WO2014102527A1

WO2014102527A1 - A bioreactor

Info

Publication number: WO2014102527A1
Application number: PCT/GB2013/051482
Authority: WO
Inventors: Simon Matthew PLUMMER
Original assignee: Micromatrices Associates Ltd
Priority date: 2012-12-30
Filing date: 2013-06-04
Publication date: 2014-07-03
Also published as: GB201223495D0

Abstract

A bioreactor (1) comprises one or more cell culture chambers (8) each containing an interchangeable scaffold (10) for the three dimensional culture of cells, one or more cell media chambers (2) for containing cell culture media, the cell media chamber (s) connected to the cell culture chamber (s) via one or more media flow channels (4, 47), and a pump (6) for pumping cell culture media through the media flow channel (s) from the cell media chamber (s) to the cell culture chamber (s). The bioreactor preferably comprises a nano wire- based detection system for the detection of target molecules using a measure of current/voltage changes in the nano wire. The bioreactor can preferably simultaneously measure several different biological indicator molecules, and enable molecular cellular responses to be monitored via simultaneous real-time measurement of multiple biological indicator molecules.

Description

A BIOREACTOR

[001] The present invention relates to a bioreactor, and in particular a bioreactor for the three dimensional culture of cells to assess the biological effects of a treatment on the cells .

[002] A challenge researchers face in drug, environmental, nutritional, consumer product safety, and toxicology studies is the extrapolation of data from in vitro cell culture assays to animals. Although some conclusions can be drawn with the application of appropriate pharmacokinetic principles, there are still substantial limitations. For example, screening assays utilize cells under conditions that do not replicate their function in their natural setting. The circulatory flow, interaction with other tissues, and other parameters associated with a physiological response are not found in standard tissue culture formats. The resulting assay data is not based on the pattern of drug or toxin exposure that would be found in an animal.

[003] Toxicity results from a complex interaction between a compound and the organism. During the process of biotransformation within the organism, the resulting metabolite can be more toxic than the parent compound. The single-cell assays used by many for toxicity screening miss these complex inter-cellular and inter-tissue effects.

[004] Consequently, accurate prediction of human responsiveness to potential pharmaceuticals is difficult, often unreliable, and invariably expensive. Traditional methods of predicting human response utilize surrogates - typically either static, homogeneous in vitro cell culture assays or in vivo animal studies. However, in vitro cell culture assays are of limited value because they do not accurately mimic the complex environment a drug candidate is subjected to within a human and thus cannot accurately predict human risk. Similarly, while in vivo animal testing can account for these complex inter-cellular and inter-tissue effects not observable from in vitro cell-based assays, in vivo animal studies are extremely expensive, labour-intensive, time consuming, and often the results are of doubtful relevance when correlating human risk. [005] Bioreactors are devices which support a biologically active environment, and can be used to grow cells or tissues. Organisms growing in bioreactors may be suspended or immobilised. Under optimum conditions, the microorganisms or cells are able to perform their desired function successfully.

[006] US 5,612,188 describes a multicompartmental cell culture system. This culture system uses large components, such as culture chambers, sensors, and pumps, which require the use of large quantities of culture media, cells and test compounds. This system is very expensive to operate and requires a large amount of space. Because this system is on such a large scale, the physiological parameters vary considerably from those found in an in vivo situation.

[007] The development of microscale screening assays and devices that can provide better, faster and more efficient prediction of in vivo toxicity and clinical drug performance have been developed, for the more accurate production of physiologically realistic parameters which more closely model the desired in vivo system being tested.

[008] For example, WO 03/027223 describes a microscale cell culture analog (CCA) device, which permits cells to be maintained in vitro under conditions with pharmacokinetic parameter values similar to those found in vivo.

[009] The present invention seeks to provide an improved bioreactor .

[0010] Thus, according to the present invention there is provided a bioreactor comprising:

one or more cell culture chambers each containing an interchangeable scaffold for the culture of cells;

one or more cell media chambers for containing cell culture media; the cell media chamber (s) connected to the cell culture chamber (s) via one or more media flow channels; and

a pump for pumping cell culture media through the media flow channel (s) from the cell media chamber (s) to the cell culture chamber (s) .

[0011] The bioreactor of the present invention can enable molecular cellular responses to be monitored via simultaneous real-time measurement of multiple biological indicator molecules .

[0012] The bioreactor of the present invention comprises one or more cell culture chambers each containing an interchangeable scaffold for the culture of cells, and one or more cell media chambers for containing cell culture media .

[0013] Each cell culture chamber and cell media chamber may be any suitable shape, for example circular, rectangular or oval. Each chamber may have a diameter of approximately 5 to 20 mm, more preferably 10 to 15 mm. The relative sizes/diameters of the cell culture and cell media chambers may be different. [0014] The bioreactor preferably comprises at least four pairs of cell culture and cell media chambers, more preferably at least eight pairs of chambers, still more preferably at least 40 pairs of chambers.

[0015] The cell culture and cell media chambers are preferably formed as part of an integral plate. The plate may be any suitable shape, for example rectangular, and size, and is preferably between 7 and 15 cm long and between 2 and 8 cm wide, more preferably between approximately 11 and 15 cm long and 6 and 8 cm wide. The plate may be formed from any suitable material, for example glass or plastic, and is more preferably formed from silica glass compatible with low auto fluorescence characteristics, required for fluorescence microscopy.

[0016] Each cell culture chamber is preferably connected to an exhaust conduit, for example through which effluent may pass to a detector. Each cell media chamber is preferably connected to a media inlet conduit. Cell culture media may be fed into the cell media chamber (s) via the media inlet conduit. The exhaust and media inlet conduits are each preferably controlled by a valve, such as a pneumatic micro valve comprising a compressed air chamber and a flexible membrane, for example a polydimethylsiloxane (PDMS) membrane. The valves are used to open and close the exaust and media inlet conduits.

[0017] Each cell culture chamber contains an interchangeable scaffold for the culture of cells, preferably the three- dimensional culture of cells.

[0018] Each scaffold is removable and replaceable within the cell culture chamber. The scaffold may be made from, for example, silica, glass, plastics, or polycarbonate. The scaffold may be any suitable shape and size, and is preferably circular with a diameter of at least 5mm, more preferably at least 15mm. The scaffold thickness may be at least 1mm, preferably between 1 and 4 mm, more preferably between 1 and 2 mm. The scaffold preferably comprises an interlinked pattern of holes or pores designed to maximise the surface area of the scaffold exposed to the cells that grow inside it. The scaffold may contain pores of any size or shape, and preferably contains hexagonal pores arranged in a honeycomb format. The scaffold pore size may be at least 1mm across, preferably between 1 and 5 mm across, more preferably between 1 and 2 mm across. The scaffold may contain at least 30 holes, preferably at least 50 holes, more preferably approximately at least 100 holes.

[0019] The scaffold may be coated with poly-dl-lactic acid or any other substance which promotes for the formation of heterogeneous three dimensional structures containing cells or mixtures of cells, such as mixtures of hepatocytes and hepatic stellate cells.

[0020] The scaffold is preferably compatible with the three dimensional culture of cell lines, preferably including primary cells, induced pluripotent stem (iPS) cells that may be of animal, plant or human origin, human embryonic stem (HES) cells, and/or mixtures of cells from these sources. The scaffold may also be used to support tissue explants cultures such as those derived from rodent or human fetal organs, e.g. testes etc. More preferably, the scaffold will be compatible with the 3D culture of rodent or human iPS cells or mixtures of iPS cells of different lineages.

[0021] The scaffold is preferably compatible with fluorescence microscopy and laser microdissection. [0022] Each cell culture chamber also preferably contains a porous membrane. The porous membrane may comprise any porous material, preferably a glass fibre material, more preferably a Millipore glass fibre filter with a thickness of 470 μΜ and a retention rating of 2.7 μΜ. The membrane acts as a filter for reducing turbulence.

[0023] The cell media chamber (s) are connected to the cell culture chamber (s) via one or more media flow channels, through which cell culture media can flow. The bioreactor of the present invention also comprises a pump for pumping cell culture media through the media flow channel (s) from the cell media chamber (s) to the cell culture chamber (s) . [0024] The device of the present invention preferably comprises first and second media flow channels, through which cell culture media may be circulated to and through the cell culture chamber (s), and back to the cell media chamber (s) by the pump respectively.

[0025] The pump may be a pneumatic peristalitic pump comprising compressed air chambers and a membrane, for example a polydimethylsiloxane (PDMS) membrane. The pump may comprise at least three compressed air chambers, more preferably at least five compressed air chambers. Cell culture media may be fed to the pump under gravity by the cell media chamber (s) being placed higher than the cell culture chamber (s) . Alternatively, the pump for pumping cell culture media through the media flow channels may comprise one or more positive displacement and/or continuous flow pumps, for example one or more piezoelectric pumps. The pump(s) may be contained within an integral plate which also contains the cell culture and/or cell media chambers, or may be separate from the plate. The pump may be able to provide a flow-rate of from 10 to 200μ1/ιηίη, for example from 50 to 150μ1/πιίη, e.g. approximately ΙΟΟμΙ/min.

[0026] The device of the present invention may further comprise a detection system for measuring a range of biological end-points, such as miRNAs, preferably in a multiplexed fashion. Thus, effluent from the cell culture chamber (s) may be diverted to the detection system, for example via an exhaust conduit. The exhaust conduit is preferably regulated by a valve, for example a pneumatic peristaltic micro valve. The valve may be controlled by software, to open and thus allow effluent fluid to be pumped to the detector. The effluent sample rate may be, for example, approximately Ο.ΐμΐ/min. The effluent conduit valve may operate in tandem with the cell culture media pump and/or valves, such that when the effluent conduit valve opens the cell culture media pump and/or valves close, and vice versa.

[0027] The detection system is preferably a nano wire-based detection system. The nano wires are preferably coated with detector molecules (molecules that bind to/pair with the target molecule in such a way as to cause a flux in current in the nano wire) that will enable the real-time measurement of the target molecules (e.g miRNAs, proteins, enzymes, substrates) using a measure of current/voltage changes in the nano wire. The nano wire detection system is very sensitive, potentially enabling the detection of single molecules in the effluent cell culture medium of the bioreactor, and the simultaneous measurement of several different indicator molecules. The detection system may measure the properties of samples at a suitable rate, for example from 0.1 to 10Hz, e.g. approximately lHz. The nano wire detection system is preferably flushed out after each measurement to reset the sensors, for example at a flow rate of approximately ΙΟΟμΙ/min. [0028] In operation, a suspension of cells and cell culture media are fed into the cell culture chamber (s) . Cell culture media are fed into the cell media chamber (s), preferably via a media inlet conduit (s) controlled by a pneumatic micro valve comprising a compressed air chamber and a flexible PDMS membrane .

[0029] The culture media chamber (s) feeds culture media through the cell culture chamber (s) and preferably back to the cell media chamber (s) via the first and second media flow channels respectively. This may be achieved under the force of gravity from the culture media chambers to the pump, and then by the pump. The pump causes media to flow up through the cell culture chamber (s) containing the scaffold and preferably a porous membrane, and back to the cell media chamber (s), such that media circulates through the bioreactor. Back pressure may be regulated through the use of a porous membrane, by allowing media to flow both through and past the porous membrane. Gases contained in the cell culture incubator in which the bioreactor will typically be housed, comprising a standard C0₂ tissue culture incubator, more preferably a microscope mounted temperature and C0₂ controlled incubator, will exchange with the cell culture media by virtue of the gas permeability of the bioreactor plate assembly.

[0030] The bioreactor of the present invention facilitates organisation of cells and cell mixtures in a three dimensional structure similar to an intact organ/tissue, and biological responses of the cells/tissue to a given stimulus more like that of a tissue/organ in vivo. For example, liver cells exposed to a non-genotoxic carcinogen (NGC) compound grown in the bioreactor may proliferate in such a way as to mirror the response to the NGC compound seen in the liver in vivo. This is valuable because it can be used in a high throughput manner to detect compounds with toxic liability and/or efficacy deficiencies at an early stage in the development process of a drug, food additive or industrial chemical. The detection of an NGC-like response caused by compounds/treatment ( s ) is facilitated by measuring changes to molecules such as miRNAs either in the cells, or in the media used to culture the cells, to which the compound is exposed . [0031] As the biological response measured in the bioreactor may be predictive of a similar response in vivo, the bioreactor can be used to develop a short-term test for toxicity which could have considerable economic benefits. The bioreactor is preferably used in conjunction with a detection system, preferably a detection system incorporating nano wires coated with detector molecules that will enable the real-time measurement of the target molecules (e.g miRNAs, proteins, enzymes, substrates) using a measure of current/voltage changes in the nano wire. The nano wire detection system is very sensitive, potentially enabling the detection of single molecules in the effluent cell culture medium of the bioreactor and the simultaneous measurement of several different indicator molecules. [0032] Simultaneous measurement of several biological indicator molecules (molecules that change in response to the stimulus in such a way as to drive the response to the stimulus) will increase the ability of the bioreactor to molecularly characterise the response in a multi-dimensional way. Hence, the measurement of combinations of indicators will facilitate the measurement of multi-dimensional biomarkers that will be informative with respect to the 'nature' (phenotype) of the biological response, and also predictive in terms of whether that response is likely to occur in vivo, either in rodents or humans. [0033] The bioreactor preferably has low light scatter/autofluorescence characteristics so as to be compatible with the measurement of cell responses using fluorescent probes or dyes in a fluorescence microscope. The dimensions of the reactor are preferably such that it will be compatible with being mounted on a movable microscope stage, such as a PALM microbeam microscope (Zeiss) and contained within a PM PALM SI incubator, to facilitate time- lapse measurement of cellular responses using live cell imaging techniques. The bioreactor scaffold containing cells may be removed and placed in a petri dish in the PM PALM SI incubator assembly to facilitate laser microdissection mediated capture on cells for the purpose of making molecular measurements and/or subculture of the cells. The device of the invention should be moveable in X-Y translations in order to change the viewing position of the microscope.

[0034] The integration of this multi-dimensional data in a software capable of visualising fluxes in the biological indicators in a single view and in parallel to perform integrated statistical processing of the different end-points (biological indicators) will facilitate the design of biomarkers, coupled with in vivo-relevant testing systems, with greater predictive potential for the complex biological responses that they are designed to predict. Hence, the use of the bioreactor in a high throughput mode in conjunction with a nano wire-based detection system and a data visualisation and statistical processing software will enable drug/chemical/food companies to select molecules with lower toxic liability and greater efficacy that will have a better chance of success in the market place.

[0035] According to the present invention there is also provided a method of monitoring molecular cellular responses via measurement of multiple biological indicator molecules using the bioreactor of the present invention. The biological indicator molecules preferably include miRNAs, proteins, enzymes, and substrates. The cells being monitored are preferably selected from primary cells, induced pluripotent stem (iPS) cells that may be of animal, plant or human origin, human embryonic stem (HES) cells, and/or mixtures of cells from these sources. [0036] The present invention will now be described in detail with reference to the accompanying drawings, in which:

Figures la and lb show schematic cross-sectional and plan views respectively of a bioreactor according to one embodiment of the present invention;

Figures 2a and 2b show cross-sectional views of the top plate and bottom plate respectively of the embodiment of the present invention shown in Figure 1;

Figure 3a shows a cross-sectional view of the middle plate of the embodiment of the present invention shown in Figure 1; Figure 3b shows a top view of a preferred scaffold for use in the present invention;

Figure 4 shows a flow diagram of the fluid scheme of an embodiment of a device of the present invention;

Figure 5 shows a flow diagram of a preferred operation of a device of the present invention; and

Figure 6 shows a device of an embodiment of the present invention in position on a microscope. [0037] Thus, Figures la and lb show schematic cross- sectional and plan views respectively of a bioreactor 1 according to one embodiment of the present invention comprising top 20, middle 12 and bottom 22 plates, cell media chamber (s) 2 connected via a bottom media flow channel 4, containing a pneumatic peristaltic pump 6, to cell culture chamber (s) 8 linked back to the culture media chamber (s) 2 via a top media flow channel 47. The direction of flow of cell culture media is indicated by arrows in Figure la.

[0038] The bioreactor 1 may be located on a microscope moveable platform inside a temperature and gas controlled chamber also mounted on a microscope platform (see Figures 5 and 6) .

[0039] The bioreactor 1 contains a removable scaffold filter insert 10 that could be made of silca or polycarbonate comprising a hexagonal interlinked pattern of holes designed to maximise the surface area of the scaffold exposed to the cells that grow inside it (see Figure 3b) . The scaffold may be used to support the three dimensional (3D) culture of primary cells, cell lines, induced pluripotent stem (iPS) cells or human embryonic stem (HES) cells and/or mixtures of cells from these sources. The scaffold 10 may also be used to support tissue explants cultures such as those derived from rodent or human fetal organs e.g. testes etc. Tissue culture medium may be circulated through the cell culture chamber (s) 8 and back to the cell media chamber (s) 2 via bottom and top media flow channels 4, 47 driven by the pneumatic peristalitic pump 6 comprising compressed air chambers 7 (see Figure 2b) and a polydimethylsiloxane (PDMS) membrane 42. Alternatively, the pump for pumping cell culture media through the media flow channels may comprise one or more positive displacement and/or continuous flow pumps, for example one or more piezoelectric pumps. The pump(s) may be contained within an integral plate which also contains the cell culture and/or cell media chambers, or may be separate from the plate. Culture media may be fed to the pump 6 under gravity facilitated by the cell media chambers 2 being placed higher than the cell culture chambers 8 in the middle bioreactor plate 12.

[0040] The scaffold (s) 10 may be designed to be compatible with fluorescence microscopy and also laser microdissection. Effluent from the cell culture chambers 8 may be diverted, via an exhaust conduit 14, regulated by a pneumatic micro valve 16 to a nano wire-based detection system (not shown in Figure la) designed to measure a range of biological end- points such as miRNAs etc in a multiplexed fashion.

[0041] As shown in Figure la, the bioreactor of this embodiment comprises top, middle and bottom plates. The top plate 20 (Figure 2a), middle plate 12 (Figure 3a) and bottom plate 22 (Figure 2b) may comprise any hard material such as glass or polycarbonate, more preferably gas porous silica glass, and may comprise a plurality of cell culture 8 and cell media chambers 2.

[0042] The middle plate 12 of an embodiment of the present invention is shown in cross-sectional view in more detail in Figure 3a. The middle plate 12 may be rectangular, and could be any size, preferably between 7 and 15 cm long and between 2 and 8 cm wide, more preferably between approximately 11 and 15 cm long and 6 and 8 cm wide. The middle plate 12 may comprise any glass or plastic and is more preferably silica glass compatible with low auto fluorescence characteristics, required for fluorescence microscopy, and may be any colour. The middle plate 12 comprises at least four pairs of cell culture 8 and cell media chambers 2, but preferably comprises at least eight pairs of chambers, more preferably approximately at least 40 pairs of chambers.

[0043] Each cell culture 8 and cell media chamber 2 may be circular, rectangular or oval in shape, may have a diameter of approximately 5-20 mm, more preferably 10 to 15 mm. The relative sizes/diameters of the cell culture 8 and cell media chambers 2 may be different.

[0044] The cell culture chamber 8 contains a porous membrane 28 and a scaffold insert 10. The porous membrane 28 may comprise any porous material, preferably a glass fibre material, more preferably a Millipore glass fibre filter with a thickness of 470 μΜ and a retention rating of 2.7 μΜ. [0045] The scaffold insert 10 may comprise a silica glass, plastic or polycarbonate material, may be any shape and size, and is preferably circular with a diameter of approximately at least 5mm, more preferably approximately at least 15mm. The scaffold 10 thickness could be between 1 and 4 mm, preferably between 1 and 2 mm. The scaffold 10 is compatible with 3D culture of cell lines, primary cells, iPS cells that may be of animal, plant or human origin. More preferably the scaffold 10 will be compatible with the 3D culture of rodent or human iPS cells or mixtures of iPS cells of different lineages. The scaffold 10 may contain pores of any size or shape, more preferably pores of hexagonal shape in a honeycomb format 32, as shown in Figure 3b. The scaffold 10 pore size could be between 1 and 5 mm across, preferably between 1 and 2 mm across. The scaffold 10 filter may contain at least 30 holes, preferably at least 50 holes, more preferably approximately at least 100 holes.

[0046] In operation, a suspension of cells and media are fed into the cell culture chambers 8 via the exhaust conduit 14, controlled by the pneumatic micro valve 16 comprising a compressed air chamber 17 (see Figure 2a) and a flexible PDMS top membrane 18. Cell culture media are fed into the cell media chambers 2 via the media inlet conduit 36, controlled by a pneumatic micro valve 40 comprising a compressed air chamber 41 (see Figure 2a) and the flexible PDMS top membrane 18. The pneumatic valves 16 and 40 are used to open and close the exhaust 14 and media inlet 36 conduits, respectively . [0047] The cell media chambers 2 feed culture media under the force of gravity, and from the cell media chambers 2 up through the cell culture chambers 8 and back to the cell media chambers 2, by the mini pneumatic peristaltic pump 6 comprising the PDMS bottom membrane 42 and at least 3 compressed air chambers, but preferably approximately at least 5 compressed air chambers. The mini peristaltic pump 6 causes media to flow up through the cell culture chambers 8 containing the cell/tissue scaffold insert 10 and the porous membrane 28, and back to the cell media chambers 2 such that media circulates through the bioreactor system (as indicated by the arrows in Figure la) . During this process gases contained in the cell culture incubator in which the bioreactor is typically housed, comprising a standard C0₂ tissue culture incubator, more preferably a microscope mounted temperature and C0₂ controlled incubator, will exchange with the cell culture media by virtue of the gas permeability of the bioreactor plate assembly.

[0048] Figure 4 shows a flow diagram to represent the fluid scheme of an embodiment of a device of the present invention. Thus, cell culture media is added to cell media chamber 2, which may be open to the atmosphere. The cell media chamber 2 feeds the cell culture chambers 8. A drug to be tested can be injected into the cell media chamber 2 via drug injector 50. Cells can be injected into the cell culture chambers 8 via cell injector 52. Cell culture media is pumped through the system using pump 6; cell culture media is drawn through the cell culture chambers 8 by the action of the pump 6, and recycled back to the cell media chamber 2 via the valve 54.

[0049] Samples of cell culture media from the cell culture chambers 8 may be drawn through the exhaust conduit 14 via the exhaust micro-valve 16. The exhaust micro-valve 16 and cell culture media recycling valve 54 are preferably operated in tandem, such that when one valve is open the other is closed, as may be controlled by suitable software. The cell culture media samples are pumped to the detection system 56 which preferably comprises a nano-wire system as described herein. After testing, cell culture media samples are disposed of via suitable waste disposal means 58. The detection system 56 may measure the properties of samples at a suitable rate, for example approximately lHz. The nano wire detection system 56 is preferably flushed out after each measurement to reset the sensors, for example by a buffer contained within a buffer reservoir 60 by a buffer pump 64, for example at a flow rate of approximately ΙΟΟμΙ/min, via a buffer valve 62.

[0050] Figure 5 shows a flow diagram of a preferred operation of a device of the present invention within an incubator chamber 80 when connected to a microscope 72 and suitable computer hardware 78 operating suitable software. Thus the bioreactor 1 of an embodiment of the present invention may be placed on a support base 70 of a microscope 72, movable in X-Y translation 74 to allow different areas of the bioreactor 1 to be observed, within an incubator chamber 80. Heat and gas control within the incubator chamber 80 may be controlled as desired, as indicated 82. The microscope 72 may comprise a camera 76 for recording images of the cell culture chambers (not shown in Figure 5) . The output of the detection system 56 may be recorded by a reader 57. The bioreactor 1, microscope 72, detection system 56, 57, and all other aspects of the operation of the device of the invention may be controlled by suitable hardware 78, operating suitable software.

[0051] Figure 6 shows a device of an embodiment of the present invention in position on a microscope 72. Thus, the bioreactor 1 is shown positioned on a support base 70 of a microscope 72. Representative bioreactor cell culture chambers 8 are shown. The bioreactor is positioned within an incubator chamber 80, the front wall of which is shown in Figure 6.

[0052] Features of the present invention include:

(1) A bioreactor comprising a cell culture chamber (s) containing a removable/interchangeable scaffold (s) permitting the three dimensional culture of cells under flow driven by a pump connected via media flow channels to a cell media containing reservoir chamber (s) where molecular cellular responses are monitored via simultaneous real-time measurement of multiple biological indicator molecules' (biomarkers) .

(2) A bioreactor according to feature (1) in which the biological indicator molecules are measured using silicon nanowire-based field effect transistors functionalised with detector molecules specific for the biological indicator molecule ( s ) .

(3) A bioreactor according to feature (1), in which cellular responses are monitored by microscope based imaging of cells using fluorescent dyes. (4) A bioreactor according to feature (1), in which the cell culture scaffolds are interchangeable such that different types of cells or tissues can be cultured. (5) A bioreactor according to feature (1), in which the media flow rate, back pressure and media components can be automatically controlled via a digital electronic control unit . (6) A bioreactor according to feature (1), in which the timing and nature of data collection via nanowire and microscope-based detection is automated via a digital electronic control unit. (7) A bioreactor according to feature (1), in which the output of data from the nanowire- and microscope-based detection systems can be visualised on a computer screen with the aid of appropriate software.

Claims

1. A bioreactor comprising:

2. A bioreactor according to claim 1 wherein each cell culture chamber and cell media chamber is circular, rectangular or oval.

3. A bioreactor according to claim 1 or 2 wherein each cell culture chamber and cell media chamber has a diameter of 10 to 15 mm.

4. A bioreactor according to any one of claims 1 to 3 comprising at least four pairs of cell culture and cell media chambers .

5. A bioreactor according to claim 4 comprising at least forty pairs of cell culture and cell media chambers.

6. A bioreactor according to any preceding claim wherein the cell culture and cell media chambers are formed as part of an integral plate.

7. A bioreactor according to claim 6 wherein the plate is substantially rectangular, and/or between 11 and 15 cm long and 6 and 8 cm wide.

8. A bioreactor according to claim 6 or 7 wherein the plate is formed from silica glass compatible with use in fluorescence microscopy.

5 9. A bioreactor according to any preceding claim wherein each cell culture chamber is connected to an exhaust conduit through which effluent may pass to a detector.

10. A bioreactor according to any preceding claim wherein 10 each cell media chamber is connected to a media inlet conduit via which cell culture media may be fed into the cell media chamber ( s ) .

11. A bioreactor according to claim 9 or 10 wherein the 15 exhaust and/or media inlet conduits are controlled by a pneumatic micro valve comprising a compressed air chamber and a flexible membrane.

12. A bioreactor according to any preceding claim wherein 20 each scaffold is made from silica glass, plastics, or polycarbonate .

13. A bioreactor according to any preceding claim wherein each scaffold is substantially circular with a diameter of

25 at least 15mm.

14. A bioreactor according to any preceding claim wherein each scaffold has a thickness between 1 and 2 mm.

30 15. A bioreactor according to any preceding claim wherein each scaffold comprises an interlinked pattern of holes or pores .

16. A bioreactor according to claim 15 wherein each

35 scaffold contains hexagonal pores arranged in a honeycomb format .

17. A bioreactor according to claim 15 or 16 wherein the scaffold pore size is between 1 and 2 mm across.

5

18. A bioreactor according to any one of claims 15 to 17 wherein each scaffold contains at least 100 holes.

19. A bioreactor according to any preceding claim wherein 10 the scaffold is coated with a substance which promotes the formation of heterogeneous three dimensional structures containing cells or mixtures of cells.

20. A bioreactor according to claim 19 wherein the 15 substance is poly-dl-lactic acid.

21. A bioreactor according to any preceding claim wherein the scaffold is compatible with the three dimensional culture of primary cells, induced pluripotent stem (iPS) cells that

20 may be of animal, plant or human origin, human embryonic stem (HES) cells, and/or mixtures of cells from these sources, and for supporting tissue explants cultures derived from rodent or human fetal organs.

25 22. A bioreactor according to any preceding claim wherein the scaffold is compatible with fluorescence microscopy and laser microdissection.

23. A bioreactor according to any preceding claim wherein 30 each cell culture chamber also contains a porous membrane.

24. A bioreactor according to claim 23 wherein the porous membrane comprises a glass fibre material.

35 25. A bioreactor according to claim 24 wherein the porous membrane comprises a Millipore glass fibre filter with a thickness of 470 μΜ and a retention rating of 2.7 μΜ.

26. A bioreactor according to any preceding claim which

5 comprises first and second media flow channels, through which cell culture media may be circulated to and through the cell culture chamber (s), and back to the cell media chamber (s) by the pump respectively.

10 27. A bioreactor according to any preceding claim wherein the pump is a pneumatic peristalitic pump comprising compressed air chambers and a membrane.

28. A bioreactor according to any preceding claim wherein 15 the pump comprises at least five compressed air chambers.

29. A bioreactor according to any preceding claim wherein cell culture media may be fed to the pump under gravity by the cell media chamber (s) being placed higher than the cell

20 culture chamber (s) when the bioreactor is in use.

30. A bioreactor according to any preceding claim which further comprises a detection system.

25 31. A bioreactor according to claim 30 wherein effluent from the cell culture chamber (s) is diverted to the detection system via an exhaust conduit.

32. A bioreactor according to claim 30 or 31 wherein the 30 detection system is a nano wire-based detection system.

33. A bioreactor according to claim 32 wherein the nano wires are coated with detector molecules for the detection of target molecules using a measure of current /voltage

35 changes in the nano wire.

34. A bioreactor according to any one of claims 30 to 33 which can simultaneously measure several different biological indicator molecules.

5 35. A bioreactor according to any preceding claim for use in the measurement of cell responses using fluorescent probes or dyes in a fluorescence microscope.

36. A bioreactor according to any preceding claim for 10 mounting on a movable microscope stage.

37. A method of monitoring molecular cellular responses via measurement of multiple biological indicator molecules using the bioreactor of any preceding claim.

15

38. A method according to claim 37 where the biological indicator molecules include miRNAs, proteins, enzymes, and substrates .

20 39. A method according to claim 37 or 38 wherein the cells being monitored are selected from primary cells, induced pluripotent stem (iPS) cells that may be of animal, plant or human origin, human embryonic stem (HES) cells, and/or mixtures of cells from these sources.