BIOREACTOR CHAMBER

Abstract

A bioreactor chamber (1) including a first end block (4), a second end block (6) and a flexible membrane (2). The flexible membrane (2) extends between the first end block (4) and the second end block (6) and defines a cavity (10) bounded by at least the flexible membrane (2). The cavity (10) is arranged to receive a substrate, for growing a culture on the substrate or a biomaterial for testing the biomaterial.

Claims

1. A bioreactor chamber, comprising: a first end block; a second end block; and a flexible membrane, extending between the first end block and the second end block, to define a cavity bounded by at least the flexible membrane; and wherein the cavity is arranged to receive: a substrate, for growing a culture on the substrate; or a biomaterial for testing the biomaterial.

2. The bioreactor chamber of claim 1, wherein the cavity is bounded by the first end block, the second end block and the flexible membrane.

3. The bioreactor chamber of claim 1, wherein the cavity has a substantially tubular shape.

4. The bioreactor chamber of claim 1, wherein the flexible membrane is at least partly transparent.

5. The bioreactor chamber of claim 1, wherein the flexible membrane is less than 100 microns thick.

6. The bioreactor chamber of claim 1, wherein the flexible membrane is sealingly connected to the first end block and/or the second end block.

7. The bioreactor chamber of claim 1, wherein the first end block and/or the second end block comprises an outer member and an inner member, and wherein the flexible member is clamped between the outer member and the inner member.

8. The bioreactor chamber of claim 7, wherein the first end block and/or the second end block comprises a ring; wherein the flexible membrane passes through an aperture in the inner member, through the ring, to be folded back on itself to surround the ring, and passes back through the aperture in the inner member; and wherein the flexible membrane, the outer member and the inner member are attached together, and the ring is clamped between the outer member and the inner member.

9. The bioreactor chamber of claim 8, wherein the inner member comprises a recessed portion, wherein the recessed portion further comprises the aperture, and wherein the recessed portion at least partially contains the ring.

10. (canceled)

11. The bioreactor chamber of claim 1, wherein the substrate comprises a scaffold and wherein the scaffold extends between the first end block and the second end block.

12. (canceled)

13. The bioreactor chamber of claim 1, wherein the substrate comprises a scaffold and wherein the scaffold comprises a plurality of substantially aligned electrospun filaments.

14. The bioreactor chamber of claim 1, wherein the first end block and/or the second end block comprise a fixing point, for connection to a mechanical actuator.

15. The bioreactor chamber of claim 1, wherein the cavity comprises an inlet for cell culture medium.

16. (canceled)

17. The bioreactor chamber of claim 15, wherein the first end block comprises the inlet and the second end block comprises an outlet, and wherein the inlet and the outlet are arranged to be positioned offset from each other on opposing sides of the cavity.

18. (canceled)

19. A bioengineering system, comprising: a bioreactor chamber as claimed in claim 1; a mechanical actuator, connected to the first end block and/or the second end block; wherein the mechanical actuator is configured to actuate the first end block and/or the second end block.

20. The bioengineering system of claim 19, wherein the mechanical actuator is a multi-directional actuator.

21. The bioengineering system of claim 19, wherein the mechanical actuator is constructed to mimic a specific joint in the human or animal body.

22. A method of growing a culture comprises: supplying cell culture medium to the cavity of a bioreactor chamber as claimed in claim 15, through the inlet, for growing a culture, wherein the bioreactor chamber comprises a substrate, arranged within the cavity, for growing the culture on the substrate; attaching the first end block and/or the second end block to a mechanical actuator; and moving the first end block and/or the second end block using the mechanical actuator, to move the substrate, so to apply mechanical stimulation to the culture being grown within the cavity.

23. The method of claim 22, wherein the mechanical actuator is a multi-directional actuator.

24. The method of claim 22, wherein the mechanical actuator is constructed to mimic a specific joint in the human or animal body.

25. (canceled)

Description

[0081] Certain preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0082] FIG. 1 is a schematic diagram showing the bioreactor chamber apparatus according to the an embodiment of present invention.

[0083] FIG. 2 is an image showing a scaffold, extending between a first end block and a second end block of a bioreactor chamber according to an embodiment of the present invention.

[0084] FIG. 3a is a three-dimensional exploded view of an outer member of an end block of a bioreactor chamber according to an embodiment of the present invention.

[0085] FIG. 3b is a schematic diagram showing an inner member and a rigid ring, which are parts of an end block of a bioreactor chamber according to an embodiment of the present invention.

[0086] FIG. 3c is an image showing two inner members, with a membrane extending between them, according to an embodiment of the present invention.

[0087] FIG. 3d is a cross-sectional view of a membrane connected within an end block of a bioreactor chamber, according to an embodiment of the present invention.

[0088] FIG. 3e is an image showing two inner members, with a membrane extending between them, together with an outer member, according to an embodiment of the present invention.

[0089] FIG. 4a is a schematic drawing showing a side view of an outer member of an end block of a bioreactor chamber according to an embodiment of the present invention.

[0090] FIG. 4b is a schematic drawing showing a view of an outer member of an end block of a bioreactor chamber according to an embodiment of the present invention, along the line A as shown in FIG. 4a.

[0091] FIG. 4c is a schematic drawing showing a view of an outer member of an end block of a bioreactor chamber according to an embodiment of the present invention, along the line B as shown in FIG. 4b.

[0092] FIG. 5 is an image showing the bioreactor chamber according to an embodiment of the present invention, connected to a humanoid robot, specifically a humanoid robotic shoulder.

[0093] FIG. 6a is a graph showing the percentage strain (x-axis) against the stress in MPa (y-axis) as tensile stress is applied to a bioreactor chamber according to an embodiment of the present invention.

[0094] FIG. 6b is a graph showing the Young's modulus of an exemplary scaffold and flexible membrane according to an embodiment of the present invention, in units of MPa.

[0095] Throughout the figures, like reference numerals have been used for like elements.

[0096] Bioreactors, or bioreactor chambers, are used in tissue engineering to provide a controlled environment, in which conditions can be controlled to meet the requirements needed for maintaining and stimulating living cells and tissues outside the body. Robotic devices may be integrated into bioreactor chambers to provide mechanical cues to a growing tissue sample which modulate the growth of the tissue. Mechanical stresses (tension, compression, torsion and shear stresses) naturally occur in vivo and are crucial to the development and maintenance of musculoskeletal tissues such as tendons (soft tissues connecting muscles and bones).

[0097] Conventional bioreactor chambers are capable of applying only unilateral forces using a linear actuator which connects directly to a cell or tissue sample. This is unsatisfactory, since it is known that tendons undergo profound modifications when deprived of certain stresses, including a reduction of their anatomical size, a rapid deterioration of their mechanical properties and a change in extracellular matrix (ECM) composition and organisation. The existing evidence suggests that mimicking physiological stresses more closely would improve the quality of tendon grafts produced, however this is not currently possible due to the structure of available bioreactor chambers. Furthermore, in current bioreactor chamber designs the actuator is contained within the bioreactor chamber, and connected directly to the tissue sample, such that the sample cannot be separated from the actuation system without opening the bioreactor chamber and therefore risking contamination.

[0098] The aim of the present invention is therefore to provide an improved bioreactor chamber. A simple schematic drawing of the bioreactor chamber 1 according to an embodiment of the present invention is shown in FIG. 1.

[0099] The bioreactor chamber 1 comprises a flexible membrane 2 extending between a first end block 4 and a second end block 6. The flexible membrane 2, together with the first end block 4 and the second end block 6 defines a cavity 10. In the example of the Figures the substrate within the cavity is a scaffold 8. The scaffold 8 extends between the first end block 4 and the second end block 6, within the cavity 10. The membrane 2 surrounds the scaffold 8 and acts as the chamber's wall (i.e. the wall of the cavity 10) to contain the scaffold 8. In use, the cavity 10 may also contain a tissue engineering medium, supported by the scaffold 8, for growing a tissue sample, the cavity created by the flexible membrane 2, the first end block 4 and the second end block 6 is therefore important to maintain sterility inside the cavity 10 for the growth of a tissue sample. The membrane 2 comprises a thin sheet of transparent polyurethane rolled into a tubular shape and sealed along the long edge.

[0100] The embodiment of the present invention thus provides a bioreactor chamber 1 which is both flexible, owing to the chamber cavity 10 being formed, at least in part, by a flexible membrane 2, and which is independent from a surrounding mechanical actuation system (since the end blocks 4, 6 can be attached to a mechanical actuator, without any part of the actuator attaching directly to the scaffold 8). This improvement enables the application of multiaxial motions by complex actuation system, since torsion, compressive and tensile forces can easily be transmitted through the flexible membrane 2. The flexible membrane 2 also allows the tissue within the bioreactor chamber 1 to be easily observed (e.g. by transporting the entire sealed bioreactor chamber to a microscope) without interfering with the inner sample.

[0101] The scaffold 8 or artificial matrix is made of parallel PCL filaments produced by electrospinning, shown in more detail in FIG. 2. In this example, a total of 200 filaments, regrouped in 5 bundles of 40 filaments each, have been stretched through the tubular membrane and fixed in the end blocks at both ends with medical grade resin. Each filament is made of aligned submicron fibres having an average diameter of 800 nm. This arrangement creates a highly anisotropic and porous scaffold.

[0102] In the example shown in the Figures, the flexible membrane 2 is sealingly connected to each of the first end block 4, and the second end block 6. It is of course possible that at another point on the boundary of the cavity 10 (i.e. in one of the end blocks) an opening may be formed forming a connection from the exterior of the cavity to the interior of the cavity.

[0103] One example of a sealing connection between the membrane 2 and each end block 4, 6 is shown in FIGS. 3a-3e. The structure of an end block will now be described with reference to the first end block 4, however it will be understood by the skilled person that any of these features may, either additionally or alternatively, be present in the second end block 6. The first end block 4 comprises an inner member 20, a rigid ring 22 and an outer member 25, as shown in the three-dimensional exploded view of FIG. 3a.

[0104] The inner member 20 and the rigid ring 22 are shown in more detail in FIG. 3b. The inner member 20 comprises a recessed portion 24, indicated in FIG. 3b by a dashed line. The recessed portion 24 is of a suitable shape and size to accommodate the rigid ring 22. In order to provide a better seal, a first rubber O-ring 26 is provided within the recessed portion 24 of the inner member 20.

[0105] The flexible member 2 is shown in FIG. 3c, connected between the inner member 20 of the first end block 4 and the inner member 30 of the second end block 6. In order to form the sealing connection between the flexible member 2 and the first end block 4, i.e. to seal the flexible membrane 2 against the inner member 20, the flexible membrane 2 is passed through an aperture 28 in the inner member 20, from the side which does not contain the recessed portion 24, through the aperture 28, which opens, on the other side, into the recessed portion 24 of the inner member 20. The flexible membrane 2 is then passed through the central aperture 23 of the rigid ring 22, which is defined by the inner edge of the rigid ring 22. The flexible membrane 2, which in this example has a tubular shape i.e. a substantially circular or oval cross-section, is then folded back on itself, so as to surround the outer edge of the rigid ring 22.

[0106] The flexible membrane 2, which is surrounding the outer edge of the rigid ring 22, is then passed back through the aperture 28 in the inner member 20. The rigid ring 22 is then inserted into the recessed portion 24, which is sized to contain it, and is pushed against the first rubber O-ring 26 to form a seal. The resulting structure is shown in FIG. 3c, and is also shown in cross-section in FIG. 3d.

[0107] The rigid ring 22 may be fixedly attached to the inner member 20, for example it may be glued in place. Alternatively, as shown, the rigid ring 22 may be clamped in position, i.e. held in the sealing position by pressure, by fixing the outer member 25 to the inner member 22, retaining the rigid ring 22 between the inner member 20 and the outer member 25. As shown in FIG. 3d, a second rubber O-ring 27 may be provided between the outer member 25 and the rigid ring 22. The second rubber O-ring is positioned so that, when the outer member 25 and the inner member 20 are bought together, with the rigid ring 22 clamped between them, the second rubber O-ring 27 contacts the flexible membrane 2, at the point 29 at which the flexible membrane 2 passes around the exterior of the rigid ring 22. This advantageously provides an improved seal and reduces wear on the flexible membrane.

[0108] The outer member 25 may optionally be attached to the inner member 20 by inserting screws through screw holes in the inner member 20, and screwing these into the outer member 25. This clamps the rigid ring 22 against the two parts of the flexible membrane 2, which are passing through the aperture 28 in the inner member 20, in order to form a seal. This stage is also illustrated in FIG. 3e, which is an image showing both of the inner members 20, 30, with the membrane 2 connected between them.

[0109] The scaffold 8 may be placed inside the flexible membrane 2 before, or after, the flexible membrane 2 is attached to the rigid ring 22 and inner member 20. Preferably, the scaffold is placed within the cavity before the outer members of both of the end blocks are attached to the inner members of the respective end blocks, so as to clamp the membrane in place. FIG. 3e shows the outer member 25, including second rubber O-ring 27, ready to be connected to the first inner member 20, in order to clamp the flexible membrane 2 in place. This arrangement largely prevents slippage of the flexible membrane and also creates a seal so as to prevent leakage out of the cavity 10 at the point where the membrane meets the end blocks. Other points on the surface of the cavity may be arranged to provide inlets or outlets to the interior of the cavity 10.

[0110] In particular, in the example shown in FIG. 4a, the outer member 25 includes resin channels 40. The outer member 25 is shown in a side profile in FIG. 4a, the side being the profile which is also shown in FIG. 1. As shown the resin channels are only visible from one of the two sides of the outer member 25. The resin channels 40 connect from the side of the outer member 25, to the inner face of the outer member 25, i.e. the surface which is adjacent to the inner member 20 when the bioreactor chamber 1 is assembled. The resin channels 40 therefore form an inlet to the cavity 10, allowing resin to be added to the cavity.

[0111] FIG. 4b shows an “end-on” view of the outer member 25, i.e. a view along the line A shown in FIG. 4a, from both an outer side (left) and an inner side (right), the inner side being the side that, when the bioreactor chamber is assembled, is adjacent to the inner member 20. The surface of the outer member 25 shown on the right of FIG. 4b is the surface to which the resin channels 40 connect. As shown in FIG. 4b, the outer member 25 includes scaffold channels 42, into which the scaffold may be inserted. The scaffold channels 42 may be blind channels, meaning that they are closed at one end and do not form a connection between the cavity 10 and the external environment. In contrast, the resin channels 40 may be “go-through” channels which connect from the external environment to the cavity 10, allowing resin to be inserted into the cavity 10. Optionally the scaffold may be fixed within the scaffold channels 42. The resin channels 40 may connect to, or feed into, the scaffold channels 42, for example the resin channels 40 may run perpendicularly to the scaffold channels 42, and may meet the scaffold channels 42 perpendicularly, allowing resin to be injected into the end of the scaffold channels 42.

[0112] The outer member 25 additionally includes a tubing channel 44. An inlet tube 46 may be connected to the tubing channel of the outer member 25 of the first end block 4, as shown in FIG. 1. An outlet tube 48 may be connected to the tubing channel of the outer member of the second end block 6. The inlet and outlet tubes 46, 48 allow perfusion of the medium during culture. In a particularly advantageous arrangement, as shown in FIG. 1, the inlet tube 46 and the outlet tube 48 are arranged to be positioned offset from each other i.e. to be not directly opposite each other on opposing sides of the cavity. This forces the perfusion medium to flow across the scaffold 8, as it passes from the inlet tube 46 to the outlet tube 48.

[0113] FIG. 4b also shows that there is an attachment member 50 arranged on the exterior surface of the outer member 25. This is more clearly visible in FIG. 4c, which shows a view along the line B, as shown in FIG. 4b. The attachment member 50 may, for example, comprise a loop, formed on the exterior surface of the outer member 25, through which a clip, or string, or other suitable connection may be passed, so as to create a mechanical connection between the outer member 25 (and therefore the bioreactor chamber 1) and an actuator.

[0114] FIG. 5 is an image, showing the bioreactor chamber 1 of an embodiment of the present invention attached to a multi-directional actuator 52. In this example, the multi-directional actuator 52 is a real-size musculoskeletal (MSK) humanoid robot. The bioreactor chamber 1 is attached to a robotic arm of the humanoid robot. MSK humanoid robots are a class of humanoids that aim to replicate the human MSK system by mimicking the inner structures of the human body such as muscles, tendons and bones. Their actuators mimic the physiologic behaviour of muscles by pulling the skeletal structure using a series of strings. In this example, the first end block 4 is attached to the humerus 54 of the humanoid robotic arm, and the second end block 6 is attached to a muscle string 56 of the humanoid robotic arm, at a location which corresponds to that of the supraspinatus tendon.

[0115] It is particularly advantageous to certain aspects of the present invention that the scaffold 8 contributes to most of the load bearing effect of the bioreactor chamber 1 at low strains, compared to the flexible membrane 2 which contributes little. This is shown in the graph of FIG. 6a. FIG. 6a represents the percentage strain (x-axis) against the stress in MPa (y-axis) as tensile stress is applied to the bioreactor chamber. The scaffold contribution 60 can be seen in the sharp peak on the left of the graph, showing that the maximum failure stress of the scaffold was at approximately 170 MPa, with a corresponding maximum strain of around 75%. The contribution of the membrane 62 can be seen in the much gentler slope, appearing on the graph only at higher strains. The flexible membrane 2 maintains its integrity until approximately 500% of strain.

[0116] The graph of FIG. 6b shows the Young's modulus of the scaffold (left) and the membrane (right) in units of MPa (y-axis). This graph highlights the large difference between the Young's modulus of the scaffold 8 and the flexible membrane 2. This ensures that any mechanical actuation which the scaffold 8 is able to withstand can be tolerated by the flexible membrane 2 without any risk of damage or yielding.

[0117] It will be appreciated by those skilled in the art that the invention has been illustrated by describing one or more specific embodiments thereof, but is not limited to these embodiments; many variations and modifications are possible, within the scope of the accompanying claims. For example, while the Figures refer primarily to growing a cell culture (e.g. a tissue structure) in the cavity, it will be appreciated that the cavity may alternatively be used for testing a biomaterial, e.g. by placing the biomaterial in the cavity (e.g. connected between the first and second end blocks).