WO2017025620A1

WO2017025620A1 - Bioreactor

Info

Publication number: WO2017025620A1
Application number: PCT/EP2016/069216
Authority: WO
Inventors: Andrea NEGRO
Original assignee: Morphodyne Sa
Priority date: 2015-08-13
Filing date: 2016-08-12
Publication date: 2017-02-16
Also published as: GB201514420D0

Abstract

The present invention relates to a bioreactor for culturing and/or growing artificial or natural tissues, comprising a top plate and a bottom plate, wherein the top and bottom plate together define a perfusion chamber for receiving and supporting an artificial or natural tissue, and wherein the top plate further comprises a plurality of inlet structures and a plurality of outlet structures, wherein the inlet and outlet structures are in fluid communication with the perfusion chamber to allow flow of at least one medium through the perfusion chamber; and wherein the bottom plate further comprises a height adjustable portion for adjusting the volume of the perfusion chamber.

Description

Bioreactor

FIELD OF THE INVENTION The present invention relates to a bioreactor for culturing and/or growing artificial or natural tissues, comprising a top plate and a bottom plate, wherein the top and bottom plate together define a perfusion chamber for receiving and supporting an artificial or natural tissue, and wherein the top plate further comprises a plurality of inlet structures and a plurality of outlet structures, wherein the inlet and outlet structures are in fluid communication with the perfusion chamber to allow flow of at least one medium through the perfusion chamber; and wherein the bottom plate further comprises a height adjustable portion for adjusting the volume of the perfusion chamber. Another aspect of the invention relates to a method of producing and/or culturing an artificial tissue or culturing a natural tissue using the bioreactor of the invention. Another aspect relates to artificial tissue obtained or obtainable using the bioreactor of the invention.

BACKGROUND OF THE INVENTION

The artificial production or fabrication of living tissues represents one of the main targets of tissue engineering. Among the key challenges in the field is the recapitulation of physiological mass transport. In order to guarantee nutrient provision and metabolite disposal, engineered tissues cannot rely on diffusion alone; convective flows must be integrated into the design of the tissue in order to overcome diffusion limitations. Although tissue design and production remains very challenging, bioreactors that are able to sustain convective flows across the tissues need to be considered as an integral part of the tissue development process. Beyond sustaining artificially produced tissues, the development of a new bioreactor might also be beneficial for in vitro tissue culture by providing support structures that are able to re-establish physiological flow conditions.

Bioreactors for tissue engineering have been described in the art, for example

• EP2254987B1 describes a perfusable bioreactor designed to produce and cultivate blood vessel tissues. This bioreactor is specific for blood vessel tissue and it is designed accordingly with the tubular geometry of the blood vessels. The patent mentions a transparent window to observe the sample. This device is limited to a single vessel structure.

• US8709793B2 describes a bioreactor with the capability to also generate the sample that will be perfused. Sample growth is guided by various stimulations i.e. mechanical and electrical. Moreover one or more optical ports allow monitoring of the stimulation process. The device is classified as millifluidic and aims to follow tissue growth in a non-invasive manner. The device does not consider perfusion of an existing vessel network nor promote the formation of one.

• WO2014102527A1 describes a bioreactor for hosting a plurality of scaffolds.

The design is focused on a multi-chamber format. Flow through the chambers is maintained by external pumping devices. Non-invasive monitoring is mentioned to follow the sample growth: measures of current and analysis of the effluxes among others.

• WO20141 1 1518A1 describes a bioreactor for soft tissue explant culture. The geometry is specifically circular and there are no references to vasculature alignment between the explant and the bioreactor. The main application for this device is the monitoring of soft tissue growth in ex-vivo condition.

In both artificial and ex-vivo tissues, where inner channels are present, the establishment of convective flows across the volume is crucial to overcome the diffusion limitation (100-200um) in order to maintain a viable tissue. Indeed, a diffusion distance exceeding the 100-200 micron range normally results in rapid necrosis of a growing tissue. Nowadays, several pumping devices can generate the abovementioned flows, however, the coupling of those devices with biological material remains an unsolved issue.

Furthermore, in order to properly host and perfuse either an artificial or natural tissue the geometry of the chamber of the bioreactor is crucial. In particular, if the chamber is too large for the hosted sample it is difficult to properly align the convective flows with the inner channels of the tissue, leading to inefficient perfusion that in turn affects tissue viability. There therefore exists a need to provide a bioreactor that not only can sustain multiple flows across the perfusion chamber but can also adapt to host a range of tissues of different geometries. The present invention addresses the need of providing convective flows across artificially produced and natural tissues by describing some innovative design features in bioreactor design and production. The invention relates to a bioreactor comprising a perfusion chamber across which it is possible to generate controllable and independent flows of different fluids. Furthermore, the described bioreactor comprises a chamber of variable geometry matching a particular tissue of interest. The chambers are sealed around the cultured tissue to guarantee proper perfusion. Two chamber configurations are presented, one in which the chamber is completely closed and one that has an open-roof geometry where the top side of the chamber presents apertures to provide nutrients or signaling molecules. In this latter configuration the chamber elements mainly maintain the tissue in the proper position to promote perfusion.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a bioreactor for culturing and/or growing artificial or natural tissues, the bioreactor comprising

a top plate and a bottom plate, wherein the top and bottom plate together define a perfusion chamber for receiving and supporting an artificial or natural tissue, wherein the top plate further comprises a plurality of inlet structures and a plurality of outlet structures, wherein the inlet and outlet structures are in fluid communication with the perfusion chamber to allow flow of at least one medium through the perfusion chamber; and wherein the bottom plate further comprises a height adjustable portion for adjusting the volume of the perfusion chamber.

In a preferred embodiment the height adjustable portion is moveable to a plurality of positions between a first upper position and a second lower position, wherein when the height adjustable portion is in the first position the volume of the perfusion chamber is minimised and wherein when the height adjustable portion is the second position the volume of the perfusion chamber is maximised. In a further embodiment of the device described herein, the bioreactor further comprises a height adjustment driver and a housing for a height adjustment driver, and the height adjustable portion comprises an inclined plane, wherein the height adjustment driver is arranged to abut the inclined plane, such that the height adjustment driver and plane together urge the height adjusting portion into a predetermined position with respect to the top plate.

In another embodiment the height adjustment driver is moveable linearly within the housing and wherein linear inward movement of the height adjustment driver abuts the inclined plane to vertically move the height adjustable portion between a plurality of positions between the first upper position and the second lower position.

Preferably, the height adjustment driver is a screw. In an alternative embodiment, the height adjustment driver is a lever. In a further alternative embodiment, the height adjustment driver comprises a gear and linear actuator.

In a preferred embodiment, the inlet and outlet structures are channels in the top plate. The channels may also be termed conduits. Such terminology is interchangeable. Preferably the channels are integral to (i.e. within) the top plate. This means that the channels are closed, in that every side of the channel is enclosed by the top plate. In an alternative embodiment, the channels or conduits are formed on the surface of the top plate, meaning that the channels are open on one side (the top side).

In one embodiment there is at least one inlet and one outlet structure. In a preferred embodiment there may be two, three, four, five, six, seven or eight inlet and outlet structures. The channels are configured to be in fluid communication with the perfusion chamber to allow the flow of at least one medium through the perfusion chamber. The medium can be any medium suitable to perfuse and maintain the viability of a sample within the perfusion chamber. Examples include a cell culture medium, a physiological solution, saline solution, nutrient solution, blood replacements, plasma, nutrient cocktails and a conditioned medium. The medium may additionally comprise at least one of growth factors, cytokines, enzymes, fixation reagents, antibodies, drugs, drug candidates and chelating agents. In an alternative embodiment, there is provided a bioreactor for culturing and/or growing artificial or natural tissues, wherein the bioreactor comprises a top plate and a bottom plate, wherein the top and bottom plate together define a perfusion chamber for receiving and supporting an artificial or natural tissue, wherein the bottom plate further comprises a plurality of inlet structures and a plurality of outlet structures, wherein the inlet and outlet structures are in fluid communication with the perfusion chamber to allow flow of at least one medium through the perfusion chamber; and wherein the top or bottom plate further comprise a height adjustable portion for adjusting the volume of the perfusion chamber.

In one embodiment, the diameter of the channels is any diameter from 0.5 to 1 .5mm inclusive. In a further embodiment, the channels are capable of receiving a volume of medium, wherein the flow rate of the volume of medium is within the range 1 nl_/min to 50ml_/min inclusive, preferably 10nL/min to 1 mL/min inclusive.

In a further embodiment the inlet and outlet structures further comprise valves to regulate the flow of a medium through the perfusion chamber. In one embodiment the flow of medium through the perfusion chamber is continuous. In an alternative embodiment, the flow of medium though the perfusion chamber is periodic.

The channels are also configured to be attachable to at least one pump. In one embodiment, the channels comprise a Luer taper for attachment to a pump.

In another embodiment the bioreactor further comprises at least one pump, wherein the inlet structures are connectable or connected to the pump and wherein the pump is configured to pump medium through the inlet structures to the perfusion chamber. The pump may be a peristaltic pump, a syringe pump, a turbine pump or a positive pressure pump. Alternatively, the pump may comprise one or more continuous flow pumps. In a further embodiment the pump can provide a flow rate of 1 nL/min to 50mL/min, preferably 10nl_/min to 1 mL/min. In one embodiment the pump is separate from the top and bottom plate.

Preferably in use the inlet and outlet structures are aligned with artificially created channels or a network of channels in an artificial tissue or with vessels in a natural tissue. In another embodiment, the top plate comprises a top surface. In one embodiment, the top surface covers the perfusion chamber. This is called the closed configuration. In an alternative embodiment the top surface comprises an opening in line with at least a portion, but preferably all, of the perfusion chamber. This is called the open configuration.

In a further embodiment, the bioreactor further comprises a lid. Preferably the lid is configured to attach to the top plate. In one embodiment the lid comprises an opening in line with at least a portion, preferably all, of the perfusion chamber.

In a further embodiment, at least a portion of the top plate and/or at least a portion of the lid is transparent or substantially transparent, such that when in use, a tissue within the perfusion chamber can be visually analysed. Preferably visual analysis can be selected from visualisation by the user, microscopy, CT (computed tomography) scan and MRI (magnetic resonance imaging) and ultrasound.

In another embodiment, the bioreactor further comprises at least one integrated sensor. Preferably the sensor monitors at least one of pH, temperature and gas pressure. Preferably the sensor is a pH meter, a thermometer or manometer.

The perfusion chamber may be any suitable shape or size, for example, rectangular, circular, oval, square. Preferably, the chamber is rectangular. The chamber may be of any diameter suitable for supporting and hosting an artificial or natural tissue. In one preferred embodiment, the chamber is 8mm by 12mm, wherein the height can be varied by the height adjustable portion. Preferably the height can vary from 0.5mm to 1 .5mm inclusive.

In a further embodiment, the bioreactor further comprises at least one efflux conduit, wherein in use the efflux conduit or conduits allow the flow and/or analysis of effluent following perfusion of the tissue. In one embodiment the efflux conduit or conduits is/are the outlet structure(s). Preferably the efflux conduit is in fluid communication with the perfusion chamber. The efflux conduit may further be connected to an efflux analyser configured to analyse the effluent. Preferably, the bioreactor further comprises a valve for regulating the flow of effluent through the efflux conduit. In another embodiment, the top plate or bottom plate or both further comprises a sealing element to hermetically seal the perfusion chamber. Preferably the sealing element is attached to the top plate. Alternatively, the sealing element is attached to the bottom plate. In either configuration the combination of top plate, bottom plate and sealing element (on either the top or bottom plate or both) together define the perfusion chamber. Preferably the sealing element is made from a deformable rubber/elastomer or rubber-like /elastomer-like material. Examples of suitable materials include: Acrylonitrile-butadiene rubber, hydrogenated acrylonitrile-butadiene rubber, fluorocarbon rubber, perfluorelastomer, ethylene propylene diene rubber, silicone rubber, fluorosilicone rubber, chloroprene (or neoprene) rubber, polyester urethane, polyether urethane, natural rubber, polyacrylate rubber, ethylene acrylic (Vamac), styrene-butadiene rubber, ethylene oxide epichlorohydrine rubber, chlorosulfonated polyethylene, butadiene rubber, isoprene rubber, butyl rubber, Tango Plus-FullCure 9XX series.

In a further embodiment, the bioreactor comprises at least one supporting structure for supporting the top and bottom plate in a coupled configuration. Preferably the supporting structure is selected from one or more clamps or a press-hold device. Alternatively, the top and bottom plate are maintained in a coupled configuration through chemical bonding or an adhesive layer on one or both of the top and bottom plates, or combinations of any of the above.

The bioreactor may be made from any suitable material. Such suitable materials will be known to the person skilled in the art, but preferably the bioreactor may be made from plastic. In a preferred embodiment the bioreactor may be made from Acrylic (polymethylmethacrylate), Butyrate (cellulose acetate butyrate), Lexan (polycarbonate), and PETG (glycol modified polyethylene terphthalate), or Tango Plus - Full Cure 720. In a second aspect of the present invention there is provided a method of producing an artificial tissue or culturing an artificial or natural tissue, the method comprising using a bioreactor of the present invention. In a third aspect of the present invention there is provided the use of a bioreactor of the present invention to produce an artificial tissue or to culture an artificial or natural tissue. Preferably the tissue is selected from brain tissue, skin tissue, ocular tissue, muscular tissue, pulmonary tissue, cardiac tissue, venous tissue, artery tissue, lymphoid tissue, mammary tissue, thymus tissue, stomach tissue, liver tissue, pancreatic tissue, intestinal tissue, kidney tissue, bladder tissue, cartilage tissue, tendon tissue and bone tissue.

In a fourth aspect of the present invention there is provided an artificial tissue produced using the bioreactor of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described in the following non-limiting Figures:

Figure 1 shows a section view of the bioreactor of the present invention and the various geometries of the perfusion chamber.

Figure 2 shows a section view of the bioreactor of the present invention, where the bioreactor has a plurality of inlet and outlet structures.

Figure 3 shows a flowchart of the various analyses that can be performed on the bioreactor to obtain different biological information on the status of the sample within the perfusion chamber.

Figure 4 shows a further view of the bioreactor of the present invention. Figure 5 shows a number of further views of the bioreactor of the preset invention.

Figure 6 shows a number of views of the bioreactor of the preset invention

Figure 7 shows a cross-section of the top and bottom plates, wherein the top plate further comprises a sealing element. Figure 8 shows a number of views of a supporting structure. DESCRIPTION OF THE INVENTION

Three-dimensional bioprinting shifts the classic tissue-engineering concept based on tissue scaffolds towards a more dynamic process. Bioprinting can reproduce tissue features in 3D by patterning scaffolding materials together with active components, i.e. cells, and supporting molecules, i.e. protein and growth factors. However, scaffold- based approaches rely on interconnected porosities to guarantee nutrient provision and disposal of metabolites across the tissue construct. On the other hand, 3D bioprinting is capable of generating controlled channel networks within the tissue-like structure. Such networks are precisely designed to overcome mass transport limitation related to diffusion.

The bioreactor represents the key device to connect a biological tissue, either naturally derived or artificially produced, and the pumping device that actually generates the perfusion flows within the bioreactor. In other terms, the bioreactor accomplishes two main tasks: holding and supporting the hosted tissue and providing the connections with the perfusion device.

Considering scaffold-based strategies, bioreactors are responsible for moving culturing medium and guaranteeing a flow across the scaffold. However, considering the geometry of the tissue constructs, the generation of movement does not have specific limitations or constraints.

Considering bioprinted samples, the inner channel networks are characterised by a precise geometry. The bioreactor is responsible for aligning inlets/outlets on the sample with the devices generating the flow, i.e. pumps. In this controlled scenario, the bioreactor can promote multiple flows across the sample achieving a higher freedom in controlling sample maturation/culture.

Compared to prior bioreactor technologies, the present invention represents an improvement on those bioreactors that are used to flow medium across scaffold-based samples. This invention introduces the concept of aligning the sample within a tight-fit chamber. By controlling channel geometry, multiple flows can be established across the samples and culturing conditions can be significantly improved. Among the various requirements, this invention focuses on three main characteristics that the bioreactor should preferably feature:

(i) The perfusion chamber should be characterised by a variable geometry.

Chamber geometry is crucial to properly host and perfuse a tissue, either artificially produced or naturally derived. The ability to modify the chamber geometry on-demand is a crucial feature to include in the design of the bioreactor. This feature is of particular relevance for naturally derived tissue, where the geometry of the sample can vary sample to sample. Moreover, this feature is also convenient to compensate the possible geometric variability in artificially produced tissues (i.e. tissue height in bioprinted samples). A further advantage of a variable geometry is the possibility to follow tissue growth/evolution within the chamber, adapting the geometry of the latter to tissue geometric variations.

The same chamber should allow for multiple perfusion flows.

The physiological complexity of living tissues imposes a wide range of requirements for the flows across the tissue itself (arterious and venous blood, lymphatic fluid). So the ability to sustain multiple flows across the perfusion chamber represents a relevant feature to include in the design process to mimic physiology. When a multitude of flows can be independently tuned (preferably by an internal or external pump) the distance between the physiological and the artificial scenario can be drastically reduced, obtaining a more representative model of the real phenomenon. Furthermore, the presence of multiple channels also translates into more flexibility in establishing a pattern of perfusion. While artificially produced tissue, can be designed to align inner channels with inlet structure within the chamber, there is no such control in naturally derived tissues. So a multitude of possible inlets in the bioreactor, allows for a better alignment with natural tissues within the bioreactor chamber.

(iii) The possibility to monitor the chamber and the behavior of the hosted sample is crucial. Integrated sensors should follow macroscopic parameters recording: pH, temperature and gas pressure. Transparent construction materials should allow direct investigation of sample behavior: microscopy, CT scan or MRI, to name a few. Finally the analysis of effluxes should give a more analytic grade to sample characterization.

Consequently, and in summary, the key advantages of the bioreactor of the present invention are as follows: · Chamber design allows fast and reliable alignment of printed vessel-like structure with fluidic inlets/outlets;

• Chamber geometry is variable to adapt to variable dimensions in bioprinted samples;

• Chamber can be sealed and the sample results isolated for better sterility;

· Multiple flows can be held in parallel fashion and independently controlled;

• Considering the controlled vessel geometry (bioprinted), the bioreactor design allows fast and effective extrapolation of convective flows across the printed channels. Shear-stress can then be evaluated from those parameters. Turning to the Figures, Figures 1A to 1 C show a schematic representation of the bioreactor according to the present invention. In particular, these section views of the bioreactor show how chamber geometry can be varied. As shown in both Figures 1A and 1 B, the bioreactor (1 ) comprises a top plate (2) and a bottom plate (3), which together define the perfusion chamber (4). In other words, the top plate provides at least the sides (or side) of the perfusion chamber, and optionally the top surface, and the bottom plate provides the bottom surface. When the top and bottom plate are coupled, as shown in Figures 1 A and 1 B, the perfusion chamber is formed. The bottom surface of the perfusion chamber is formed by the height adjustable portion (6), which acts as a moveable stage to adjust the volume of the perfusion chamber. The height adjustable portion also comprises a downward inclined plane (18). In this embodiment, the top plate further comprises a plurality of inlet and outlet channels (5) to maintain the perfusion status within the chamber. In this scenario, the position of such channels is fixed within the bioreactor and allows for faster and more effective alignment with tissue features.

The bottom plate further comprises a height adjustable driver (not shown) and a housing for the height adjustable driver (7). As previously explained, the height adjustable portion is moveable between a plurality of positions between a first upper position where the volume of the perfusion chamber is minimized (Figure 1A) to a second lower position where the volume of the perfusion chamber is maximized (Figure 1 B). Movement between these two positions is achieved by the height adjustable driver. The height adjustable driver allows the user to adjust the position of the height adjustable portion, and thus the volume of the perfusion chamber, at any time during use of the bioreactor. Preferably, the height adjustable driver is a screw but other means that achieve the same result would be apparent to the skilled person. The height adjustable driver is arranged to abut the inclined plane (18) of the height adjustable portion such that movement of the driver towards the inclined plane (18) moves the adjustable portion to the first upper position, or positions in-between. Conversely, movement of the driver away from the inclined plane (18) moves the adjustable portion to the second lower position, or positions in-between. Figure 1 C shows a top (plan) view of the bioreactor of the present invention (1 ), showing the plurality of inlet and outlet structures (5), the perfusion chamber (4) and the housing for the height adjustable driver (7). In an alternative embodiment, the top plate can comprise the necessary elements, such as a height adjustment portion and driver, to control the chamber volume.

Figure 2 shows a section of the bioreactor (1 ). In this embodiment the bioreactor contains eight inlet and/or outlet structures (5) (this could be considered as four inlet and four outlet structures). The inlet and/or outlet structures are channels or conduits within the top plate through which medium can flow from a pump (not shown) through the perfusion chamber (4). In one embodiment (not shown) the medium flowing through the outlet structures (the effluent) can be analysed as described herein. Alternatively, the medium can be discarded. In one further embodiment (also not shown) the medium from the outlet structure can be recycled and re-fed to the inlet structures thereby creating a closed-loop system. A closed loop system is advantageous as it can be used to maintain a sterile environment that is free from microorganism contamination. In this embodiment the bioreactor may further comprise a reservoir containing fresh or treated and recirculating medium. In this embodiment, effluent may first be fed through the reservoir before being re-fed to the perfusion chamber through the at least one inlet structure.

Figure 3 shows a flow chart of some of the analyses that can be carried out while the sample is in the bioreactor (1 ) and preferably being perfused by at least one appropriate medium. In this Figure the perfusion chamber is fed by a number of inlet structures or channels (5). Integrated sensors in the bioreactor (not shown) allow the measurement of a number of parameters such as pH, temperature and gas pressure. The ability to evaluate the behavior of the hosted sample is essential to assess artificial tissue maturation or to investigate naturally derived tissue responses to specific inputs, such as drugs, drug candidates, proteins, small molecules, enzymes, growth factors and any other compound that can be useful for conditioning tissue behavior. In one embodiment at least the perfusion chamber, preferably the top plate, is made from a transparent material, such as Acrylic (polymethlamethacrylate), Butyrate (cellulose acetate butyrate), Lexan (polycarbonate), and PETG (glycol modified polyethylene terphthalate), or Tango Plus - Full Cure 720. This also allows the sample to be directly observed by a user. Alternatively, the sample can be directly investigated or analysed using various imaging techniques, such as microscopy, MRI scan, CT scan and ultrasounds. Where such techniques are to be employed it may be useful to perfuse the sample (through the inlet channels) with contrast agents/mediums or staining solutions. Where an MRI scan is to be employed the bioreactor may further comprise suitable elements to produce or interact with the magnetic fields during image recording. Finally, the bioreactor also contains a number of outlet channels. In one embodiment these also act as the efflux conduit. The efflux conduit or outlet channels allow the collection of effluent following perfusion of the sample within the chamber. This effluent can subsequently be analysed by a number of independent methods, such as spectrographic investigation, HPLC (High performance liquid chromatography), HNMR (proton nuclear magnetic resonance) and separation columns. The possibility to analyse the effluxes coming out of the culture sample is a powerful method to evaluate tissue behavior and evolution. Figure 4 shows a further view of the bioreactor (1 ), comprising the top plate (2), the bottom plate (3), the perfusion chamber (4), a plurality of inlet and outlet structures (5) , the height adjustable portion (6) and the housing for the height adjustable driver (7). The top plate may further comprise a sealing element (not shown) which hermetically closes the perfusion chamber (4). The design can be also used when it is turned upside-down.

Figure 5 shows a number of further views of the bioreactor. Figures 5A and 5B are cross-sectional and top views respectively of the bottom plate (3) showing the height adjustable portion (6) and the housing for the height adjustment driver (7). Figure 5C is an axonometric view where the housing for the height adjustment driver (7) is clearly depicted. In one embodiment the height adjustment driver can be a screw (not shown), such as a M2 screw, which is used to control the volume of the perfusion chamber (4) by interacting with the downward inclined plane (18) on the height adjustable portion (6).

Figure 6 shows two alternative embodiments of the top plate. In one embodiment (Figure 6A) the top plate (2) comprises a top surface (9) that completely covers the perfusion chamber (4). This is called the closed configuration. In a second alternative embodiment (Figure 6B), the top surface (9) comprises an opening (10) in line with at least a portion, preferably all, of the perfusion chamber (4). This is called the open configuration. In this open configuration the ring of material that is the top surface (9) ensures that the sample is held in position for perfusion. Figure 7A shows a cross-section of the bioreactor comprising the top (2) and bottom (3) plate and the height adjustable portion (6). In this embodiment, the top plate (2) further comprises a sealing element (1 1 ). The sealing element (1 1 ) represents a crucial element for the overall design. This soft ring is attached on the non-movable element of the bioreactor (in one embodiment this is the top plate (2)), in order to be aligned with inlet and outlet structures (not shown). When coupled with the bottom plate (3) the sealing element on the top plate (2) abuts a recess (12) in the bottom plate (3) leading to deformation of the sealing element (1 1 ) (as shown in Figure 7B) and sealing of the perfusion chamber (4). The seal is also used to direct the tissue in its proper position to promote perfusion through its internal fluidic network. The sealing element (1 1 ) may be any suitable material but preferably the material should allow perfusion of a medium through the material. In one embodiment the sealing element (1 1 ) is any flexible material, preferably a deformable rubber-based material. In another embodiment the flexible material may be selected from one or more of the below listed materials:

Table 1 : List of possible sealing materials

As a general rule, these are all classified as elastomers.

The following table refers principally to materials used for o-ring production i.e. the sealing element (1 1 ).

Chemical Abbreviation

Description ASTM D 1418 ISO/DIN 1629

Acrylonitrile-butadiene rubber NBR NBR

Hydrogenated acrylonitrile-butadiene rubber HNBR (HNBR)

Fluorocarbon rubber FKM FPM

Perfluoroelastomer FF M (FFPM)

Ethylene propylene diene rubber EPDM EPDM

Silicone rubber VMQ VJV

Pluorosilicone rubber FVMQ FVMQ

Chloroprene (or Neoprene) rubber CR CR

Polyester urethane AU AU

Polyether urethane EU EU

Natural rubber NR NR

Polyacrylate rubber ACM ACM

Ethylene Acrylic (Vamac®) AEM AEM

Sryrene-butadiene rubber SIR SBR

Ethylene oxide epichlorohydrine rubber ECO ECO

Chlorosulfonated polyethylene CSM CSM

Butadiene rubber BR BR

Isoprene rubber IR IR

Butyl rubber 111 IIR

{ ) = not listed in the standard.

Further candidate materials are the Tango Plus - Full Cure 9XX series, which is defined as elastomer and they are specifically used in 3D printing application. Figure 8 shows a number of views of one embodiment of a supporting structure (13,14) of the present invention. Such supporting structures (13,14) guarantee bioreactor mechanical stability. Clamp-like devices fit the bioreactor (not shown) geometry and ensure closure of the perfusion chamber. Among the possible strategies to hold together the bioreactor elements are one or more clamps; press-and-hold devices; chemical bonding; adhesive layer or combinations of the above mechanisms. Ideally, if a supporting structure/holding device is deployed, the device should be reusable for cost-efficiency purposes. In this embodiment the supporting structure is comprised of two parts - a top supporting structure (13) and a bottom supporting structure (14). The top and bottom supporting structures can be clamped or slotted together and together define a supporting structure for the bioreactor. Figures 8A, B and C show a top view, side view and isometric view of the top supporting structure (13). Figures 8D, E and F similarly show a top view, side view and isometric view of the bottom supporting structure (14). The top supporting structure (13) is substantially planar and comprises an opening (15) which in use will be in line with at least a portion, preferably all, of the perfusion chamber (not shown). The bottom supporting structure comprises a base (16) and a plurality of recessed arms (17) configured to couple with the top supporting structure (13). Figure 8G shows the assembled supporting structure (13,14) comprising the top (13) and bottom (14) supporting structures.

Additional elements of the bioreactor according to the present invention are summarised below: 1 . The bioreactor of the present invention is designed to be capable of sustaining convective blood-like flows across artificially produced or naturally derived tissues.

2. The perfusion is established across the perfusion chamber, which represents the core of the device.

3. The perfusion chamber is formed by multiple elements, preferably assembled vertically (one of top of the other), namely at least one top plate and at least one bottom plate. Top and bottom plates are coupled by seal elements, which guaranteed the tight closure of the perfusion chamber.

4. Perfusion chamber geometry can be modified to fit the hosted tissue geometry. 5. Geometry variation of the chamber is achieved by moving parts in either the top or bottom plates. The geometry adaptation is user-controlled and it is accessible at any time.

6. The perfusion chamber can be characterized by either a closed or open roof. In the first embodiment, the sample is fully surrounded by the chamber. In the open scenario (second embodiment), the roof is at least, partially open, in order to promote a free-exchange with the environment, while holding the sample inside the chamber to ensure perfusion.

7. The seal elements, preferably made from rubber-like material, maintain the sample in the proper position within the chamber and guarantee proper connection between the inlet/outlet structures and the sample channel network.

8. Multiple inlet/outlet channels serve the perfusion chamber.

9. The ability to vary chamber geometry increases the odds of proper perfusion in naturally derived tissues.

10. The ability to vary chamber geometry also allows the generation of multiple flows across the hosted tissues.

1 1 . The connection between the bioreactor and a pumping device can be obtained by commercially available elements.

12. The bioreactor can be held together mechanically, chemically, by adhesive layers or combination of the above.

13. The chamber and holding elements can be produced by, for example, by 3D printing, molding or injection molding.

14. The chamber is monitored by integrated sensors that measure, for example, pH, temperature and gas pressure.

15. Hosted tissues can be investigated by various techniques and the bioreactor is compatible with those. Effluxes can be analyzed after extraction. The sample can also be imaged by, for example, microscopy, CT scan or MRI scan, or any combination of the above.

16. Artificial tissues can be produced by 3D bioprinting.

17. Artificial tissues can also be produced by liquid handling techniques.

18. The internal microfluidic network can be obtained by sacrificial material approaches.

19. The internal microfluidic network can be generated by etching strategies, specifically laser etching. Naturally derived tissues are characterized by natural vessel networks and they can be obtained by surgery or biopsy.

Claims

CLAIMS:

1 . A bioreactor (1 ) for culturing and/or growing artificial or natural tissues, the bioreactor comprising

a top plate (2) and a bottom plate (3), wherein the top (2) and bottom plate (3) together define a perfusion chamber (4) for receiving and supporting an artificial or natural tissue,

wherein the top plate

(2) further comprises a plurality of inlet structures and a plurality of outlet structures (5), wherein the inlet and outlet structures (5) are in fluid communication with the perfusion chamber (4) to allow flow of at least one medium through the perfusion chamber (4); and

wherein the bottom plate

(3) further comprises a height adjustable portion (6) for adjusting the volume of the perfusion chamber (4). 2. The bioreactor (1 ) of claim 1 , wherein the height adjustable portion (6) is moveable to a plurality of positions between a first upper position and a second lower position, wherein when the height adjustable portion (6) is in the first position the volume of the perfusion chamber (4) is minimised and wherein when the height adjustable portion (6) is the second position the volume of the perfusion chamber (4) is maximised.

The bioreactor (1 ) of claim 1 or 2, wherein the bioreactor (1 ) further comprises a height adjustment driver and a housing for a height adjustment driver (7), and the height adjustable portion (6) comprises a downward inclined plane (18), wherein the height adjustment driver (7) is arranged to abut the inclined plane (18), such that the height adjustment driver and plane (18) together urge the height adjusting portion (6) into a predetermined position with respect to the top plate (2).

4. The bioreactor (1 ) of claim 3, wherein the height adjustment driver is moveable linearly within the housing (7) and wherein linear inward movement of the height adjustment driver abuts the inclined plane (18) to vertically move the height adjustable portion (6) between a plurality of positions between the first upper position and the second lower position.

5. The bioreactor (1 ) of claim 3 or 4, wherein the height adjustment driver is a screw.

6. The bioreactor (1 ) of any preceding claim, wherein the inlet and outlet structures (5) are conduits in the top plate.

7. The bioreactor (1 ) of any preceding claim, further comprising a pump, wherein the inlet structures (5) are connectable to the pump and wherein the pump is configured to pump medium through the inlet structures (5) to the perfusion chamber (4).

8. The bioreactor (1 ) of any preceding claim, wherein in use the inlet and outlet structures (5) are aligned with artificially created channels or a network of channels in an artificial tissue or with vessels in a natural tissue.

9. The bioreactor (1 ) of any preceding claim, wherein the top plate (2) further comprises a top surface (9), and wherein the top surface (9) completely covers the perfusion chamber.

10. The bioreactor (1 ) of claim 9, wherein alternatively the top surface (9) comprises an opening (10) in line with at least a portion of the perfusion chamber (4).

1 1 . The bioreactor (1 ) of any preceding claim, wherein at least a portion of the top plate (2) is transparent or substantially transparent.

12. The bioreactor (1 ) of any preceding claim, wherein the bioreactor (1 ) further comprises at least one integrated sensor.

13. The bioreactor (1 ) of claim 12, wherein the integrated sensor monitors at least one of pH, temperature and gas pressure.

14. The bioreactor (1 ) of any preceding claim, wherein the bioreactor (1 ) further comprises at least one efflux conduit (5), wherein in use the efflux conduit (5) allows analysis of effluent following perfusion of the tissue.

15. The bioreactor (1 ) of any preceding claim, wherein the top plate (2) or bottom plate (3) further comprises a sealing element (1 1 ) to hermetically seal the perfusion chamber (4).

16. The bioreactor (1 ) of any preceding claim, wherein the perfusion chamber (4) is rectangular.

17. The bioreactor (1 ) of any preceding claim, wherein the bioreactor (1 ) further comprises at least one supporting structure (13, 14) for supporting the top (2) and bottom plate (3) in a coupled configuration.

18. A method of producing an artificial tissue or culturing an artificial or natural tissue, the method comprising using a bioreactor (1 ) according to any of claims 1 to 17.

19. The method of claim 18 wherein the tissue is selected from brain tissue, skin tissue, ocular tissue, muscular tissue, pulmonary tissue, cardiac tissue, venous tissue, artery tissue, lymphoid tissue, mammary tissue, thymus tissue, stomach tissue, liver tissue, pancreatic tissue, intestinal tissue, kidney tissue, bladder tissue, cartilage tissue, tendon tissue and bone tissue.