one opening—for example, multiple openings—in the tubular member, the thickness of each layer of the biomaterial can be precisely controlled.

[0051] Advantageously, providing at least one opening—for example, multiple openings—in the tubular member allows for purging of gelling / crosslinking agents that need to be added to the biomaterial to initiate gelling and / or crosslinking.

[0052] For example, if the biomaterial is a hydrogel, such as a low-viscosity hydrogel, a gelling agent and / or crosslinking agent can be added following the formation of a layer of the biomaterial. During use, following the addition of the gelling agent and / or crosslinking agent, the tubular member may be rotated to distribute the gelling agent and / or crosslinking agent onto the biomaterial, for example, to distribute it substantially uniformly. By providing at least one opening in the tubular member, a path is provided for the escape of excess gelling agent and / or crosslinking agent, thus avoiding or reducing the risk of unreacted residual gelling agent and / or crosslinking agent being present on the surface of the biomaterial before a new layer of the biomaterial, such as a hydrogel, is added, and as a result, undesirable premature crosslinking and / or gelation is avoided.

[0053] However, it will be understood that, depending on the properties of the biomaterial, gelation and / or crosslinking may not require the addition of a separate gelling and / or crosslinking agent. In such cases, the thickness of each layer of the biomaterial can still be precisely controlled by providing at least one, for example, multiple, openings in the tubular member. The openings may be circumferentially arranged on the tubular member, for example, on the tubular wall. The openings may be arranged 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, for example, a second end.

[0054] By positioning the opening, the discharge of excess biological material can be limited to a specific location and recovered upon discharge. This is particularly useful when the material used is expensive and reusable, as it reduces waste of biological material. This is especially true compared to other approaches that involve forming a layer on the outer surface of the rotating rod.

[0055] The openings may be arranged symmetrically around the circumference of the tubular member. This allows for even distribution of excess biomaterial flow across the circumference of the tubular member, providing a consistent layer thickness on the inner surface of the tubular member.

[0056] The size of the openings may be substantially the same. This can further help to evenly distribute the flow of excess biomaterial around the circumference of the tubular member and provide a consistent layer thickness on the inner surface of the tubular member.

[0057] N openings may be provided, arranged circumferentially at approximately 360° / N relative to each other. For example, three openings may be provided, arranged at approximately 120° relative to each other. Four openings may be provided, arranged at approximately 90° relative to each other. Five openings may be provided, arranged at approximately 72° relative to each other. Six openings may be provided, arranged at approximately 60° relative to each other.

[0058] Multiple sets of openings may be provided, and each set of openings is arranged symmetrically around the circumference of the tubular member.

[0059] A first set of openings may be provided, arranged symmetrically around the circumference of a tubular member, the first set of openings located near a 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 arranged circumferentially at approximately 360° / N1 relative to each other.

[0060] A second set of openings may be provided, arranged symmetrically around the circumference of the tubular member, the second set of openings 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 arranged circumferentially at approximately 360° / N2 relative to each other.

[0061] Typically, at least one opening, for example, a first set of openings and / or a second set of openings, may be substantially circular. This may help in the discharge of biological material.

[0062] At least one opening, for example, a first set of openings and / or a second set of openings, can constitute a chamfer on the inner surface of the tubular member, for example, the tubular wall. This may help or facilitate the discharge of biomaterial and may help achieve a consistent thickness of biomaterial.

[0063] The number of openings may depend on the location where biological material is supplied or intended to be supplied.

[0064] If the biological material is intended to be supplied near the end of the tubular member, for example, near the first end, i.e., the proximal end, a pair of openings may be provided. The openings may be located near the second end, i.e., the distal end, of the tubular member. By providing them in this manner, during rotation, the biological material can be coated onto at least the inner surface of the tubular member between the supply location and the pair of openings.

[0065] If the biological material is intended to be supplied near the opposite end of the tubular member, for example, near the second end, i.e., the distal end, a pair of openings may be provided. The openings may be located near the first end, i.e., the proximal end, of the tubular member. By providing such an arrangement, during rotation, the biological material can be coated onto at least the inner surface of the tubular member between the supply location and the pair of openings. With this configuration, the biological material can be supplied or dispensed near the second end, i.e., the distal end. Advantageously, this can minimize the insertion of a supply device, such as a syringe or nozzle, toward the first or proximal part of the tubular member. In other words, this can help reduce the overlap between the tubular member and the supply device, and thus help reduce the risk or contact between the tubular member and the supply device during rotation.

[0066] If the biomaterial is intended to be supplied away from either end of the tubular member, for example near the central region, then one set of openings or multiple sets of openings, for example two sets of openings, may be provided near the first or second end of the tubular member. A first set of openings may be provided near the first end, and a second set of openings near the second end. This arrangement allows the biomaterial to be coated onto the inner surface of the tubular member between at least the first set of openings and the second set of openings during rotation.

[0067] An advantage of the present invention is that, when using low-viscosity materials such as hydrogels, multilayer structures of biomaterials can be formed. These types of hydrogels are useful from the standpoint of tissue engineering and biological research because they typically mimic the properties of innate tissues. Collagen is an example of such a material. Unfortunately, these materials often cannot maintain their three-dimensional shape when transitioning from liquid to gel form, making it difficult to form biologically appropriate structures (such as microscale multilayer structures) with existing techniques. The present invention makes it possible to form multilayer structures that mimic the macroscale and microscale structures of innate tissues using low-viscosity biomaterials such as hydrogels.

[0068] The biomaterial may contain or may not contain hydrogels. The biomaterial may also contain alginic acid, collagen, etc. In this specification, the term “biomaterial” will be understood to mean the use of tubular structures in biological applications. However, the biomaterial may, in some cases, consist of synthetic materials suitable for biological applications, such as homo- or copolymers of polyethers, like Pluronic® F-127.

[0069] The biological material may include cellular material, such as cells. Therefore, the apparatus and method of the present invention enable the preparation of multilayer cellular structures.

[0070] The device is connected to a first control unit configured to control the rotation of a tubular member. The control unit may be configured to control the operation of a rotatable support and / or motor.

[0071] The first control unit may be configured to control the direction of rotation.

[0072] The first control unit may be configured to control the rotational speed. Typically, the first control unit may be configured to control and / or adjust the rotational speed to a range of approximately 1,000 to 15,000 rpm, for example, approximately 4,000 to 10,000 rpm.

[0073] The device may be configured to be connected to a second unit configured to control the supply of biological material.

[0074] The first control unit and the second control unit may be the same or they may be different.

[0075] The apparatus, for example, the first control unit and / or the second control unit, may be operated or controlled manually.

[0076] The apparatus—for example, a first control unit and / or a second control unit—can be operated or controlled automatically by, for example, a computer.

[0077] This invention offers a cost-effective approach to tissue engineering, particularly for researchers attempting to create biological tissues in the laboratory, compared to bioprinting equipment, which can sometimes be an economic barrier. The speed of layered tissue preparation using the methodology of this invention offers further advantages to researchers, especially compared to techniques that require long maturation times, such as cell sheet engineering.

[0078] A second embodiment provides a method for manufacturing a tubular biological structure. The method comprises the steps of (i) providing an apparatus comprising a tubular member configured to rotate, the tubular member comprising a tubular wall extending from a first end and a second end, the tubular member comprising at least one opening penetrating the tubular wall configured to allow for the discharge of excess biological material during use; (ii) supplying a first amount of biological material to the inner surface of the tubular member; and (iii) rotating the tubular member.

[0079] The tubular member may be a tubular member as described in relation to the first embodiment.

[0080] The apparatus may be one of those described in relation to the first aspect.

[0081] The method may include providing a tubular member on a rotatable support configured to rotate. The method may also include mounting the tubular member on the support.

[0082] The method may include mounting the tubular member onto a support such that the tubular member extends substantially horizontally.

[0083] The method comprises supplying a predetermined amount of biomaterial. It will be understood that the amount of biomaterial supplied to the tubular member may depend on the size of the tubular member, the desired thickness of each layer of biomaterial, and / or the rotational speed to be applied. While the thickness of each layer is controlled primarily by the rotational speed and the viscosity of the material, it will be understood that the amount of material supplied to the tubular member should be sufficient to create a complete layer of material on the inner surface of the tubular member. Typically, the amount of material, e.g., volume, should be sufficient to induce a fringe flow on the inner surface of the tubular member, and any excess material can exit through at least one opening—e.g., a drain hole. Thus, it will be 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 have a step of supplying approximately 0.002 to 4 ml, e.g., approximately 0.01 to 1 ml of biomaterial.

[0084] The method may include a step of supplying biological material through the opening of the stopper in the second part of the tubular member.

[0085] The method includes the steps of rotating a rotatable support and rotating a tubular member.

[0086] The method may include the step of forming a layer of biological material, such as a first layer, on the inner surface of a tubular member, such as a tubular wall.

[0087] The method may include the step of operating a motor coupled to a support.

[0088] The method may include a step of rotating the tubular member on a substantially horizontal axis. This makes it possible to maintain a constant layer thickness of the biological material.

[0089] The method may include a step of controlling the rotational speed of a rotatable support and / or tubular member. This may help to achieve a desired layer thickness of a biological material.

[0090] The method may include a step of rotating the tubular member at a speed of approximately 1,000 to 15,000 rpm, for example, approximately 4,000 to 10,000 rpm.

[0091] Step (ii) may be performed before step (iii). In other words, the first amount of biological material may be supplied to the tubular member before the tubular member is rotated. In such a case, step (ii) can be performed while the tubular member is stationary and / or not rotating. This minimizes the risk of spillage and / or damage during the supply step.

[0092] Steps (ii) and (iii) may be performed simultaneously. In this case, the first amount of biomaterial can be supplied to the tubular member while the tubular member is rotating. In this case, step (ii) can be performed while the tubular member is already rotating. This may improve the uniformity of the layer thickness within the tubular member.

[0093] The method may include a step of maintaining the same rotational speed during step (ii).

[0094] Alternatively, the method may include the step of supplying a first amount of biological material at a first rotational speed, then setting a second rotational speed, for example increasing the rotational speed, to form each layer of material on the inner surface of a tubular member.

[0095] The method may include a step of enabling the discharge of excess biological material during rotation through at least one opening—for example, multiple openings, such as drain holes.

[0096] The method may include a step of forming a layer of biological material on the inner surface of a tubular member, for example, the wall of a tubular structure.

[0097] The method may include the step of forming a layer of biological material between the first end of the tubular member and at least one opening, for example, a first set of openings.

[0098] The method may include the step of supplying a predetermined amount of biological material near a first end of a tubular member. In such an example, a pair of openings provided near a second end of the tubular member may be provided. The method may also include forming a layer of biological material between the first end and the pair of circumferentially located openings near the second end.

[0099] The method may include supplying a predetermined amount of biomaterial at a location away from either end of a tubular member—for example, near its central region. In such an example, one set of openings may be provided near a 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 a first end, and a second set of openings may be provided near a second end. The method may include forming a layer of biomaterial between a set of circumferentially located openings near the first end and the second end, or between a first set of openings and a second set of openings.

[0100] The method may further include (iv) adding or supplying a first amount of gelling / crosslinking agent inside a tubular member. Such supply may cause the gelling / crosslinking agent to initiate the gelation and / or crosslinking of a first amount or first layer of the biological material.

[0101] It will be understood that certain types of biomaterials, such as alginates, require the addition of crosslinking or gelling agents to gel or crosslink. However, other types of biomaterials, such as collagen or agarose, may not require the addition of crosslinking or gelling agents for gelation or crosslinking, and therefore may not require this additional step after the formation of each discrete layer of the biomaterial.

[0102] The method may include supplying a gelling agent and / or a crosslinking agent through an opening in the stopper of the second part of the tubular member.

[0103] The method may include (v) a step of rotating the tubular member at a speed of, for example, about 1,000 to 15,000 rpm, or for example, about 4,000 to 10,000 rpm.

[0104] Preferably, the method may include adding or supplying a first amount of gelling / crosslinking agent into the interior of the tubular member while the tubular member is rotating. The method may include a step of performing steps (iv) and (v) simultaneously. Advantageously, this can prevent or reduce localized or non-uniform crosslinking associated with the addition of the gelling / crosslinking agent.

[0105] In other words, the biomaterial may be supplied either before or during the rotation of the tubular member, but the addition of the gelling / crosslinking agent may preferably be carried out during the rotation of the tubular member in order to improve the homogeneity of the crosslinking process.

[0106] The method may include a step that allows for the discharge of excess gelling agent and / or crosslinking agent during rotation through at least one opening, for example, multiple openings, for example, a drain hole.

[0107] Advantageously, the method may allow for substantially uniform distribution of the gelling agent and / or crosslinking agent onto the biomaterial. Advantageously, the method may also allow for flushing or removal of excess gelling or crosslinking material from the surface of the first layer of the biomaterial. This may avoid or reduce the risk of unreacted residual gelling and / or crosslinking agent being present on the surface of the first layer of the biomaterial before a fresh layer of the biomaterial, such as a hydrogel, is added, thus avoiding undesirable premature crosslinking and / or gelling.

[0108] Therefore, the method may include a step of forming a first gelled or cross-linked layer of biological material on the inner surface of the tubular member.

[0109] The method may include a step of activating crosslinking by applying a stimulus such as irradiation (e.g., UV light) or temperature.

[0110] The method may include forming a second layer of a second biological material on a first layer. The second biological material and the first biological material may be the same or different.

[0111] The method may include the steps of supplying a second amount of a second biological material to the inner surface of the first layer and rotating a tubular member.

[0112] The supply of a second quantity of the second biological material may be carried out before or simultaneously with the rotation of the tubular member.

[0113] The method may preferably involve adding or supplying a second amount of gelling / crosslinking agent into the tubular member while rotating the tubular member. Such supply allows the gelling / crosslinking agent to initiate the gelation and / or crosslinking of the second amount or layer of the second biomaterial.

[0114] The method may include repeating this step to form a multilayer biological structure on the inner surface of the tubular member.

[0115] The method may include a step of manually operating or controlling a device, such as a control unit.

[0116] The method may include a step of automatically operating or controlling a device, such as a control unit.

[0117] The method may include a step of separating the tubular member from the rotatable support.

[0118] The method may include the step of removing the stopper.

[0119] The method may include a step of removing the biological structure from the tubular member.

[0120] The method may include a step of cutting the structure, for example, in the longitudinal direction, to obtain a planar biological structure.

[0121] Therefore, the initial structure produced using the apparatus and method may be in the form of a tubular structure, but the final structure does not have to be tubular; for example, it may be planar.

[0122] According to a third aspect, a method for manufacturing a tubular multilayer biological structure is provided. The method includes (i) providing an apparatus comprising a tubular member configured to be rotated, the tubular member comprising a tubular wall extending from a first end and a second end, the tubular member comprising at least one opening penetrating the tubular wall configured to allow for the discharge of excess biological material during use; (ii) supplying a first amount of biological material to the inner surface of the tubular member; (iii) rotating the tubular member to form a first layer; (iv) optionally adding or supplying a first amount of a gelling / crosslinking agent into the interior of the tubular member to cause gelling and / or crosslinking of the first layer; and rotating the tubular member; and (v) repeating steps (ii) to (vi) to form one or more additional layers. It has.

[0123] According to the fourth aspect, a biological structure obtained by or obtained by the method according to the second or third aspect is provided.

[0124] The biological structure may be tubular.

[0125] The biological structure may be planar after, for example, a tubular structure has been cut.

[0126] Features described in relation to any aspect of the present invention may also be applicable to any other aspect and will not be repeated for the sake of brevity. For example, features described in relation to an apparatus may be applicable in relation to a method, and vice versa. [Brief explanation of the drawing]

[0127] Embodiments of the present invention will be described with reference to the accompanying drawings.

[0128] [Figure 1] This is an apparatus for manufacturing a tubular biological structure according to the first embodiment. [Figure 2] (a) and (b) are apparatus for manufacturing tubular biological structures according to the first embodiment. [Figure 3] This is an apparatus for manufacturing a tubular biological structure according to the first embodiment. [Figure 4] (a) and (b) are apparatus for manufacturing tubular biological structures according to the first embodiment. [Figure 5] (a) and (b) are apparatus for manufacturing tubular biological structures according to the first embodiment. [Figure 6] These are multilayer alginate hydrogel tubes with different inner diameters, manufactured using the apparatus shown in Figures 1-5(b). [Figure 7] Figure 6 is a bright-field microscope image of the tube wall, showing a discrete alginate layer (the scale bar in the lower right is 500 μm). [Figure 8] (a)-(e) are bright-field microscope images of tube walls formed using 1% (w / v) alginate at various rotation speeds, and (e) is a graph showing the relationship between rotation speed and layer thickness. [Figure 9] (a)-(e) are bright-field microscope images of tube walls formed using alginate compositions of various 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. [Figure 10] (a) to (e) are combined bright-field and fluorescence microscope images of tube walls formed using HEK cells encapsulated in 1% (w / v) alginate, and graphs showing live cells (green) and dead cells (red) on day 1, b) day 4, c) day 7, and d) day 10, and (e) cell viability. [Figure 11] (a) to (d) are bright-field and fluorescence microscope composite images of the tube wall showing the location of pre-labeled red and green high-density HEK293 cells in adjacent pattern layers formed with 1% w / v alginate. [Figure 11] (e) to (f) are combinations of bright-field and fluorescence microscopy images of the tube wall showing the location of five (Figure 11(e)) or ten (Figure 11(f)) noncellular layers between the red and green high-density HEK293 cell layers formed using 1% w / v alginate. [Figure 12] (a) to (i) are studies of tubular structures created using human vascular smooth muscle cells (hSMCS) embedded in collagen hydrogel. (a): Macroscale collagen tube structure with an opaque appearance, (b): Microscale acetolar collagen layer formation, (c): viability of hSMCs in collagen with live cells (green) and dead cells (red) 1 day after biofabrication, (d): viability of hSMCs in collagen with live cells (green) and dead cells (red) 4 days after biofabrication, (e): viability of hSMCs in collagen with live cells (green) and dead cells (red) 7 days after biofabrication, (f): viability of hSMCs in collagen with live cells (green) and dead cells (red) 10 days after biofabrication, (g): orientation of hSMCs 4 days after lamination (yellow arrows indicate circumferential direction), (h) confocal stack showing the position of hSMCs with spacing between 20 cell-free layers, and (i) confocal stack showing the position of hSMCs with spacing between 10 cell-free layers. [Figure 13] These are the distances between human smooth muscle cell layers, resulting from the insertion of a certain number of cell-free layers between two populations. These correspond to the double vertical lines in Figures 12(h) and 12(i). [Figure 14] (a) to (e) are bright-field microscope images of tube walls formed using 1% agarose (w / v) at various rotation speeds, and (e) is a graph showing the relationship between rotation speed and layer thickness. [Figure 15] (a) to (e) are bright-field microscope images of tube walls formed using agarose compositions of various concentrations (w / v): a) 8%, b) 4%, c) 2%, and d) 1%, and (e) is a graph showing the relationship between concentration and layer thickness. [Figure 16]This is an apparatus for manufacturing tubular biological structures according to another embodiment. [Figure 17] This is a comparison of layer thicknesses obtained using manual and automated approaches. [Figure 18] This is a comparison of cell viability measured using manual and automated approaches. [Modes for carrying out the invention]

[0129] In this disclosure, many terms are referenced, and unless the context indicates otherwise, these terms have the meanings set forth below. The nomenclature used herein to define compounds, and in particular compounds according to the present invention, is generally based on the rules of the IUPAC Organization for compounds, in particular the "IUPAC Compendium of Chemical Terminology (Gold Book)". To avoid misunderstanding, if the rules of the IUPAC Organization conflict with the definitions set forth herein, the definitions herein shall prevail. Furthermore, if the structure of a compound conflicts with the name provided for that structure, the structure shall prevail.

[0130] The term "comprising" or its variation thereof is understood in this specification to mean including the described components, integers or steps, or groups of elements, integers or steps, but not to mean excluding other elements, integers or steps, or groups of elements, integers or steps.

[0131] The term "consisting" or its variations is understood to mean including the listed elements, integers, or steps, or groups of elements, integers, or steps, and excluding other elements, integers, or groups of elements, integers, or steps.

[0132] In this specification, the term "approximately" is used to refer to a value that is within ±5% of a specified value when modifying a number or value. For example, if a temperature is specified as approximately 5 to approximately 13°C, it includes temperatures of 4.75 to 13.65°C.

[0133] Unless otherwise indicated in the context, references to the physical state of matter (such as liquid or solid) refer to the state of matter at 25°C and atmospheric pressure.

[0134] Figures 1-5(b) show an apparatus 105 for manufacturing a tubular biological structure according to the first embodiment, and related methods for manufacturing the tubular biological structure.

[0135] As shown in Figure 1, the device 105 includes a motor 110 configured to actuate a rotatable support 120 in the form of a cylinder.

[0136] The tubular member 150 defines an open pipe having a circular cross-section.

[0137] 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 away from the support 120.

[0138] The tubular member 150 has an outer surface 153 on its outside and an inner surface 154 on its inside.

[0139] In this embodiment, the support 120 is a cylinder configured to engage with the inner surface 154 of the tubular member 150 in a sealable manner.

[0140] The device 105 is configured to fit inside the second end 152 of the tubular member 150 and includes a stopper 155 configured to engage in a sealable manner with its inner surface 154 at the second end 152 to prevent the discharge of biological material during use.

[0141] The stopper 155 is configured to allow the supply of the biological material 160 within the tubular member 150, typically on its inner surface 154.

[0142] As best shown in Figure 2(a), the stopper 155 has an opening 157, for example, a slot or hole, near its central region. Such a provision allows the opening 157 to supply the biological material 160 via a supply device 162, in this embodiment a syringe, without impairing the stopper 155's ability to prevent the discharge of the biological material 160 when it is rotated into the tubular member 150.

[0143] The tubular member 150 includes an opening 156, in this embodiment two openings, located near the second end 152 of the tubular member 150, and is configured to allow for the discharge of excess biological material during use, as will be described later.

[0144] Figure 2 shows a first step of supplying a first amount, typically between 2 μl and 4 ml, of biomaterial 160 onto the inner surface 154 of the tubular member 150. As described above, the stopper 155 has an opening 157, e.g., a slot or hole, near its central region. Such provision allows the opening 157 to supply the biomaterial 160 via a supply device 162, in this embodiment a syringe, without impairing the stopper 155's ability to prevent the discharge of the biomaterial 160 when the tubular member 150 is rotated. As shown in Figure 2(b), in this embodiment, a predetermined amount of biomaterial 160 is supplied near the central region of the tubular member 150. However, as described above, in other embodiments, the biomaterial 160 may be supplied to different regions of the tubular member 150, e.g., near the ends of the tube 150 opposite the stopper 155 and / or the opening 156.

[0145] As shown by the rotational arrows in Figures 1-3, in this embodiment the tubular member is continuously rotated. However, as described above, in other embodiments the biological material 160 may be supplied under static conditions, and the tubular member 150 may be rotated following the supply step.

[0146] Afterward, the supply device 162 is removed.

[0147] As shown in Figure 3, the tubular member 150 is rotated at a predetermined rotational speed. Advantageously, by providing an opening 156 in the tubular member 150, excess biological material 160 can be discharged from the tube 150, thereby allowing for precise control of the thickness of the layer of biological material 160 formed on the inner surface 154 of the tube 150.

[0148] In this embodiment, there are two openings 156 arranged opposite each other. However, it will be understood that the openings may be any number N, preferably arranged symmetrically in a circumferential manner, for example, at an angle of about 360° / N relative to each other.

[0149] 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 help or facilitate the discharge of biomaterial and may help achieve a consistent thickness of biomaterial.

[0150] Advantageously, the positioning of the opening 156 allows excess biomaterial 160 to be contained in a specific location and recovered upon discharge. This is particularly useful when the material used is expensive and reusable, as it reduces waste of biomaterial 160. This is especially true compared to other approaches that form a layer on the outer surface of the rotating rod.

[0151] If the biological material 160 requires a gelling agent or crosslinking agent to be added to gel or crosslink the material between successive layers, such as a hydrogel like alginate, the method comprises a crosslinking step, as shown in Figure 4. In this step, a first amount of crosslinking agent or gelling agent 170 (100 mM CaCl in this embodiment) is supplied to the inner surface of the first layer 160 of the material via a supply device 172, in this embodiment a syringe, which is inserted from a stopper 155 through a stopper opening 157. As shown in Figure 4(b), in this embodiment, a predetermined amount, typically 2 μl to 4 ml, of crosslinking agent 170 is supplied near the central region of the tubular member 150. The tube 150 is rotated to coat the surface of the biological (alginate) material 160 with the crosslinking agent or gelling agent 170 and cause its crosslinking or gelling. The addition of the first amount of gelling / crosslinking agent 170 to the interior of the tubular member 150 occurs while the tubular member 150 is rotating. Advantageously, this promotes homogeneous crosslinking of the biomaterial 160.

[0152] Advantageously, the opening 156 also allows for purging of excess gelling / crosslinking agent 170, thus avoiding or reducing the risk of unreacted residual gelling and / or crosslinking agent 170 forming on the surface of the biomaterial 160 before a new layer of biomaterial is added, and as a result, undesirable premature crosslinking and / or gelling is avoided.

[0153] This process forms the first layer 165 of the cross-linked or gelled biomaterial.

[0154] To construct a further layer, the same process is repeated as shown in Figure 5, in which a second biomaterial 180 is supplied onto the inner surface of the first layer 165 via a supply device 182, and the tubular member 150 is rotated to form the second layer.

[0155] Depending on the required application and the desired biological structure, the second material 180 (and any subsequent materials used for additional layers) may be the same as or different from the first material 160.

[0156] An advantage of the present invention is that, using low-viscosity materials such as hydrogels, multilayer structures of biomaterials can be formed. These types of hydrogels often mimic the properties of innate tissues, making them useful from the perspective of tissue engineering and biological research. Collagen is an example of such a material. Unfortunately, these materials often cannot maintain their three-dimensional shape when transitioning from liquid to gel form, making it difficult to form biologically appropriate structures (such as microscale multilayer structures) using existing techniques. The present invention makes it possible to form multilayer structures that mimic the macroscale and microscale structures of innate tissues using low-viscosity biomaterials such as hydrogels.

[0157] Figure 6 shows an example of a multilayer alginate hydrogel tube formed using the method described above in Figure 1-5(b), using 150 tubes of different diameters (in this case, 3 mm, 5 mm, 7 mm, and 10 mm, respectively).

[0158] Figure 7 is a bright-field microscope image of one tube wall from the tube in Figure 6, showing a discrete alginate layer (scale bar in the lower right is 500 μm).

[0159] Figure 16 shows an apparatus for manufacturing tubular biological structures according to another embodiment. Apparatus 205 in Figure 15 is largely similar to apparatus 105 in Figures 1-5, with similar parts indicated by similar numbers, but increased by "100". However, in this embodiment, apparatus 205 is operated automatically.

[0160] Therefore, in this embodiment, the computer 290 controls the following: - Rotation of the tubular member 250, e.g., operation, speed and duration - Operation of the material supply device 295 - which comprises multiple circulation units (such as a pneumatic system or pumps) that control the supply of biological material 260 stored in the first container 263 via supply device 262, and the supply of crosslinking agent or gelling agent 270 stored in the second container 273 via supply device 272 -

[0161] It will be understood that multiple control units or computers may be provided, each configured to control the operation of a separate component of the apparatus—for example, the operation of the tubular component 250 and the material supply device 295. Furthermore, multiple containers or additional containers for supplying or dispensing additional material may also be provided.

[0162] In addition, in the embodiment shown in Figure 16, the device 205, instead of having a compressible stopper 155 as shown in Figure 1, has a blocking portion 255 comprising a protrusion or shoulder extending radially inward in the second, or distal, portion 252, and a tubular blocking portion that engages with the protrusion or shoulder and partially extends into the tubular member 250 from the second or distal portion 252. Similar to the stopper 155 in Figure 1, the blocking portion 255 has an opening in its central region, which allows the supply devices 262,272 to extend into the tubular member 250 without interfering with the tubular member 250, even when rotating. [Examples]

[0163] [Examples 1 and 2: Investigation of the relationship between rotation speed and layer thickness] [Example 1] Multilayer tubes were prepared using 1% (w / v) alginate at various rotation speeds according to the methods described in Figures 1-5. At each stage, 30 μl to 100 μl of the alginate was deposited into the tube before rotation.

[0164] Layered tubes were fabricated at rotational speeds of 4500, 6000, 7500, and 9000 rpm. Figures 8(a) to 8(d) are bright-field microscope images of the tube walls of each tube. Figure 8(e) is a graph showing the relationship between rotational speed and layer thickness.

[0165] Thus, it was found that the layer thickness is a function of the rotation speed. Analysis of the layer thickness revealed that, as shown in Figure 8(e), thinner layers are formed as the rotation speed increases.

[0166] [Example 2] Similar experiments were conducted using 1% (w / v) agarose at various rotation speeds. However, the addition of a crosslinking agent was not necessary for the preparation of the agarose-based structures. In each stage, 30 μl to 100 μl of material was submerged in the tube before rotation. Layered tubes were prepared at speeds of 4500, 6000, 7500, and 9000 rpm. Figures 14(a) to (d) are bright-field microscope images of the tube walls of each tube, respectively. Figure 14(e) is a graph showing the relationship between rotation speed and layer thickness.

[0167] Thus, it was found that the layer thickness is a function of the rotation speed. Analysis of the layer thickness revealed that, as shown in Figure 14(e), thinner layers are formed as the rotation speed increases.

[0168] [Examples 3 and 4: Investigation of the relationship between concentration and layer thickness] [Example 3] Multilayer tubes were fabricated using (w / v) alginate solutions of various concentrations formed at a rotation speed of 9000 rpm, according to the methods described in Figures 1-5.

[0169] Layered tubes were prepared using alginate concentrations of (w / v) (a) 4%, (b) 2%, (c) 1%, and (d) 0.5%.

[0170] Figures 9(a) to (d) are bright-field microscope images of the tube walls of each tube, respectively. Figure 9(e) is a graph showing the relationship between alginate concentration and layer thickness.

[0171] As shown, the layer thickness was found to be a function of alginate concentration. Analysis of the layer thickness revealed that, as shown in Figure 9(e), thicker layers were formed with increasing concentration.

[0172] While we do not wish to be bound by theory, it is thought that higher concentrations of alginate result in higher viscosity, and that there is a positive correlation between layer thickness and viscosity. High-viscosity materials are thought to require greater speed, and therefore greater centrifugal force, to transition to rimming flow and thus reach a certain thickness.

[0173] When attempting to use low-viscosity hydrogels such as low-concentration alginate or collagen, researchers often find that conventional bioprinting methods cannot form microscale features. A compromise between degradability and printability is frequently required. This system enables the formation of microscale multilayer structures using low-viscosity biomaterials (such as alginate) by forming a stable layer before adding the gelling agent.

[0174] [Example 4] Similar experiments were conducted using an agarose composition, and tubes were formed at a rotation speed of 9000 rpm. However, the addition of a crosslinking agent was not necessary for the preparation of the agarose-based structure.

[0175] Layered tubes were prepared with (w / v) agarose concentrations of (a) 8%, (b) 4%, (c) 2%, and (d) 1%.

[0176] Figures 15(a) to (d) are bright-field microscope images of the tube walls of each tube, respectively. Figure 15(e) is a graph showing the relationship between agarose concentration and layer thickness.

[0177] As shown in the alginic acid composition described above, the thickness of the agarose layer was found to be a function of the agarose concentration. Analysis of the layer thickness revealed that, as shown in Figure 15(e), thicker layers were formed as the concentration increased.

[0178] [Example 5: Investigation of the viability of living cells using the method of the present invention] Multilayer tubes were prepared using HEK cells encapsulated in 1% (w / v) alginate formed at a rotation speed of 9000 rpm, according to the method described in Figures 1-5. In this example, the concentration of HEK cells in the biological material was 2.8 × 10⁻⁶. 6 The value was cells / ml.

[0179] Figures 10(a) to 10(d) combine bright-field and fluorescence microscope images of tube walls formed using these materials, showing live cells (green) and dead cells (red) on days a) 1, b) 4, c) 7, and d) 10.

[0180] The cell viability data after layer encapsulation is shown in Figure 10(e). Error bars represent ±SD, n=3, and the white scale bar represents 100 μm.

[0181] HEK cells encapsulated in a 1% (w / v) alginate layer showed a survival rate of 97.5% ± 0.4 on day 1. As shown in Figure 10(e), the percentage of viable cells subsequently decreased to 60.4% ± 1.2 on day 10. While we do not wish to be bound by theory, the decrease in survival rate is thought to be due to the formation of a necrotic core as the cells proliferated and transitioned into larger spheroid bodies.

[0182] This data shows that the trypsinization, pelletization, suspension in alginate, and overall formation protocol of this process result in high cell viability at a similar percentage to that achieved in extrusion bioprinting. Throughout the current process, cells are subjected to centrifugal force from the rotation of the tube and shear force from the fluid flow. From a simple calculation of centrifugal force, the force acting on a single cell is equal to the standard centrifugal force used during pelletization (1.22 × 10⁻¹⁰). -10 1.55 × 10 for N -11It was found to be an order of magnitude lower than N). Shear force is also thought to have a detrimental effect on cell viability, and Mironov et al. proposed that in their closed cylindrical system, shear force resulting from cell migration rather than centrifugal force is the cause of cell death. The viability data presented here show that the shear force is low enough for cell survival, and that cell encapsulation is possible at rotation speeds higher than those shown here.

[0183] [Example 6: Examination of cell layer positioning] Multilayer tubes were prepared using HEK293 cells encapsulated in 1% (w / v) alginate at a rotation speed of 9000 rpm, according to the method described in Figures 1-5. This composition consisted of a high-density cell population.

[0184] Figures 11(a)–(d) combine bright-field and fluorescence microscope 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 create these structures was 1.0 × 10⁶. 8 The value was cells / ml.

[0185] As shown in Figures 11(a) to 11(d), the label layers were assembled using various patterns.

[0186] Figures 11(e) to 11(f) show bright-field and fluorescence microscope images of the tube wall, indicating the location of labeled red and green high-density HEK293 cell layers, formed using 1% w / v alginate and consisting of 5 (Figure 11(e)) or 10 (Figure 11(f)) cell layers. The cell concentration in the biological material used to create these structures was 2.7 × 10⁻⁶. 6 The value was cells / ml.

[0187] These results confirmed that each layer is formed individually and separate from adjacent layers, indicating the possibility of forming highly diverse complex layered structures. High-density cell layers were observed to be thicker than cell-free and low-density layers.

[0188] These results also demonstrate that high cell density encapsulation is possible because a small volume of hydrogel is required for assembly. In contrast, conventional tubular biofabrication techniques generally require large volumes for immersion, spraying, and extrusion.

[0189] Laminated tissues vary greatly depending on the anatomical structure of mammals, with various combinations of cell type, matrix, and anatomical thickness of the microlayer. Biofabrication methods that can account for this variation through controlled layer deposition containing high-density cells can play a valuable role in the future development of highly representative three-dimensional tissues. Precisely spatially separating independent cell populations may also have applications beyond tissue engineering, such as the study of intercellular signaling.

[0190] [Example 7: Formation of vascular collagen cell structures] Multilayer tubes were prepared using hydrogel collagen and encapsulated human vascular smooth muscle cells (hSMCS) according to the methods described in Figures 1-5. Formation was performed at a rotation speed of 4500 rpm at 37°C. The gelation time for each collagen layer was 3 minutes, without the addition of alginate ion agent.

[0191] Figures 12(a) to 12(f) show an investigation of such structures.

[0192] Figure 12(a) shows a macroscale collagen tube structure with an opaque white appearance prepared by this method.

[0193] A cellulose tube was also formed. Figure 12(b) shows the microscale cellulose collagen layer formation in this structure. This provides evidence of microscale layer formation when viewed under a bright-field microscope. However, visualization of the layers was more difficult than in the alginate tube due to the opacity of the gelled collagen, and only a layer of about 15 μm was visible at the outer edge of the wall (Figure 12(b)). Since each layer needs to be gelled for 3 minutes, the biofabrication time is long, requiring about 1.5 hours for 25 collagen layers.

[0194] Figures 12(c) to (f) show the viability of hSMC cells in collagen produced by this method, with live cells (green) and dead cells (red) at 1, 4, 7, and 10 days after bio-assembly. The cell concentration in the biological material used to prepare these structures was 7.4 × 10⁻⁶. 6 The values ​​were cells / ml. These images show that hSMC, added as a monolayer within a cell-free layer, remained viable for up to 10 days after biological tissue formation.

[0195] Figure 12(g) shows the orientation of hSMCs 4 days after stratification, with the yellow arrows indicating the circumferential direction. The cell concentration in the biological material used to prepare this structure was 0.6 × 10⁻⁶. 6 The concentration was cells / ml. This visualization revealed that the hSMC network was aligned circumferentially. This cell orientation in a microscale layer faithfully mimics the microscale anatomical structures observed in natural vascular tissue. Cell orientation after encapsulation is a challenge for tissue engineering strategies. While we do not wish to be bound by theory, it is thought that the circumferential flow of liquid-phase collagen caused by the rotation of the molded tube and the static tension induced by compression inward on the mandrel contribute to the orientation of hSMCs. In viability and positioning experiments, monolayer-encapsulated hSMCs did not show visible compressive forces or discernible alignment on the macroscale tube, suggesting that tensile forces can be identified as a major contributing factor.

[0196] Figures 12(h) and 12(i) show confocal stacks of hSMCs positioned at cell-free layer intervals of 20(h) and 10(i). Phalloidin staining and visualization of F-actin by confocal microscopy revealed that the hSMCs were spread across two separate, independent, narrow-scale micro-layers. The distance between cell layers in the 10-layer and 20-layer aggregates increased proportionally, with an average layer thickness of 12.5 ± 1.7 μm. This distance is in close agreement with previously published measurements for the thickness of the intrinsic inner lamellar unit, which were measured at 13.9 ± 1.2 μm (rat) and 13.2 μm (human). Assembling living hSMCs concentrically into anatomically accurate layers represents a significant advance for vascular biofabrication techniques. Bioprinting studies often only mention the macrolayers of the intima, media, and adventitia, oversimplifying the complex micro-scale layered structures within these structures, such as the inner lamellar unit. This system makes it possible to encapsulate vascular cells in anatomically precise, concentric inner lamellar units within the original ECM material, which is composed of collagen.

[0197] Figure 13 shows the distances between layers of human smooth muscle cells. These correspond to the double vertical lines in Figure 12(h) (20 layers, therefore large gaps) and Figure 12(i) (10 layers, therefore small gaps).

[0198] [Example 8: Examination of automation of the device] Using the automated apparatus shown in Figure 16, the layer thickness and cell viability were investigated in the same manner as in the experiments shown in Figure 8 (Example 1) and Figure 10 (Example 5).

[0199] Figure 17 shows a comparison of the layer thicknesses obtained by the manual and automated approaches. The automated approach yields similar results and is therefore a reliable alternative.

[0200] Figure 18 shows a comparison of cell viability measured using manual and automated approaches. Similar results were obtained with the automated approach, demonstrating its reliability as an alternative.

[0201] This embodiment is provided for illustrative purposes only, and it will be understood that various modifications can be made to this embodiment without departing from the scope of the invention. The apparatus 105 for manufacturing a tubular biological structure 165 comprises a tubular member 150 configured to rotate. The tubular structure 150 has a tubular wall extending from a first portion 151 to a second portion 152. The tubular member 150 is configured to receive biological material 160 on its inner surface 154. The tubular member 150 has at least one opening 156 penetrating the tubular wall 160, configured to allow the release of excess biological material 160 during use.

Claims

1. An apparatus for manufacturing tubular biological structures, It comprises a tubular member configured to rotate, The tubular member has a tubular wall extending from the first portion to the second portion, The tubular member is configured to receive biological material on its inner surface, The tubular member has at least one opening that penetrates the tubular wall, which is configured to allow the release of excess biological material during use. Device.

2. The apparatus according to claim 1, comprising a rotating support configured to rotate, wherein the tubular member is configured to be placed on the rotating support or attached to the rotating support.

3. The apparatus according to claim 1 or 2, wherein the tubular member defines an open pipe having a first opening and a second opening.

4. An apparatus according to any one of claims 1 to 3, comprising a motor having the function of starting the rotation of the tubular member or the rotating support.

5. The apparatus according to any one of claims 1 to 4, comprising a blocking unit configured to prevent the release of the biological material in the second part, i.e., the far part, when in use.

6. The apparatus according to claim 5, wherein the blocking portion is provided to the second portion, i.e., the far portion, of the tubular member.

7. The apparatus according to claim 5 or 6, wherein the blocking portion is configured to enable the supply of the biological material onto the inner surface of the tubular member inside the tubular member.

8. The apparatus according to any one of claims 5 to 7, wherein the blocking portion has an opening inside the blocking portion.

9. An apparatus according to any one of claims 1 to 8, wherein the at least one opening penetrates the wall of the tubular member from the inner surface to the outer surface of the tubular member.

10. The apparatus according to any one of claims 1 to 9, wherein the tubular member has a plurality of openings configured to allow the release of excess biological material during use.

11. The apparatus according to claim 10, wherein the plurality of openings are arranged around the periphery of the tubular member.

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

13. An apparatus according to any one of claims 1 to 12, wherein the biological material includes a hydrogel.

14. An apparatus according to any one of claims 1 to 13, wherein the biological material includes a cellular material.

15. An apparatus according to any one of claims 1 to 14, further comprising a control unit configured to control the rotation of the tubular member.

16. The apparatus according to claim 15, wherein the control unit is configured to control the rotational speed of the tubular member and / or the rotating support.

17. The apparatus according to claim 16, wherein the control unit is configured to control and / or adjust the rotational speed to a speed within the range of approximately 4,000 to 10,000 rpm.

18. A method for producing tubular biological structures, A step of providing a device comprising a tubular member configured to rotate, a tubular wall extending from a first portion to a second portion, and at least one opening penetrating the tubular wall configured to allow the release of excess biological material during use, The steps include supplying a first amount of biological material onto the inner surface of the tubular member, The step of rotating the tubular member, A method of having.

19. A method according to claim 18, further comprising the steps of adding or supplying a first amount of gelling / crosslinking agent inside the tubular member, and rotating the tubular member.

20. A method for producing a tubular multilayer biological structure, (i) Providing a device comprising a tubular member configured to rotate, a tubular wall extending from a first portion to a second portion, and at least one opening penetrating the tubular wall configured to allow the release of excess biological material during use; (ii) A step of supplying a first amount of biological material onto the inner surface of the tubular member, (iii) A step of generating a first layer by rotating the tubular member, (iv) A step of optionally adding or supplying a first amount of gelling / crosslinking agent inside the tubular member to cause gelling and / or crosslinking of the first layer, and a step of rotating the tubular member, (v) A step in which one or more additional layers are generated by repeating (i) to (iv). A method of having.

21. A method according to any one of claims 18 to 20, further comprising the step of cutting the structure—optionally in the longitudinal direction—to obtain a planar biological structure.

22. An apparatus according to any one of claims 18 to 21, the resulting biological structure.

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