METHOD FOR MANUFACTURING A VASCULAR STRUCTURE

Abstract

A method for manufacturing a vascular structure comprising: a) the fabrication of elements comprising at least one core comprising at least one biopolymer, and endothelial cells and / or endothelial progenitor stem cells on its outer periphery; b) the placement of said elements in a fluidic chamber so that they form at least one stack; c) the establishment of a fluid flow ensuring nutrient and oxygenation supply through the stack so as to create channels between the elements along the flow lines. No abstract figure.

Description

Title of the invention: METHOD FOR MANUFACTURING A VASCULAR STRUCTURE

[0001] TECHNICAL FIELD AND PRIOR TECHNOLOGY

[0002] The present invention relates to a method of manufacturing a vascular structure, for example to manufacture a vascularized tissue, or even a vascularized organ, for example to carry out an in vivo implantation of this organ.

[0003] In the medical field, the aim is to create organs-on-a-chip in order to study their reaction to drugs, for example. To this end, medical teams are trying to manufacture a vascular network whose behavior closely resembles that of organs in vivo.

[0004] The human vascular network is composed of blood vessels including arteries, arterioles, veins, venules, and capillaries. It originates from the heart and distributes throughout the body. The vascular network is responsible for transporting blood, which delivers oxygen, nutrients, hormones, blood cells, immune cells, fluids, and heat to the tissues while collecting waste products. In vivo, tissues are perfused, supplied by an entire vascular network, and more specifically by capillaries within the tissues.

[0005] The vascular system is complex and branched. The internal diameter of blood vessels can range from a few microns in diameter, on the order of 5 µm, to several millimeters for arteries, on the order of 25 mm. The characteristics of these vessels are varied, as is their composition, depending mainly on their final function.

[0006] The manufacture of an artificial vascular network is therefore very complex.

[0007] There are various techniques for attempting to manufacture a structure that approximates of a vascular structure of a human body.

[0008] One technique consists of coating the wall of a channel made of a plastic or glass substrate with hydrogel and endothelial cells. A flow of liquid passes through the channel, providing nutrients and oxygen, allowing the cells to grow and form tubes.

[0009] Another technique consists of placing the endothelial cells in a medium favorable to their development, which will then self-arrange until they form three-dimensional tubular structures.

[0010] However, the structures obtained by the first technique are complex to implement and do not allow for the creation of endothelialized hollow tubes with diameters smaller than approximately 100 µm. The second technique, based on the self-assembly of Endothelial cells allow for a more physiological capillary network. However, the formation of this network takes more than a week. Other cell types potentially involved in the construction are not nourished during this period and undergo necrosis, preventing the formation of thick tissue. This is why devices using this technique do not exceed one hundred micrometers in thickness.

[0011] There is also a bioprinting technique that involves printing the vascular structure using an ink containing endothelial cells. The extrusion bioprinting method makes it possible to form vessels several hundred micrometers in diameter, for example, between 600 and 700 micrometers. However, this method is time-consuming and requires the printing of scaffolds to maintain the three-dimensional structure. Furthermore, it places significant stress on the cells. Indeed, during ink preparation and printing, the endothelial cells are not oxygenated. In addition, the resolution limitations of the bioprinting technique do not allow for achieving the size of capillaries.

[0012] These techniques do not allow the fabrication of thick vascularized tissues. Non-vascularized tissue models have been designed and implanted in vivo (skin, cartilage). A tissue-engineered pulmonary artery was also transplanted into a 4-year-old child with pulmonary atresia. This is described in Shin'Oka, 2001, Transplantation of a tissue-engineered pulmonary artery. A network of hollow tubes was also bioprinted and implanted in mice and showed anastomosis with its vascular system (Sooppan, 2015, In vivo anastomosis and perfusion of a 3D printed construct containing microchannel net Works). Description of the invention

[0013] It is therefore an object of the present invention to provide a method for manufacturing a complex vascular structure, in particular one which may include capillaries for the purpose of producing thick tissues or organs on a chip, or for the purpose of in vivo implantation of tissues or organs.

[0014] The above-mentioned objective is achieved by a manufacturing method comprising the production of elements comprising a biopolymer core and at least one outer layer comprising endothelial cells and / or endothelial progenitor stem cells, the stacking of the elements in a fluidic chamber comprising an inlet orifice and an outlet orifice and the application of a fluid flow through the stack of elements, the fluid comprising at least nutrients and ensuring oxygenation.

[0015] The method according to the invention makes it possible to produce thick fabrics incorporating capillaries, for example fabrics several hundred µm thick, whereas the methods of the prior art do not make it possible to produce both fabrics equipped with capillaries and having sufficient thickness.

[0016] Advantageously, the flow is such that it applies shear forces to the cells, promoting the attachment of the cells to each other, and their elongation along the streamlines.

[0017] By choosing the size of the elements, it is possible to manufacture a capillary network or arterioles and / or venules, by adapting the inter-element spacing which will define the maximum size of the blood vessels.

[0018] Very advantageously, by using several sizes of elements it is possible to manufacture a structure comprising arterioles and venules connected by capillaries.

[0019] Advantageously, the biopolymer core contains cells from an organ to be manufactured. Thus, a vascularized structure of a given organ is directly manufactured.

[0020] The biopolymer can be retained in the structure or eliminated.

[0021] The method according to the invention offers great freedom in the type of structure which can be manufactured by choosing the type(s) of biopolymer(s), the type(s) of cell(s), and the flow characteristics.

[0022] In other words, a stack of biopolymer capsules is created, which can contain one or more types of cells, the capsules being at least partially covered with endothelial cells and / or endothelial progenitor stem cells, and a supply of nutrients and oxygenation is ensured by fluid circulation through the stack. Channels then form along the flow lines.

[0023] The present invention relates to a method for manufacturing a vascular structure comprising: a) the manufacture of elements comprising at least one nucleus comprising at least one biopolymer, and endothelial cells and / or endothelial progenitor stem cells on its outer periphery, b) placing said elements in a fluidic chamber so that they form at least one stack, c) the establishment of a fluid flow ensuring a supply of nutrients and oxygenation through the stack so as to provide channels between the elements along the flow lines.

[0024] For example, the fluid flow has a flow rate such that the elements undergo an average shear stress of between 0.1 dyne / cm2 and 100 dynes / cm2, preferably between 1 dyne / cm2 and 10 dynes / cm2.

[0025] In one example, during step a), cells are introduced into the nucleus. The nucleus may comprise at least a core containing cells and an outer layer containing cells, the cells of the core and the outer layer being of the same type or not. The outer layer may contain pericytes.

[0026] For example, in step a), the nucleus is made with a mixture of biopolymers, at least one of which offers good affinity with endothelial cells and / or endothelial progenitor stem cells

[0027] Preferably, the elements have a largest dimension less than 1 mm, advantageously less than 500 pm.

[0028] The elements are advantageously arranged in a culture medium before step b) to allow the proliferation of endothelial cells and / or endothelial progenitor stem cells and the total or partial covering of the nuclei.

[0029] During step b), it may be planned to put in place elements of different dimensions and / or shapes. Elements of different sizes may be distributed in several successive stacks in the direction of the flow.

[0030] For example, a stack of smaller elements having an inter-element spacing on the order of the diameter of the capillaries is arranged between two stacks of larger elements having an inter-element spacing on the order of the diameter of the arterioles or venules.

[0031] Advantageously, prior to the placement of the elements in the fluidic chamber, endothelial cells and / or endothelial progenitor stem cells cover the walls of the chamber and / or the fluid inlet and / or outlet channels.

[0032] The present invention also relates to a system for manufacturing a vascular structure for implementing the manufacturing method according to the invention comprising n fluidic chambers, n > 1, provided with a fluid supply port and a fluid discharge port, a cavity disposed between the supply port and the discharge port and configured to receive the elements, and means for maintaining the elements in the cavity, a fluid reservoir connected at least to the supply inlet and circulation means to ensure the circulation of the fluid through the cavity.

[0033] The reservoir is advantageously connected to the discharge port so as to form a closed circuit.

[0034] According to an additional feature, the system may include means for isolating the fluidic chamber before stopping the circulation means.

[0035] According to another additional feature, the manufacturing system includes means for oxygenating the fluid.

[0036] Each fluidic chamber may include a removable part to allow access to the cavity and removal of the vascular structure.

[0037] In an advantageous example, the cavity comprises a central part and upstream and downstream ends considering the direction of fluid flow, the upstream and downstream ends having a cross-section decreasing towards the supply and discharge ports.

[0038] In one example, n is greater than or equal to 2, and the manufacturing system comprises a first channel connecting the fluidic chambers in parallel and a second channel connecting the fluidic chambers in parallel, the first channel and the second channel having diameters on the order of those of arteries and veins. Brief description of the drawings

[0039] The present invention will be better understood on the basis of the following description and the accompanying drawings in which:

[0040] [Fig. 1 A] is a schematic representation of an example of an element that can be implemented in the manufacturing process.

[0041] [Fig.1B] is a schematic representation of another example of an element that can be implemented in the manufacturing process.

[0042] [Fig.lC] is a schematic representation of another example of an element that can be implemented in the manufacturing process.

[0043] [Fig.1D] is a schematic representation of another example of an element that can be implemented in the manufacturing process.

[0044] [Fig.2] is a schematic representation of the fabrication of the biopolymer cores,

[0045] [Fig.3] is a schematic representation of a fluidic chamber for the implementation of the manufacturing method.

[0046] [Fig.4] is a schematic representation of a fluidic circuit adapted for the implementation of the manufacturing method.

[0047] [Fig.5] is a view of the chamber of [Fig.3] comprising elements.

[0048] [Fig.6] is a schematic representation of an example of stacking elements enabling the production of capillaries, venules and arterioles.

[0049] [Fig.7] is a view similar to that of [Fig.5] on which the fluid flow is shown.

[0050] [Fig.8] is a schematic representation of endothelial cells self-organized into channels.

[0051] [Fig.9] is a photograph of a slice of a vascular structure obtained by the manufacturing method.

[0052] [Fig. 10] is an enlarged view of [Fig. 9].

[0053] [Fig. 11] is a schematic representation of an example of a body-on-a-chip manufacturing system.

[0054] DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0055] In the present invention, "vascular structure" means a structure comprising one or more channels, the dimensions of each channel being of the same order of magnitude as an artery, vein, arteriole, venule or capillary.

[0056] The term "vessel" refers to a channel through which blood is intended to circulate; a vessel is an artery, vein, arteriole, venule, or capillary. The channel has walls covered with a porous endothelium that allows exchange with the surrounding environment. In the case of a capillary, the endothelium forms the wall; in the case of an artery or vein, the endothelium forms an inner layer of the channel covering one or more outer layers. The outer layer(s) are also porous, allowing exchange with the surrounding environment.

[0057] The term "vascular network" means a network comprising arterioles and venules connected by capillaries, the arterioles being able to be connected to an artery and the venules being able to be connected to a vein.

[0058] The term “tissue” means a functional group of cells and the term “organ” means a group of tissues performing a specific function.

[0059] The method for manufacturing a vascular structure comprises at least: a) The manufacture of elements of at least one type, comprising on their external surface endothelial cells and / or endothelial progenitor stem cells, b) The stacking of said elements, c) Establishing a fluid flow through the stacking by creating channels along the flow lines.

[0060] Each of the steps will now be described in detail.

[0061] An example of El elements manufactured in step a) is schematically represented on [Fig.1A]. They comprise at least a central part or core 2 in biopolymer and an outer layer 4 comprising endothelial cells and / or endothelial progenitor stem cells.

[0062] Endothelial cells are constituents of the endothelium, which forms the innermost layer of blood vessels. Arteries and veins have three layers, including the endothelium, while capillaries have only one layer formed by the endothelium.

[0063] Endothelial cells are differentiated cells, i.e., they are specific to a given organ. For example, we speak of lymphatic endothelial cells and pulmonary endothelial cells.

[0064] Endothelial progenitor stem cells are not yet differentiated.

[0065] Endothelial cells are chosen according to the application, for example HMECs (Human Microvascular Endothelial Cells in Anglo-Saxon terminology) or liver endothelial cells or LSECs (Liver sinusoidal endothelial cells in Anglo-Saxon terminology) in co-culture with hepatocytes.

[0066] For the sake of simplicity, endothelial cells and / or endothelial progenitor stem cells will be referred to in the rest of the description as "endothelial cells"

[0067] The core 2 comprises one or more biopolymers. A biopolymer is a collection of biological macromolecules. The usable biopolymers are well known to those skilled in the art and may be natural or synthetic. They are chosen, for example, from alginate, collagen, fibrin, elastin, and hyaluronic acid.

[0068] The biopolymer(s) is / are chosen according to the vascular structure to be fabricated, the endothelial cells covering the nucleus, and the tissue or organ to be fabricated. Thus, by creating elements comprising several layers of biopolymers, it is possible to form both an environment favorable to endothelial cells and one or more environments favorable to tissue cells that would have a greater affinity for a biopolymer different from the one with which the endothelial cells have a good affinity.

[0069] The biopolymer(s) may or may not be resorbable depending on the structure to be fabricated. The biopolymer(s) are gelled when they form the components. For example, it may be possible to reconvert the biopolymer(s) into a liquid once the vascular structure is complete in order to remove the biopolymer(s).

[0070] In one embodiment, the core comprises only biopolymer(s).

[0071] In another example, the nucleus comprises biopolymer(s) and cells typical of the organ that one wishes to produce.

[0072] The cells in the nucleus can be of any cell type, can be clusters of cells or organoids. For example, typical cells are hepatocytes in order to develop a liver-on-a-chip, or islets of Langerhans in order to mimic a pancreas-on-a-chip.

[0073] The endothelial cells cover at least part of the nucleus 2. In this example the endothelial cells are seeded on the outer surface of the nucleus.

[0074] Depending on the biopolymer(s) and endothelial cells, it is advantageous to functionalize the nucleus to facilitate the attachment of endothelial cells to the nucleus. For example, in the case of alginate, which has little affinity for endothelial cells, a protein that improves the anchoring of endothelial cells to the nucleus can be used. Alternatively, the nucleus is made with a mixture of biopolymers, one of which provides mechanical strength to the nucleus, such as alginate, and the other ensures affinity with endothelial cells, such as collagen.

[0075] Advantageously, the nucleus also contains a vascular endothelial growth factor (VEGF) that promotes the proliferation of endothelial cells around the nucleus. The growth factors can be added either directly to the perfusion medium or by co-culturing fibroblasts that will secrete growth factors. Preferably, the fibroblasts are placed close to and in contact with the endothelial cells. For example, the fibroblasts are located on the outer layer of the nucleus like the endothelial cells, or in a layer between the nucleus and the endothelial cells. Alternatively, the fibroblasts are placed in other components, releasing the factors and allowing proliferation.

[0076] In [Fig.IB], another example of E2 elements can be seen in which the nucleus It comprises Cl cells, for example typical of the organ to be produced, and the outer layer 4 includes an encapsulating layer 8 of endothelial cells 6. Preferably, the encapsulating layer comprises collagen. Alternatively, the encapsulating layer comprises a mixture of collagen and alignment.

[0077] In [Fig.IC], another example of E3 elements can be seen in which the The nucleus contains Cl cells of one type at its core and C2 cells of another type at its periphery. The Cl and C2 cells are encapsulated in the same biopolymers or different biopolymers. In this example, the endothelial cells are also encapsulated.

[0078] Stacking layers containing different cell types makes it possible to recreate stratified tissues.

[0079] The number of layers containing different types of cells is not limited to two. Thus, it is possible to provide all the cell types necessary for the proper functioning of the tissue or organ that one wishes to produce.

[0080] For example, C2 cells are pericytes. Indeed, pericytes are cells that spread across the outer wall of capillaries. In vitro, they help to mature the microvascular network. Thus, by creating elements comprising a nucleus containing the tissue cells of interest and pericytes in its outer layer, and endothelial cells on the surface of the nucleus, the architecture of blood capillaries is preserved, with blood flow circulating within a wall composed of endothelial cells, covered by pericytes.

[0081] In [Fig. 1D], another example of element E4 can be seen, comprising a functionalization layer 10 to facilitate cell attachment to the nucleus. Alternatively, this could be a biopolymer layer, typically collagen, chosen according to the tissue to be produced.

[0082] It will be understood that any type of combination between biopolymers, typical organ cells and endothelial cells is conceivable in order to get as close as possible to the tissues or organs that one wishes to reproduce.

[0083] In the examples shown, the elements have substantially spherical shapes. Other shapes are conceivable, for example the ovoid shape.

[0084] Preferably, the dimensions of the elements are such that the cells contained within the nucleus can be perfused. Indeed, the diffusion distance of nutrients in the biopolymer is approximately 200 pm. The dimensions of the elements are therefore chosen so that the cells are located at most 200 pm from the outer surface of the nucleus. For example, in the case of a spherical element, it preferably has a radius of approximately 200 pm. It will be understood that the diffusion distance can vary and depends on the type of biopolymer, its density, and the type of nutrients. For example, the elements have a diameter of less than 500 pm.

[0085] An example of manufacturing the El elements will now be described.

[0086] The manufacture of the cores can be carried out according to the method described in the document Morimoto Y, Onuki M, Takeuchi S. Mass Production of Cell-Laden Calcium Alginate Particles with Centrifugal Force. Adv Health v Mater. Jul 1, 2017;6(13).

[0087] For example, we wish to produce an alginate core. Alginate gels by diffusion of calcium ions. Alginate droplets of a given diameter are produced, which then fall into a highly concentrated calcium bath to allow rapid gelation of the droplets.

[0088] Figure 2 shows a schematic representation of the realization of biopolymer cores.

[0089] The formation of the droplets 12 is, for example, achieved using a needle 14 mounted on a vial 16. The free end of the needle is positioned above the calcium bath 18, at a distance from the free surface of the bath. The vial 16 and the needle 14 are rotated, generating a centrifugal force. The biopolymer then enters the needle, and droplets of biopolymer detach from the needle tip and fall into the bath. As they fall, the biopolymer, being liquid, takes on a spherical shape. Upon entering the calcium bath, the droplets gel. This yields biopolymer microspheres. The gelled droplets can then be used for seeding with endothelial cells.

[0090] In cases where the nucleus contains cells specific to the tissues or organs to be produced, the cells can be integrated into the nucleus or into a layer external to the nucleus using several techniques. One technique consists of mixing the specific cells with the biopolymer constituting the nucleus before being placed in the needle.

[0091] In the case of multi-layered elements, one technique involves creating initial elements which, once gelled, can be mixed with a second biopolymer and additional cells, then reinjected into a larger needle for a second encapsulation. According to another technique, two co-flowing polymer streams (flow focusing) are sheared by an oil flow. For example, a needle containing the core biopolymer is inserted into a larger needle containing the biopolymer of the future outer layer, this biopolymer potentially containing cells. Centrifugation allows the formation of two layers in a single step and can create an element with a gelled outer layer and a liquid core.

[0092] Alternatively, elements are produced by co-flow, allowing for the creation of sectorized elements, with the different zones containing identical or different cells. Manufacturing methods for producing elements by co-flow are described, for example, in the document Agarwal et al. One-step microfluidic generation of pre-hatching embryo-like core-shell microcapsules for miniaturized 3D culture of pluripotent stem cells, LabChip, 2013, 13, 4525-4533 and in the document Ma et al. Core-shell Hydrogel Microcapsules for Improved Islets Encapsulation, Adv Health Mater. 2013 May; 2(5) pp. 1-12.

[0093] The nuclei and endothelial cells are then suspended in a cell culture medium to allow the endothelial cells to adhere to the surface of the nuclei. Preferably, after the cells have adhered, they are left in culture to proliferate and cover the surface of the nuclei in a substantially uniform manner. Proliferation can last, for example, from one day to several days. Alternatively, the nuclei and endothelial cells are directly injected into a fluidic chamber, which will be described below, in which cell proliferation on the nuclei and vessel generation take place.

[0094] It will be understood that the culture medium is also chosen to ensure the survival of cells inside the nucleus, and in the case of several types of cells, the culture medium is chosen so as to be compatible with the different types of cell.

[0095] In the case of a biopolymer, such as alginate, which has little affinity for endothelial cells, functionalization of the outer surface of the nuclei is advantageously carried out to facilitate endothelial cell adhesion. This functionalization is performed prior to suspending the nuclei in the culture medium containing the endothelial cells.

[0096] For example, this functionalization is achieved by coating the nuclei with a biopolymer that has a good affinity for endothelial cells, for example, collagen. To do this, the nuclei are placed in a cold collagen bath. to prevent gelation. Then the nuclei are collected and placed in a medium at a temperature that allows the collagen to gel without damaging the cells, for example between room temperature and 37°C.

[0097] Step b) will now be described.

[0098] The elements produced in step a) are then placed in a fluidic chamber 20 shown schematically in [Fig.3], so as to form a porous stack.

[0099] The fluidic chamber 20 is, for example, formed in a microfluidic card and comprises a cavity 22 for receiving the elements El, for example, a fluid inlet 24 located on one side of the cavity, and a fluid outlet 25 located on the other side of the cavity so that the cavity is traversed by the fluid flow. The cavity has an upstream end located on the side of the inlet and a downstream end located on the side of the outlet.

[0100] The fluid provides nutrients to the cells and also ensures their oxygenation. Fluid circulation through the cavity is achieved, for example, by means of a peristaltic pump. The use of a peristaltic pump allows for precise calibration of the fluid flow rate through the fluid chamber and thus control of the effect of the fluid flow on the endothelial cells.

[0101] The fluidic chamber includes, on the downstream end side of the cavity, means 26 preventing the elements El from being evacuated through the evacuation orifice 25.

[0102] In the example shown, the means 26 comprise a wall 28 extending transversely from the lower bottom 29 of the cavity over a portion of the cavity's height upstream of the discharge orifice. The distance between the free end 28.1 of the wall 28 and the upper bottom 31 of the cavity is less than the minimum dimension of the elements EL. In the case of spherical elements, the distance d is less than the diameter of the elements, and in the case of ovoid elements, the distance d is less than the smallest dimension of the elements. Alternatively, the wall extends from the upper bottom of the chamber, or from a lateral wall of the chamber, allowing the fluid to flow below and above the wall. The wall 28 may be solid or perforated. Preferably, the wall is arranged to promote a homogeneous flow.

[0103] Alternatively, means 26 comprise a grid with holes smaller than the minimum dimension of the elements, and pillars spaced apart to prevent the elements from passing towards the discharge outlet. Any other means allowing the elements El to be retained in the cavity while permitting fluid flow between the inlet and outlet ports may be implemented.

[0104] In addition, the cavity is also delimited on its downstream end to contain the elements and ensure the stability of the stack. In this example, it It is also a wall 27 extending from the lower bottom of the chamber to the upper bottom over part of the height of the cavity.

[0105] Advantageously, all or part of the chamber is transparent to allow visualization of the elements during the manufacturing process.

[0106] Also highly advantageous, the upper base 31 is removable, allowing the fabricated vascular structure to be recovered. Sealing means can be provided between the upper base and the rest of the fluidic chamber.

[0107] In the example shown, the upper base 31 is a film having adhesive properties, which is laminated onto the cavity, for example made of COC (cyclo-olefin copolymer), and which can then be removed to recover the generated tissue or organ.

[0108] Alternatively, the upper bottom 31 is made of rigid material, for example COC (cyclo-olefin copolymer) coupled to the rest of the chamber for example by magnetization and thus able to be removed.

[0109] In the example shown, the supply and discharge ports are located in the upper bottom of the chamber. Alternatively, one or both of the ports are located in the lower bottom and / or in one or more side walls.

[0110] Figure 4 shows a representation of an example of a circuit ensuring the circulation of the fluid in the fluidic chamber. In this example, the circuit is advantageously a closed circuit allowing a continuous flow to be maintained for several days.

[0111] The circuit includes a fluid reservoir 30, for example a bottle, the fluidic chamber, a peristaltic pump 34 and a waste bin 35. The circuit also includes a first fluidic connection 36 between the reservoir 30 and the inlet port 24 of the fluidic chamber, on which the pump 34 is provided. A second fluidic connection 38 is provided between the outlet port 25 and the reservoir 30.

[0112] Advantageously, a two-way valve 40 is provided on the second fluid connection to allow or permit circulation between the outlet port and the reservoir, and a three-way valve 42 is provided on the first fluid connection 36, allowing the reservoir 30 to be connected either to the inlet port 24 or to the waste container 35 via a third fluid connection 44. The valves prevent backflow into the fluid chamber when the pump is stopped. Specifically, by switching the three-way valve 42 to connect the reservoir 30 to the waste container 35 and switching the two-way valve 40 to interrupt the connection between the outlet port 25 and the reservoir 30 before stopping the pump, backflow is prevented.

[0113] The first 36, second 38 and third 44 fluidic connections are for example made by microfluidic tubes

[0114] Preferably, the reservoir 30 includes a filter 45 allowing oxygenation of the culture medium while maintaining the circuit's sterility. Alternatively, all or part of the circuit and / or the circuit board is oxygen-porous, ensuring oxygenation of the culture medium. Alternatively, a suitable gas mixture is bubbled into the culture medium, which is then injected into the chamber. For example, the chamber is placed in an incubator that allows for the regulation of exchanges by establishing, for example, an atmosphere of 5% CO2 and approximately 80% to 85% humidity.

[0115] Preferably the circuit is maintained at a temperature of 37°C, for example by placing it in an INC incubator.

[0116] It will be understood that other devices for precisely controlling the flow rate of the fluid through the fluid chamber can be implemented, for example by using a syringe pump or gravity flow between two connected inlet and outlet reservoirs with different levels. In the case of the syringe pump, the operator must manipulate the fluids to maintain a continuous flow. In the case of gravity flow, either the reservoirs are large enough that fluid manipulation between the upstream and downstream reservoirs is not required, or the operator transfers liquid between the two reservoirs to maintain the flow.

[0117] Figure 5 shows the fluidic chamber containing the E2 elements. In this example, the stack comprises elements of different sizes and may contain cells of different types for co-culture and / or different endothelial cells. The elements are, for example, injected or inserted into the fluidic chamber to form a relatively compact stack.

[0118] Preferably, the elements have a maximum dimension of less than 500 µm. Depending on the stacking and the different dimensions of the elements, or even the different shapes of the elements, the inter-element spacing can be adjusted according to the architecture of the vascular structure to be fabricated. The spacing between the elements can range from a few µm to a few tens of µm, or even a hundred µm. These spacing values ​​correspond to the diameters of the capillaries, arterioles, and venules that one wishes to reproduce.

[0119] The invention allows the elements to be stacked according to their dimensions in order to create a structured network. Advantageously, the elements can be distributed according to their size to reproduce a vascular network comprising arterioles and venules connected by capillaries. Indeed, the use of elements of different dimensions makes it possible to obtain different stacking arrangements, in order to achieve even smaller inter-element pores.

[0120] A layer of large elements can be stacked, then a layer of small elements, then again a layer of large elements to form the dimensions of arteriole pores, then capillaries, then venules.

[0121] Stacking elements of different sizes within the same layer can make it possible to obtain inter-element pores of even smaller dimensions.

[0122] We can therefore create stacks with small pores, in order to approximate the structure of blood capillaries, and then stacks with larger pores, in order to approximate the structure of larger diameter arterioles / venules

[0123] In [Fig.6], we can see an example of the distribution of elements enabling the manufacture of such a network.

[0124] The elements arranged in the fluidic chamber for perfusion are distributed in three stacks I, II, and III. The elements in stack I are those with the smallest diameter, resulting in the smallest inter-element spacing. This advantageously allows for the formation of capillaries. The nuclei, for example, contain tissue cells and pericytes and are covered with endothelial cells.

[0125] Stack I is arranged between stacks II and III.

[0126] Stacks II and III have larger diameter elements and offer larger inter-element spacings.

[0127] Stack II comprises, for example, elements comprising a nucleus covered by a thick layer of muscle cells covered by endothelial cells, and stack III comprises, for example, elements comprising a nucleus covered by a thin layer of muscle cells covered by endothelial cells.

[0128] This distribution can be arranged so that layers II, I, and III are positioned one above the other, allowing for better stability during stacking. Preferably, separate fluid connections are provided for each of the stacks I, II, and III to inject the fluid into each stack separately. Preferably, an injection first takes place in stack II so that its elements form a first vascular structure and become lodged against the filter; then an injection takes place in stack I, which forms a second vascular structure that becomes lodged against stack II; and finally, an injection takes place in stack III, forming a third vascular structure that becomes lodged against the second structure.

[0129] Furthermore, by choosing the dimensions of the elements to increase the spacing between the elements at the upstream and / or downstream ends of the chamber, and thus create vessels of a larger diameter, it is possible to promote anastomosis.

[0130] Step c) will now be described.

[0131] After the elements are placed in the cavity, a fluid flow is established through the stack between the inlet and outlet ports. The flow lines are schematically represented by arrows F in [Fig.7].

[0132] The flow creates preferential pathways within the stack. Perfusing the stack of elements will cause the endothelial cells to reorganize, forming hollow tubes, then blood vessels and capillaries. The flow ensures cell alignment along the flow lines. Figure 8 shows the self-assembly of endothelial cells that define capillaries.

[0133] Advantageously, the fluid flow rate is chosen so as to induce sufficient shear at the surface of the elements to stimulate the attachment of endothelial cells to one another and promote endothelium formation. For example, the flow rate is chosen so that the shear stresses applied to the elements are between 1 dyne / cm² and 10 dynes / cm², i.e., between 10 N / cm² and 10 N / cm².

[0134] It will be understood that the shear stresses vary within the stack. The calculated shear value is therefore an average value within the stack.

[0135] The appearance of vessels in the stack of elements may appear after several hours of perfusion, for example one day.

[0136] Vascularized tissues are then obtained that can be used alone, for example in organ-on-a-chip applications, in which case the tissues can be stored in the fluidic chamber. Alternatively, the vascularized tissues can be assembled for cell reconstruction applications, in which case the tissues are removed from the chamber.

[0137] Depending on the composition of the nuclei and the cells they may contain, the nuclei can either be resorbed or remain intact and participate in the vascular structure. To remove the biopolymer(s), a step to degel the biopolymer(s) can be included, which, in a liquid state, are then carried away by the fluid flow. For example, at the end of the fabrication of the vascular structure, one or more substances causing the degelation and liquefaction of the biopolymer(s) are added to the fluid; for example, sodium citrate can be introduced, which degels alginate by chelating the calcium. By maintaining the fluid flow, the biopolymer(s) are carried by the fluid to a waste container. The fluid can be injected into the vascular structure, for example, into the capillaries. The citrate, passing through the porous capillary walls, reaches the biopolymer components and causes their dissolution.Alternatively, the chamber has openings in its walls to allow citrate to be injected into the cavity outside the vascular structure and in direct contact with the elements to be dissolved.

[0138] The cavity walls can be lined with endothelial cells to more closely mimic the conditions of vascularized tissue. This prevents the flow from occurring primarily along the cell-free walls. Furthermore, the flow is only in contact with the endothelium, as is the case in an in vivo system. This also allows for the possibility of perfusing with blood, as the endothelium prevents the formation of blood clots.

[0139] For example, the entrance and exit of the cavity are formed by channels, for example, made of plastic or glass. Advantageously, these channels are lined with endothelial cells. Under perfusion, the endothelial cells will assemble and form vessels whose diameters correspond to those of the channels. These vessels are intended to connect the vascular structure to a vascular system, for example, to an artery and a vein.

[0140] In the example shown, the cavity is rectangular in shape. As a result, the vascularized tissue produced is also rectangular in shape. In order to approximate the structure of the blood network at the level of the subdivision of arteries into arterioles and then capillaries, and their branching into venules and then veins, it is advantageous to use a cavity having upstream and downstream ends that narrow towards the inlet and outlet respectively, for example in the shape of a diamond or an ellipse when viewed from above, or even in the shape of an organ.

[0141] These shapes have the advantage of facilitating the anastomosis of the capillaries with the wall of the microfluidic channels covered with endothelial cells as explained above.

[0142] As explained above, the application of fluid flow exerting sufficient shear force is favorable to channel formation by endothelial cells. We will now describe how the shear forces applied by the flow to the elements in the fluidic chamber can be estimated.

[0143] When a fluid passes through a microfluidic channel, a shear force is applied to the channel walls, proportional to the viscosity of the liquid, its velocity in the conduit, and the cross-sectional area of ​​the channel. For a porous medium, the fluid velocity is variable and depends on the structural properties, such as porosity, which makes its determination complex. Indeed, solving the continuity equations of matter and the Navier-Stokes equations is very complex in such media due to the complexity of the geometry. Thus, studies have been carried out and models have been established in a semi-empirical manner in order to characterize the flow within porous media.

[0144] Porous media are defined by a set of solid particles between which pores are found, forming the voids. The void fraction e is called Porosity. Porous media are often modeled as stacks of undeformable spheres contained in a circular channel.

[0145] All the parameters used for the theoretical calculations are presented in the table below. [Table 1] Microfluidic Parameters Notation Unit Flow Rate Q [qL / s] = [mm³ / s] Velocity upstream of the stack uo [mm / s] Velocity in the stack U [mm / s] Microfluidic channel cross-section A [mm²] Cross-section diameter D [mm] Stack length H [mm] Porosity s Sphere diameter D [mm] Hydraulic diameter Dh [mm] Dynamic viscosity of the fluid [Po] = [dyn.s / cm²] Kinematic viscosity of the fluid N [m² / s]

[0146] The following formula relating the average shear stress to the flow rate in the chamber is established from the Hagen-Poiseuille equations:

[0147] [Math.l] T= 12 X q During the manufacturing process, the fluid is at 37°C and has a composition close to that of water. We therefore consider the dynamic viscosity of our medium to be equal to that of water at 37°C, i.e., 6.92 x 10³ dyn.s / cm². The cross-section of the chamber used is known, and the fluid flow rate is controlled by means of a peristaltic pump. The diameter of the elements can be determined using image processing software such as ImageJ®. It should be noted that the elements produced using the technique described above have an ellipsoidal shape; the largest and smallest diameters are measured, and an average is calculated to obtain a mean radius.

[0148] The porosity of the stack is to be determined.

[0149] Theories on sphere packing show that the minimum porosity achievable by a rigid sphere packing is 26%, obtained for a compact hexagonal structure. However, when the placement of the spheres is random, as in the case of the present invention, the minimum achievable porosity is 36%. To verify this, a count of the elements in the fluidic chamber is performed. For example, a chamber with a cross-section of 1.8 x 1 mm² is used, filled with elements not covered by endothelial cells. Using uncovered elements allows observation of the entire stack because the elements are transparent.

[0150] The elements measure 302.5 pm ± 14.4 pm. In a volume of 1.8 x 2.4 x 1 mm³, there are 219 elements, resulting in a porosity of:

[0151] [Math.2] £=1 -Calvings / e - 2 6.5% We thus obtain a compactness close to that of the hexagonal close-packed structure, which is not achievable with the random packing of rigid spheres. This is because the elements are actually deformable, allowing for a more compact packing.

[0152] The shear stress can then be calculated using the formula established above, resulting in a stress: [Math 3] for a flow rate of 50 pL / min.

[0153] The velocity and shear field in the stack was measured by particle image microvelocimetry (pPIV), applying a flow rate of 50 pl / min. The average shear is 0.4 to 0.5 dyn / cm².

[0154] Consequently, the shear formula established above is verified. This can therefore be used to calculate the fluid flow rate to be applied through the chamber to obtain a given average shear exerted on the elements.

[0155] An example of the manufacture of vascular tissue will now be given.

[0156] In step a), a mixture of alginate and collagen is used, for example with a 50:50 ratio. The ratio actually depends on the initial concentration of the biopolymers. It's a 50:50 ratio with an alginate solution initially at 4% and a collagen solution initially at 6 mg / mL.

[0157] This mixture has the advantage of combining the rigidity offered by alginate once gelled and the affinity of collagen with endothelial cells.

[0158] Endothelial cells are HMEC-GFP cells. These endothelial cells permanently express the GFP protein, which allows their observation by fluorescence.

[0159] The elements are manufactured according to the method described above.

[0160] The collagen mixture is placed in a vial fitted with a needle, to which a centrifugal force is applied. The resulting droplets fall into a calcium bath, which causes the alginate to gel. Preferably, the alginate bath is cold to prevent the collagen from gelling. The nuclei are formed.

[0161] The nuclei are then placed in a collagen solution so that a layer of collagen forms on the nuclei to improve the adhesion of endothelial cells to the nuclei. The solution is then heated to 37°C to induce gelation of the collagen.

[0162] In a subsequent step, the nuclei are suspended with the endothelial cells in a cell culture medium to allow the cells to adhere to the surface of the nuclei. Once the cells have adhered, the elements are left in culture so that the endothelial cells proliferate and cover the surface of the nuclei, for example, for two days. The endothelial cells then form a substantially uniform layer on the nuclei.

[0163] The elements are ready to be arranged in the fluidic chamber.

[0164] In step b), the elements are injected into the fluidic chamber.

[0165] In step c), fluid circulation at a given flow rate is established through the chamber fluidic.

[0166] For example, the flow rate is chosen to apply an average shear stress between 1 dyne / cm2 and 10 dynes / cm2.

[0167] The dimensions of the elements were previously measured by image processing. Since the elements have ovoid shapes, the large and small diameters are measured.

[0168] An average diameter of 338.5 + / -21.0 pm and a sphericity of 92% are obtained.

[0169] The elements have a diameter of less than 400 pm, so any point inside the element is less than 200 pm from the surface. Thus, all cells encapsulated in the elements would have access to nutrients transported by the future blood network.

[0170] The elements are, for example, injected into the fluidic chamber using a pipette. The circuit shown in [Fig. 4] is used, for example. The peristaltic pump is set to deliver the fluid at the given flow rate to generate the desired shear stresses in the stack. The perfusion takes place at 37°C, for example, in an oven. The pump is started with the two-way and three-way valves positioned to allow the fluid to circulate in a closed loop.

[0171] Fluid circulation is established.

[0172] The porosity determined for elements not covered by endothelial cells is approximately 0.26 due to the deformability of the elements. However, elements covered by endothelial cells are more rigid, so the resulting stacking is less dense. Therefore, a porosity value of e = 0.36 is chosen, corresponding to the maximum compactness of a stack of undeformable spheres. A flow rate of 1 ml / min is then selected, which generates an average shear stress of 1.21 dyn / cm².

[0173] Under the influence of the fluid flow, the cells align and elongate in the direction of the flow. The endothelial cells proliferate by forming bridges between the elements. After three days, the endothelial cell layer becomes homogeneous and gaps between the cells are virtually no longer observable.

[0174] Figure 9 shows a cross-section of the structure obtained using the method according to The invention after 85 hours of perfusion. The structure is sliced ​​into 10 µm slices. The photo in [Fig. 9] is a wide-field view of a slice.

[0175] The elements and the layer of endothelial cells that has formed between the elements can be seen.

[0176] Figure 10 shows an enlarged view of an inter-element area. The image shows empty areas of varying sizes. Image processing allows us to measure the diameters of these areas. They range from approximately ten micrometers to one hundred micrometers. These diameters are characteristic of the physiological diameter of blood capillaries. Furthermore, during perfusion, lectin was added to the fluid. This lectin was observed within the stack of elements, having bound to the endothelial cells.

[0177] Thus the manufacturing method makes it possible to manufacture networks of hollow tubes, covered with endothelial cells and perfusable within a stack of elements with diameters less than 400 pm.

[0178] The process for manufacturing vascular structures has the advantage of applying little stress to the cells. Indeed, they are rarely in a state of asphyxiation. The cells, whether endothelial cells or typical tissue or organ cells, are mostly found in an oxygenated environment. Thus, the chances of obtaining living vascularized tissues are very high.

[0179] Furthermore, the manufacturing process offers considerable freedom regarding the dimensions of the vessels formed, allowing for a closer approximation of the actual tissue structure. Moreover, unlike bioprinting techniques, it allows for the creation of channels the size of capillaries.

[0180] The manufacture of vascularized tissues according to the invention involves few steps and these are relatively simple to implement.

[0181] The manufacturing process also offers the possibility of either producing a tissue or organ on a chip which can be used to carry out experiments, or producing tissues or organs that can be transplanted onto a human being, for example.

[0182] Figure 11 shows an example of a body-on-a-chip manufacturing and analysis system.

[0183] In this example, several organs can be manufactured simultaneously in order to create a body-on-a-chip, allowing the interaction between the different organs of a body, for example a human body, to be studied.

[0184] The manufacturing method employs a device comprising fluidic chambers 20.1, 20.2, 20.3 having supply ports 24.1, 24.2, 24.3 and discharge ports 25.1, 25.2, 25.3 respectively, as in the case of the fluidic chamber 20 of [Fig. 3], allowing the establishment of fluidic flows through each chamber and the fabrication of vascularized organs as described above. The supply and discharge ports are connected to channels 46, 48 linked to the fluid source in the case of a closed circuit. Valves 50 are provided on the channels.

[0185] The device also includes a channel 52 for supplying the organs in parallel and a channel 54 for draining them in parallel. Valves 56, 58 are provided on channels 52, 54 between each organ. Channel 52 functions as an artery, and channel 54 functions as a vein. Channels 52, 54 have diameters corresponding to those of veins and arteries.

[0186] Thus, a system can be produced that reproduces at least in part the organs and their connections.

[0187] This system offers the advantage of eliminating the need to handle the organs between their manufacture and their use for medical analysis. Switching from the manufacturing phase to the operational phase simply requires switching the valves. The risks of deterioration and / or pollution are significantly reduced.

[0188] The process according to the invention offers the advantage of not being limited in size. Indeed, it can be easily adapted to produce large fabrics, for example fabrics of several cm³.

[0189] The manufacturing method can be used in the field of organs-on-a-chip, for example, to create platforms for testing drugs or molecules for cosmetic applications, such as skin reconstruction. The invention makes it possible to create adaptable systems for testing new drugs, for example, chemotherapy molecules tailored to the patient. Furthermore, manufacturing several organs on the same chip allows for the study of multi-organ interactions.

[0190] The manufacturing method can also be used to perform tissue grafts. The method allows for a graft in which revascularization with the patient's system would be more efficient and therefore improved.

[0191] The manufacturing method also allows for the development of organs. For example, the method can generate a vascularized pancreas using elements containing pancreatic islets that release insulin according to the blood glucose level, and in which the vascular network around the periphery of the elements would be connected to the patient's vascular network. Similarly, the method according to the invention can produce a liver with elements containing hepatocytes. Liver transplantation would thus be facilitated.

Claims

Demands

1. Method of manufacturing a vascular structure comprising / a) the manufacture of elements comprising at least one nucleus comprising at least one biopolymer, and endothelial cells and / or endothelial progenitor stem cells on its outer periphery, said elements not comprising any embryonic stem cells of human origin, b) placing said elements in a fluidic chamber so that they form at least one stack, c) establishing a fluid flow ensuring a supply of nutrients and oxygenation through the stack so as to provide channels between the elements along the flow lines, the fluid flow having a flow rate such that the elements experience an average shear stress of between 0.1 dyne / cm2 and 100 dynes / cm2, preferably between 1 dyne / cm2 and 10 dynes / cm2.

2. A manufacturing method according to claim 1, wherein in step a) cells, excluding human embryonic stem cells, are introduced into the nucleus.

3. A manufacturing method according to claim 2, wherein the nucleus comprises at least a core containing cells and an outer layer containing cells, the cells of the core and the outer layer being of the same type or not.

4. A manufacturing method according to claim 3, wherein the outer layer contains pericytes.

5. A manufacturing method according to any one of claims 1 to 4, wherein the elements have a largest dimension less than 1 mm, advantageously less than 500 pm.

6. A manufacturing method according to any one of claims 1 to 5, wherein the elements are arranged in a culture medium prior to step b) to permit the proliferation of endothelial cells and / or endothelial progenitor stem cells and the total or partial covering of the nuclei.

7. A manufacturing method according to any one of claims 1 to 6, wherein in step b) elements of different dimensions and / or shapes are put in place.

8. A manufacturing method according to any one of claims 1 to 7, wherein in step b), elements of different dimensions are distributed in several successive stacks in the direction of the flow.

9. A manufacturing method according to claim 8, wherein a stack of first elements having an inter-element spacing on the order of the diameter of the capillaries is arranged between two stacks of second elements having an inter-element spacing on the order of the diameter of the arterioles or venules.

10. A manufacturing method according to any one of claims 1 to 9, wherein, prior to the placement of the elements in the fluidic chamber, endothelial cells and / or endothelial progenitor stem cells cover the walls of the chamber and / or the fluid inlet and / or outlet channels.

11. A system for manufacturing a vascular structure for implementing the manufacturing method according to any one of the preceding claims comprising n fluidic chambers (20), n > 1, provided with a fluid supply port (24) and a fluid discharge port, a cavity disposed between the supply port and the discharge port (25) and configured to receive the elements, and means (26) for retaining the elements in the cavity, a fluid reservoir (30) connected at least to the supply inlet and circulation means for ensuring the circulation of the fluid through the cavity, the fluid circulation means being such that they establish a fluid flow having a flow rate such that the elements undergo an average shear stress of between 0.1 dyne / cm2 and 100 dynes / cm2, preferably between 1 dyne / cm2 and 10 dynes / cm2.

12. Manufacturing system according to the preceding claim, in which the reservoir (30) is connected to the discharge port (25) so as to form a closed circuit.

13. Manufacturing system according to the preceding claim, comprising means (40, 42) for isolating the fluidic chamber before stopping the circulation means.

14. A manufacturing system according to any one of claims 11 to 13, comprising means for oxygenating the fluid.

15. A manufacturing system according to any one of claims 11 to 14, wherein each fluidic chamber has a removable part to allow access to the cavity and removal of the vascular structure.

16. A manufacturing system according to any one of claims 11 to 15, wherein the cavity comprises a central part and upstream and downstream ends considering the direction of fluid flow, the upstream and downstream ends having a cross-section decreasing in the direction of the supply and discharge ports.

17. A manufacturing system according to any one of claims 11 to 16, wherein n is greater than or equal to 2, and wherein the manufacturing system comprises a first channel connecting the fluidic chambers in parallel and a second channel connecting the fluidic chambers in parallel, the first channel and the second channel having diameters on the order of those of arteries and veins.

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