WO2021018365A1

WO2021018365A1 - Micro-bioreactor comprising an organ layer

Info

Publication number: WO2021018365A1
Application number: PCT/EP2019/070184
Authority: WO
Inventors: Danill GOLUBEV
Original assignee: Swiss Medical Union Sa
Priority date: 2019-07-26
Filing date: 2019-07-26
Publication date: 2021-02-04

Abstract

The present invention is directed to bioreactors and methods for testing the effect of medical products on organs. The bioreactor (1) comprises a central control system (1b) and a biochip (1a) that comprises an organ layer (20) comprising a plurality of cavities (21, 23) for housing organoids. The biochip (1a) comprises also an adaptation plate (10) comprising inlets for medical products. The bioreactor comprises a self-contained circulation system (200) for circulating a fluid through different fluidic connected cavities. The biochip (1a) comprises as least one spectrometer (220) configured to perform spectroscopic measurements during or after operation of the bioreactor (1). The invention relates also to a method to test the effect of medical product on tissues that are arranged in said biochip (1a).

Description

MICRO-BIOREACTOR COMPRISING AN ORGAN LAYER

Technical Field

The invention relates to the field of testing the effect of chemical and medical compounds or compositions on the behavior of organs or groups thereof and also the testing of the pharmacokinetic behavior of such compounds. More precisely, the invention is related to the field of alternative methods to avoid the need of testing on animals.

More precisely the invention relates to the miniaturization of a bioreactor that is configured to test the effect of chemical and medical compounds or compositions on the behavior of organoids that are arranged in such a bioreactor.

The invention also relates to micro-bioreactor that is based on a stack of layers comprising a micro structured layer and an integrated fluidic circulation system.

Background of the art

There is an increasing trend to seek for solutions to avoir animal experimentation in the search and testing of medical substances on human organs and therefor a lot of development efforts has been done to replace animal drug testing with in vitro methods based on human cells ans organs. New devices are being investigated so as to change the medical industry in the long term. For example, in the European Union (EU), animal testing for cosmetic development has been prohibited since 2013. The movement toward reduction and prohibitiion of animal testing will continue and should extend to new drug discveries in the future.

There is a trend to seek solutions to develop and test new medicines and more accurate treatments by using miniaturised reactors in which parts of organs, defined as organoids, can be introduced and tested. This not only to avoid the use of tests on animals but also to be able to test the effect on a plurality of organs at the same time. Systems have been proposed in the past to perform such tests in a simpler way and also at a higher speed, which increases the efficiency , the reliability of the tests and their cost. So there is also a need to improve considerably the automatisation of such testing. In the past, cell-based assays have been effective means for preliminary testing of the effect of medical products, for exemple by using celles cultured on petri dishes and multi-well plates but therie reponsivity and functionaliyt are not very realiable nor stable and there may be important differences between the predictions made by tests performed on animals or performed by in vitro-tests.

For this reason, techniques such as organ-on-a-chip that are based on microtechnology have been proposed. But such systems are still limtited by their low level of integration or the possibilitiy to test in a reliable way the effect of medical products on a plurality of organs.

The following paper decribes an organ-on-a chipo system configured for the testing of drugs: Hiroshoi Kimura et al. “Organ/boy-on-a-chip based on microfluidic technology for drug discovery”. The system(s) described in Kimura et al relates to micro-chips that are configured to test the effect of a medical product on a limited number of human organ cells and limited to the use of a single medical product. The system described in Kimura et al is a step towards a more sophisticated miniaturisation of a multi-organ bio-chip, but would not be able to provide a solution that would provide real-time interactions between organs. A practical in vitro model should be a system that can observe and record a variety of physiological responses to a variety of specific biological stimuli. To satisfy these requirements there is a need for improved systems that not only can evaluate biological responses but can also perform biochemical analysis in the space of a micro-bioreactor. The complicated handling requierements to use organ-on-a chip-device affects greatly throughput and usability of existing devices.

Summary

The device of the invention provides an improved micro bio-reactor that addresses the problem of improved testing of effects of drugs on a plurality of human organs. The micro bio-reactor provides a system that allows to make biochemical analysis in situ in the micro-space of the bio-reactor and provides also solutions to the handling issues of such devices. The micro bio-reactor provides also an automated and programmable sub-system that comprises cell manipulation and assays in a single system. The subsystem provides also means to control local fluidic pressures and means to control and gather spectroscopic date in situ from a plurality of organoids that are introduced in the bio-reactor. The micro-bioreactor of the invention allows also, by its single central subsystem, to control a plurality of organ-on-a chip devices.

More precisely, a micro-bioreactor is provided for testing the effects of at least one medical product on a plurality of human organ samples, and comprises:

a baseplate to provide a support for a stack of plates;

- an adaptation plate comprising at least one inlet adapted to introduce said at least one substance,

- an organ plate comprising a plurality of cavities for housing an organoid of a human organ, at least one of said cavities being connected with at least one inlet.

The central control system and the biochip are in fluidic and electric connection The micro-bioreactor comprises also a closed fluidic circulation system comprising at least one circulation pump and a fluidic conduct configured to circulate, in operation of the bioreactor, a liquid between at least two of said plurality of cavities, said fluidic circulation system comprising at least one addressable valve.

Said organ plate comprises at least a portion of said closed fluidic circulation system so that at least two of said plurality of cavities are in fluidic communication and so that, in operation of the bioreactor, a fluid is circulated from at least one of said plurality of cavities to at least another of said plurality of cavities.

The micro-bioreactor comprises at least one spectrometer being in fluidic connection with at least one of said cavities and so that, in operation of the bioreactor, a spectral analysis may be performed of the portion of said fluid and/or organoid that is facing or is in contact with at least a portion of said spectrometer.

The bio-reactor comprises an electronic system comprising electrical commands to open or close said at least one addressable valve and configured to control the circulation of said liquid in said fluidic circulation system , the bio-reactor comprising also electronic means to operate said at least one spectrometer and comprising a data processing system configured to process data provided by said spectrometer.

In an embodiment the bio-reactor comprises at least one micro spectrometer that is arranged at least partially into said organ plate. In an advantageous embodiment any of said plates comprises electrical contacts configured to receive and send electrical signals to a control and/or data processing system situated outside said stack of further plates.

In an embodiment said control and/or data processing system is integrated at least partially into said stack of plates.

In an embodiment at least two cavities are each connected each to at least one spectrometer. In an embodiment said at least one spectrometer is integrated onto or in said organ plate. In an advantageous embodiment at least one of said spectrometers is an impedance spectrometer. In a variant of execution said impedance spectrometer comprises at least two gold electrodes.

In an embodiment at least one of said spectrometers is an optical spectrometer. In a variant said optical spectrometer comprises an optical waveguide configured to guide an optical light beam. In a variant of execution at least a portion of said optical waveguide is arranged onto or into said organ plate. The optical waveguide may be arranged so that , in operation of said bio-reactor, at least a portion of it provides to at least one of said cavities an evanescent wave so that the properties of said optical light beam are altered by the presence and/or reaction of an organoids present in said cavity. In an embodiment at least one of said optical spectrometer comprises an optical emitter and an optical detector.ln variants at least one of said optical spectrometers comprises at least one Mach-Zehnder interferometer waveguide structure.

In an embodiment at least one of said cavity is connected to at least one impedance spectrometer and at least one optical spectrometer.

In an embodiment said further layers comprise a nutrition layer arranged between said adaptation layer and said organ layer and so that at least one of said inlets is connected to at least two cavities. In an embodiment the micro-bio-reactor comprises mechanical means adapted to remove said adaptation plate away from said organ plate so as to allow to provide a direct access to said cavities to place or remove organoids.

In an embodiment the micro-bio-reactor comprising an actuator layer configured to comprises a multiplicity of actuators arranged and configured to regulate pressure forces to at least one of said cavities and/or a portion of said circulation system. In an embodiment said circulation system comprises micro channels realized inside or in at least one of the surfaces of said organ plate. In an embodiment the micro-bio-reactor comprises a micropump integrated onto or into one of said stack of layers.

In an embodiment bio chip comprises an optical inspection system configured to detect, in operation, the color of at least a portion of the surface of one of said organoids. In an embodiment said cavities comprise a mechanical structure for holding and keeing in place organoids. In an embodiment at least one of said impedance spectrometers comprises a hollow electrode. In variants said hollow electrode comprises a portion that is postionned inside a cavity when the bioreactor is in operation.

The invention is also acheived by a method to test the effect of medical product on tissues comprising the steps of:

- providing a bioreactor as described ;

- introducing in at least two of said cavities an organoid ;

- starting a pump of the bioreactor so as tro circulate a circulation fluid into said fluidic circulation system;

- introducing a medical product into at least one of said inlets;

- performing a test on at least one of said organoids to identifiy the effect of said medical product.

In an embodiment the method comprising the further steps of:

- providing by the circulation fluid a nutrition layer for cells of the organoids;

- Leaving said at least one organoid into a cavity until at least 10 million cells are formed.

In an embodiment the method comprises the further steps of:

- starting a measurement by said at least one of said spectrometers, controlled by a control signal of said central subsystem 1 b;

- providing spectral data;

- gathering said spectral data by a software.

In an embodiment the method comprises the steps of: - circulating the circulation fluid for at least 7 days to allow the medical product to reach the tumor cells of the targeted organoid;

- opening the biochip after a predetermined period and verifying the color of the organoid;

- testing if a contrast liquid produces a different color to the organoids or not at all;

- deciding that, if the contrast liquid has provoqued a different color to at least a portion of the organoid, the therapeutic agent has had a therapeutical effect worked on the organoids, within the size measured by one of said spectrometers.

In an embodiment identical but different shaped organoids and spheroids are used in said biochip 1a. In an embodiment an optical inspection is performed by using an optical inspection system configured to observe an organoid through a window in said biochip 1a. In an embodiment , during a test, a measurement is made of the composition of the circulation fluid. In an embodiment the circulation fluid comprises at least a fraction that is blood from a patient.

Brief description of the drawings

Further details of the invention will appear more clearly upon reading the following description in reference to the appended figures:

- Figure 1 illustrates a exploded view of a microchip, an integrated spectrometer and a fluidic circulation system of a bioreactor of the invention ;

- Figure 2 illustrates a vertical cross section of an adaptor plate and an organ plate;

- Figure 3 illustrates a top view of an organ plate comprising at least two cavities connected to an impedance spectrometer;

- Figure 4 illustrates a vertical cross section of a microchip comprising an optical waveguide and illustrates a leaky wave defined partially in the cavities; Figure 5 illustrates a top view of an micro-chip comprising a light emitter and a light detector;

Figure 6 illustrates a top view of an organ plate comprising a Mach Zehnder interferometer;

Figure 7 illustrates a vertical cross section of a microchip comprising an adapter plate, a nutrition layer and an organ layer;

Figure 8 illustrates a top view of an organ plate comprising a plurality of cavities linked by microfluidic channels;

Figure 9 illustrates a top view of an adapter plate;

Figure 10 illustrates a 3D view of a cavity comprising a plurality of electrodes of impedance spectrometers;

Figure 11 illustrates a bioreactor of the invention comprising a central control system and comprising a plurality of micro bioreactors;

Figure 12 illustrates a fluidic pressure control and regulation system comprising a plurality of pressure regulators and a plurality of valves;

Figure 13 illustrates a 3D view on a micro bioreactor chip comprising means to slide an adaptor plate laterally relative to an organ plate;

Figure 14 illustrates a 3D view on a micro bioreactor chip having an organ plate comprising cavities that can be accessed by the bottom surface by using pivotable closures.

Figure 15 illustrated an embodiment comprising a hollow electrode.

Detailed description and embodiments of the invention

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to the practice of the invention. It is to be noticed that the term“comprising” in the description and the claims should not be interpreted as being restricted to the means listed thereafter, i.e. it does not exclude other elements.

Reference throughout the specification to “an embodiment” means that a particular feature, structure or characteristic described in relation with the embodiment is included in at least one embodiment of the invention. Thus appearances of the wording “in an embodiment” or, “in a variant”, in various places throughout the description, are not necessarily all referring to the same embodiment, but several. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a skilled person from this disclosure, in one or more embodiments. Similarly, various features of the invention are sometimes grouped together in a single embodiment, figure or description, for the purpose of making the disclosure easier to read and improving the understanding of one or more of the various inventive aspects. Furthermore, while some embodiments described hereafter include some but not other features included in other embodiments, combinations of features if different embodiments are meant to be within the scope of the invention, and from different embodiments. For example, any of the claimed embodiments can be used in any combination. It is also understood that the invention may be practiced without some of the numerous specific details set forth. In other instances, not all structures are shown in detail in order not to obscure an understanding of the description and/or the figures.

The term“fluid” includes here all types of fluids that are usually used in medical or biological applications, such as water or a fluidic plasma .

The term“microfluidic” is defined very large and means here that the fluid is transported in or through a structure that has a lateral dimension smaller than typically 2mm, defined transversely to the flow of the liquid.

The wording “chip” relates to planar elements that may comprise microstructures, but also may be a plate that may comprise a thin layer or coating.

The layers in the device of the invention define a lower surface and a top surface that have no relation with gravity but are arbitrary and only relative to the representation of the annexed figures. The wording“inlet” and“outlet” are defined broadly and are structures that are configured to introduce or extract substances and/or liquids or both and are typically small and thin tubes or apertures realized in a plate or layer.

The wording“plate” is defined broadly and may define a layer and vice-versa. For example an organ plate may be in reality a thick layer configured on another plate or layer.

The wording“micro bio-reactor” refers to the fact that the bio reactor comprises a small biochip reactor and comprises micro structured parts. The micro bio-reactor of the inventions comprises as well a central control system 1 b as well as at least one biochip 1 a, as illustrated in Fig.11.

It is also understood that the bio-reactor of the invention may be used to study the effects of medical products on animal organs. The wording“organoid(s)” is defined broadly here and encompasses organoids but also parts of organs and /or tissues and/or other parts of the body such as bone samples.

The micro bio-reactor is also defined as a multi-organ system that is preferably, but not exclusively, intended to mimic the basic functions of the blood system of a higher organism such as the one of animals and preferably a human organism. The bioreactor 1 of the invention mimics the blood system of a higher organism and comprises the interconnection between different organoids by a circulation system. This allows to test not only the effect of a medical product on a single organ but also to deduce side effects to organs that are situated further away in the circulation system. The system of the invention 1a is not limited to the testing of different organs but might also be configured to test the effect on several samples of the skin. It is also understood that the bioreactor may be configured to test the effect of at least one medical product on a plurality of similar organoids or other portions of the body that may be bone samples for example.

Prior art bioreactors do not allow to interact and cross-talk between different organs on a chip and at the same time provide detailed in-situ data on these organs. Also, prior art bioreactors are to complicated to be used and rely mainly on tedious manual introduction of organ samples. The same can be said for the extraction or the exchange of organ samples. The bioreactor of the invention provides a considerable improvement of these issues compared to devices of prior art. Compared to devices of prior art the bioreactor of the invention comprises in situ testing of the organoids by integrated test subsystems such as micro spectrometers. As further described each cavity arranged to contain an organoid may comprise several microspectrometers or other test subsystems to make reliable tests of the effect of a medial product on an organoid.

The invention includes the following embodiments.

Fig. 1 illustrates a view on the general concept of the invention. The micro bioreactor 1 of the invention is configured preferably for testing the effects of at least one medical product S1 -Sn on a plurality of human organ samples and comprises at least:

a baseplate 40 to provide a support for a stack of plates 10, 20, 30, said plates defining a plane x-y and a vertical direction z as illustrated in Fig.6 ;plates 10, 20, 30 are piled into the z-direction and may have each a typical thickness of 50m to 1 mm and may be made of any material, typically a polymer..

- an adaptation plate 10 comprising at least one inlet 12 adapted to introduce said at least one substance S1 -Sn, n may be any number, and is typically between 1 and 10

- an organ plate 20 comprising a plurality of cavities 21 , 23 for housing an organoid 01 , 02 of a human organ, at least one of said cavities 21 , 23 being connected with at least one inlet 12, 12’, 12”. Inlets may have a conical form 12’b inside said adapter plate 10 as illustrated in Fig.2

The micro-bioreactor 1 of the invention comprises a closed fluidic circulation system comprising at least one circulation pump 210 and a fluidic conduct 200 configured to circulate, in operation of the bioreactor 1 , a fluid between at least two of said plurality of cavities (21 , 23) , said fluidic circulation system comprising at least one addressable valve. Said fluid is preferably a biocompatible liquid such as water or a liquid that has similar properties as blood. The fluidic conduct 200 comprises and inlet duct portion 201 and an outlet duct portion 203 connected to said biochip 1a. The flow of the fluid is indicated by the symbol F in the figures.

In a preferred embodiment said organ plate 20 comprises at least a portion of said closed fluidic circulation system. The micro-bioreactor 1 comprises at least one test subsystem being in fluidic connection with at least one of said cavities 21 , 23 and so that, in operation of the bioreactor 1 , an analysis may be performed of the portion of said fluid and/or organoid that is facing or is in contact with at least a portion of said test subsystem.

In a preferred embodiment said test subsystem comprises at least one spectrometer 220, 222, 224 that may be any type of spectrometer of which at least a portion may be integrated in or on any of the layers 10, 20, 30, 40 of the biochip 1a of the bioreactor 1. Said at least one spectrometer 220, 222, 224 is directly connected to said cavities 21 , 23 by a spectrometer connection 240 that may be any connection such as an optical, electric, fluidic, mechanical connection. Typically said connection 240 is a duct such as an aperture that comprises at least partially an electrode connected into said cavities 21 , 23. A spectrometer connection 240 may comprise for example also more than one electrical wire or may comprise en electronic circuit. Said spectrometer connection 240 may also be a structured area 22a, 22b, 22c, 22d arranged inside or on one of the surfaces of said organ layer 20.

Fig.3 illustrates an biochip 1a arrangement comprising 2 cavities 21 , 23, each cavity comprising a pair of electrodes 223a, 223b, 222c, 222d that are part of an impedance spectrometer that comprise electrical connections 223a, 223b, 223c, 223d arranged at a surface of the organ plate. Fig. 3 may be an example of a cross section according to the vitual plane A-A of Fig.2

The bio-reactor 1 comprises an electronic system comprising electrical commands to open or close said at least one addressable valve and configured to control the circulation of said liquid in said fluidic circulation system. The bio-reactor 1 comprises also electronic means to operate said test subsystem such as at least one spectrometer 220, 222, 224 and comprises a data processing system configured to process data provided by said spectrometer 220, 222, 224.

In an embodiment the bio-reactor 1 comprises at least one micro spectrometer 220, 222, 224 that is arranged entirely into said organ plate 20. In an advantageous embodiment any of said stack of plates plates 10, 20, 30 comprises electrical contacts configured to receive and send electrical signals to a control and/or data processing system situated outside said stack of plates 10,20,30. An exemple has been discussed above under the embodiment of Fig.3 In an embodiment said control and/or data processing system is integrated at least partially into said stack of plates 10,20,30.

In an embodiment at least two cavities 21 , 23 are each connected each to at least one spectrometer 220, 222, 224. So, a single spectrometer 220, 222, 224 may be connected to more than one cavity 21 , 23.

In an embodiment said at least one spectrometer 220, 222, 224 is at least partially integrated onto or into said organ plate 20 .

In an embodiment at least one of said spectrometers 220, 222, 224 is an impedance spectrometer.

In an embodiment said impedance spectrometer comprises at least two gold electrodes 22a, 22b .

In an advantageous embodiment, at least one cavity may be connected to a 3D arrangement of electrodes in order to obtain 3D information on the organoid. IN variants at least two pairs of electrodes may be addressed with different frequencies.

In an embodiment at least one of said spectrometers 220, 222, 224 is an optical spectrometer.

In an embodiment said optical spectrometer comprises an optical waveguide 300 configured to guide an optical light beam 302.

In an embodiment at least a portion 20”a of said optical waveguide is arranged onto or into said organ plate 20.

In an embodiment said optical waveguide 24 is arranged so, in operation of said bio-reactor, at least a portion of it provides to at least one of said cavities 21 , 23, an evanescent wave so that the properties of said optical lightbeam 302 are altered by the presence and/or reaction of an organoids 01 , 02 present in said cavity 21 , 23 . This is illustrated in Fig.4 and Fig.5 in which a leaky waveguide 300 is illustrated. The leaky waveguide guides an optical beam and is configured to provide in the cavity 21 , 23 an evanescent wave that interacts with the biological sample. The use of evanescent waves for biosensing applications is well known and is not further describes here. Reference is made to the following articles:

- H. Mukundan et al. , “ Waveguide-based Biosensors for Pathogen Detection”,

Sensors 2009, 9, pp. 5783-5809; - K.Schmitt et al. , ” Evanescent field Sensors Based on Tantalum Pentoxide

Waveguides- A review”; Sensors 2008, 8, pp.711 -738;

- S.Grego et al.,“Wavelength interrogation of grating-based optical biosensors in the input coupler configuration”.

In variants the optical waveguide may be configured on the substrate layer or directly into or on the organ layer 20. In an embodiment a portion of the optical waveguide 300 may be arranged on or into said organ layer and another portion may be arranged on any of the adapter or nutrition plate, the two portions being connected by an optical connection interface or an optical connector.

In an embodiment at least one of said optical spectrometer comprises an optical emitter E and an optical detector.

In an embodiment at least one of said optical spectrometer comprises at least one Mach-Zehnder interferometer waveguide structure 320 . Fig.6 illustrates the portions of waveguides 301 ,303, 321 , 323 that constitute the optical branches of the Mach-Zehnder interferometer waveguide structure 320. One branch 323 is arra nged at least partially in one of the cavities 21 , 23 and is in optical contact with an organoid in operation of the system, and another branch 321 is the reference branch the Mach- Zehnder interferometer waveguide structure 320.

In embodiments at least one of said cavities 21 , 23, 25 is connected to at least two different types of spectral analysis subsystems.

For example, in an advantageous embodiment at least one of said cavities 21 , 23 is connected to at least one impedance spectrometer and at least one optical spectrometer. The advantages to use more than one type of spectral analysis subsystem to one or several cavities is to provide more reliable and/or redundant data on the effect of the medical product on the organoids. For example, an impedance spectrometric analysis may be used to deduce structural changes such as volume changes of the organoids and an optical spectrometer may provide optical spectral data of the treated organoid.

In advantageous embodiments the cavities of the microchip 1a may be divided in at least two chambers, one of the chambers being configured to contain an organoid and the other chamber may be empty, both chamber comprising a portion of a spectrometer. The empty chamber is the used to make a reference measurement. Fig. 15 illustrates a realization of a cavity comprising a first chamber in which a first pair of test electrodes are integrated , and a second cavity comprising a second chamber in which a second pair of reference electrodes is integrated. When the system 1 is in operation said first pair performs a test on the treated organ while the second pair performs a reference measurement in the second chamber that contains only the circulation fluid. This allows to improve considerably the test capability of an organ in the cavity.

In an embodiment, illustrated in Fig. 7 said further layers comprise a nutrition layer 30 arranged between said adaptation layer 10 and said organ layer 20 and so that at least one of said inlets 12’-12^v is connected to at least two cavities 2T-21^V. A nutrition layer comprises apertures or tubes so that an inlet of the adapter plate may be connected to a single or a plurality of cavities. For example, in the realization of Fig. 7 and inlet 12 is connected to three different cavities 12, 12’, 12”.

In an embodiment the micro-bio-reactor 1 comprises mechanical means adapted to remove said adaptation plate 10 away from said organ plate 20 so as to allow to provide a direct access to said cavities 21 , 23 to place or remove organoids. In a variant this may be realized by a rotation axis that links said adaptation plate 10 and said organ plate 20. In a variant illustrated in Fig. 13 the adaptation plate 10 and the organ plate 20 may comprise means so that the two plates can slide laterally so as to provide access to said cavities and to place organoids into these cavities.

In an embodiment the micro-bio-reactor 1 comprises a bio reactor chip 1a comprising an actuator layer configured to comprises a multiplicity of actuators arranged and configured to regulate pressure forces to at least one of said cavities and/or a portion of said circulation system.

In an embodiment said circulation system 500 comprises a first portion of micro fluidic channels realized inside or in at least one of the surfaces of said organ plate 20, and a second portion of microfluidic channels realized in said substrate 40, adaptation plate 10 or said nutrition plate 20, said first and second portions being in fluidic connection when the microchip is closed, meaning that all the layers 10, 20, 30, 40 are in contact. Such a variant may provide design flexibilities. For example the inlet of the circulation fluid into the biochip 1a may be provided by a conduct realized in said substrate 40 and the outlet of the circulation fluid from the biochip 1a may be realized in said nutrition plate 30 and/or said adaptation plate 10.

In an embodiment the micro-bio-reactor 1 comprises a micropump integrated onto or into one of said further layers 10, 20, 30.

In an advantageous embodiment the micro bio-reactor comprises as well a central control system 1 b as at least one biochip 1a, as illustrated in Fig.11. The control system comprises at least one central pump and driver electronics as well as electronic circuits that are configured to address the valves of the system. Said control system comprises also electronic interfaces and data handling means that collect data from the spectrometers integrated into the biochip 1a and may comprise a central computer.

In embodiments an optical inspection system configured to measured the optical aspect of an organoid after the treatment period may be integrated into the biochip. More precisely the optical inspection system may be configured to detect if a portion of the surface ot the organoid has a brown color.

In an embodiment a part of the control means and data handling means may be integrated in at least one of said further layers 10, 20, 30, 40.

Preferable materials for any of said layers comprise glass or polymers but may also comprise metals or semiconductors such as silicon (Si). The organ layer 20 can be made by any material but is preferably made of polymethysiloxane (PDMS), or one of the following materials or a combination thereof: glass, Si02, polycarbonate (PC), Polyimide (PI), Polyamide (PA), POlyetheretherketoe (PEEK), epoxide resin, unsaturated esters, polytetrafluoroethylene (PTFE)

In embodiments a plurality of sensors may be arranged in at least one of the layers 10, 20, 30, 40.

In advantageous embodiments the following sensors may be arranged onto or into said organ layer 20 and/or said substrate 40 and/or said nutrition layer, said sensors being in direct mechanical , electrical or optical contact with said cavities or may be entirely integreated in said cavities:

- temperature sensors;

- pH sensors;

- pressure sensors;

- oxygen sensors; - ion sensors;

- flow sensors;

- surface acoustic wave sensors.

The mentionned list of sensors is not limitative and other sensors that may be integrated into the biochip 1a are also possible, for example capacitance sensors.

In an embodiment the bioreactor chip 1a may comprise sensors that are at least partially integrated in said cavities may be interconnected. This interconnection may be realized in the form of a neural network.

In an embodiment said organ plate 20 and/or substrate 40 is made of a stransparent material and at least one microcamera is integrated said organ plate 20 and/or substrate 40, and faces at least one of said cavities.

In advantageous embodiments the biochip 1a may comprise actuators. At least some actuators may be configured to mimic heart beat. Actuators may also serve to mimic peristaltic movement or air-flow in order to mimic breathing. These actuators may be connected through the micro bio reactor chip 1a mimicing a predetermined protocol, advised by a physician d and configured by a skilled technician person. Served as master pumps in the micro bioreactor, they push the circulation liquid by a predetermined frequency pressure, also defined as pulsation, through the chip device 1a.

In an embodiment a subsystem is integrated on or into said biochip 1a and is configured to provide a gas under pressure to at least one of said cavities 21 , 23 when the system is in operation. This may be used to mimic the function of lungs. Actuators may also be integrated to mimic the removal of substances such as to remove uiring from a cavitiy arranged to contain kidney organoids.

In embodiments the organ layer 20 is fabricated by using Mems technologies so that it can be easily mass produced. Mems technologies allow not only to provide the microstructuring of the cavities, the microfluidic channels but allow also to provide with the same process sensors and/or actuators.

Fig. 8 and Fig.9 illustrate a cross section of an examplary organ plate (Fig.8) and an adapter plate (Fig.9). In the fig. 9 cavities 31a, 31 b, 32a, 32b, 33d illustrate the connection ducts to the cavities 23a, 23b, 22a, 22b of the fig.8 in order to transvase and provide the liquids or gas into the cavities27a, 27d, 27’d, 27’c illustrate the connectors from the micro bioreactor and the valves in order to adapt the circulation in the biochip upon a given treatment protocol.

The examplary organ plate of Fig.8 comprises an impedance spectrometer comprising two electrodes 223a, 223b arranged into a cavity 23c. Some cavities may have a specific shape such as cavities for intestine organas 25a, 25b.

Cavities 23a, 23b and cavities 22a, 22b may be the cavities where the organ simulations could be placed to perform an effect analysis by therapeutic agent.

Cavities 27a, 27b, 27c, 27 d may be cavities with membrane valves in order to let the liquid or gas pass through to enrich the cavities 23a, 23b, 22a and 22b.

In embodiments at least one of said cavities may comprise strcutures such as spikes to hold and fix different organoids for different analysis.

In advantageous embodiment a cavity may comprise more than 2 electrodes configured to obtain 3D data of organoids. Fig. 10 illustrates a cavity 21 comprising 3 pairs of opposing electrodes 231 , 232, 233, 234, 235, 236.

One of the issues in systems of prior art is the difficulty to keep samples in place in a biochip 1 a and particularly to perform in real time spectrocopic or othe analysis such as visual inspection analysis. Viuasl inspection anaylsis may be perfoemd for example by a high magnification microscope that may observe a particular region of an organooid thourgh a window arranged in at least the organ plate. Therefor an advantageous embodiment is proposed that is illustrated in Fig.15. The system in Fig.15 comprises an articulation axis to pivit the adapter plate 10 to the organ plate 20. The organ plate comprises one bottom electrode 223 and the adapter plate comprises a top electrode 225. The top electrode may have a shape as illustrated to hold firmly the organoid in place during the meaurement. The top electrode, or bottom electrode, may have a hollow shape as illustrated so that the medical product S1 may pass through the inside of the electrode. For electrical isolation reasons the hollow electride may comprise an electrically isolated wall 227.

Another of the issues in systems of prior art is the difficulty to indroduce samples in a biochip 1 a. In an embodiment illustrated in Figure 13 a micro bioreactor chip 1 a comprises means to slide an adaptor plate 10 laterally relative to an organ plate 20. This allows to introduce organoids in the cavities for example by an automated delivery system. Figure 14 illustrates another embodiment of a biochip 1a that comprises an organ plate comprising cavities that each can be accessed by the bottom surface by using pivotable closures (250, 240).

The invention is also achieved by a method to test the effect of at least one medical product on at least one organoid.

Generally the standard operation of the bioreactor is performed as follows. In operation, the biochip of the bioreactor 1 system receives a placement of a tumor resection on the biochip glass and cavities in order to multiply itself up to 10-20 million of cells on the chip. Those cells are alimented with neutral culture and nutriments, circulated by the micro bioreactor connected to the pumps, which will provide an optimal environment for cell to install themselves and favor the division. The therapeutical agent is added in the cavities and circulated by the micro bioreactor in order to take effect and reach the tumor cells during the period of 7-14 days. During the process the spectrometer is measuring the cell actions and growth with electrodes, connected to each cavity. In the end of the process, the biochip is opened and a contrast liquid is added in order to identify if the therapeutical agent has had an effect.

Organoid transport is normally done by a culture liquid, such as a liquid full of nutriments for the cells to multiply themselves. However it should be a neutral liquid in order not to affect results. In a variant cells may be transported by a gaz without any liquid influence. It is also understood that the reactor may be configured to have sections that are connected by conducts to conduct a gas and others that are configured to conduct a fluid.

In embodiments the method of the invention comprises the steps of :

- providing a bioreactor as described herein;

- introducing in at least two of said cavities 21 , 23 an organoid;

- starting a pump of the bioreactor so as to circulate a circulation fluid into said fluidic circulation system;

- introducing a medical product into at least one of said inlets;

- performing a test on at least one of said organoids to identifiy the effect of said medical product

In embodiments said test is an optical test. In variants the test may be an electrical test that may be combined with an optical test or any other physiological test. In embodiments tests may be performed in real time during the circulation of the medical product in the cavities of the biochip.

In embodiments tests may be performd to test the secondary effects of a medical product. For example a cavity may compris an organoid onto which the effect of a medical product has te be measured after the treatment cycle, and in the mean time another cavity may comprise another type of organoid to test the secondary effect of the medical product in real time.

In embodiments said circulation fluid is a nutrition layer for cells of the organoids an organoid is left into a cavity until at least 10 million cells are formed.

In an embodiment a step is performed consisting of starting a measurement by said least one of said spectrometers, controlled by a control signal of said central subsystem 1 b.

In an embodiment the liquid is circulated for at least 7 days to allow the medical product to reach the tumor cells of the targeted organoid. In some cases it may be necessayr to controil during the test, the quality or composition of the circulation fluid. This may de done with a subsystem integrated into or onto said biochip or by a subsystem that is connected to said circulation system 200 by for example using a duct that is connected to said circulation system 200 by a valve.

In an embodiment the biochip is opened after a predermined periond after which the color of the organoid is verified. This may be done by a human eye or by an optical inspection system. Such an optical inspection system may be integrated into the biochip. In an example, if a brown color appears by applying a contrast liquid, the agent worked and if the original color remains, the agent did not work.

In an embodiment the circulation fluid comprises at least a fraction that is blood from a patient.

Before the process of analysis, the tumor resection is placed on a biochip in order for cells to cultivate up to 10-20mio operational cells. The biochip is hermetically closed after the cell placement and the neutral culture is added in order to begin enriching the cells.

In variants the central system 1b of the micro bioreactor 1 is connected to the biochip 1a of the invention in order to circulate a neutral culture and enrich preidentified cavities with or without open valves. In a step a therapeutic agent is added to the neutral culture to circulate with it and affect the living organoids. The protocol of enriching is defined by a predetermined length and intensity of such an enrichment .

In advantageous embodiments the impedance of an impendance spectrometer is permanently connected to the biochip to read the data of the living cells.

In embodiments, after the period of circulation of culture products and results from the spectrometer, the biochip is opened in order to add a contrast liquid. In a variant, with a hand of spectrometer and microscope the cells and analysed in order to see the relevant shape, structure and the way the therapeutical agent did or did not affect the cells. Results are collected and given to a medical expert or medical analysis equipment for diagnosis.

Practical realisation of a micro bioreactor of the invention

A practical realisation as illustrated in Fig. 11 and 12 is now described

Central operating system 1 b

The central operating system 1 b, of which a portion is illustrated in Fig.12, is composed of at least:

- a support module 550 and 552 for the temperature regulation and analysis of temperature data; The module measuring the heating in the both tubes 505 and 506 in order not to overheat.

- a module of visual data integration and software matrix, tubes of air and vacuum circulation 200”, 200”, 200’”, 200””;

- a tube for compressor connection 504;

tubes of air and vacuum regulation 506 and 505;

- a safety system of distribution of air and vaccum 515, 511 , 516 and 523 towards the biochip 1a;

- a mother board, not shown in the figures, configured as a central computer, for gathering and using the data input from the visual screen,

- a module of electricity connection 514 to said mother plate, by a connector 522.

- a locking system 1a” to the the biochip 1a;

- a tube connecting system 10”between a biochip and a micro bioactor;

- connections tubes 509, 513 between support modules 550, 552 and 522; - a connection module 512 to the vacuum device

- connection modules 501 , 504, 508, 507 to the air compressor;

interlock changing tubes 502, 503 for gas distribution;

- a power supply cable 1000.

The system is operating from an electricity source by distributing air and vacuum from the compressor and distributing it to several chips at once (8 max up to date). The system has safety control and electronic failsafe in case of air overload or overheating. Equally an external person can parameter the protocol on the operating system on the screen of the micro bioreactor.

Pressure regulation system 500

The pressure regulation system 500 controls and allows the circulation of the liquid and/or gases through the biochips 1a’-1a”” connected to the micro bioreactor 1a . The tubes 1 b’ are connected to the chip 1a” and 10” in order to push and pull the pressure of the liquid towards and into the chip 1a. safety connection 10’, 10” protects the microbio chip 1a from getting any liquid inside. A secondary safety system 200’,- 200”’ is provided in order to avoid any kind of liquid going to be introduced in the micro biochip 1a apart from a liquid medical product introduced by inlets . The medical treatment according to a protocol is inputted through a mother plate configured as a computer, gathering and using the data input from the visual screen, connected to the mother plate inside connected to the micro biorector.The micro bioreactor may be operable from a regular electricity supply 1000 and air compressor 501. The electric supply may be a DC battery 504. The vacuum system 512, 552 is giving the vacuum pressure through the system and the biochip after the protocol input on the mother plate and circulated through the biochip by tubes 513, 515. The air system 507, 508, 506, 550 is distributing the air pressure through the biochip by pushing it by through tubes 509, 511.

Organ layer 20

The organ layer 20 is configured to allow to introduce air and vacuum from the micro bioreactor into the cavities 27b and is distributed through the valves 27a. When the valves are opened the concentration is circulated throught the organ cavities 21a- 25b with a specific protocol, given by a medical core.

In an embodiment the chip 1a is designed with a polycarbonate material which can easily circulate any liquid culture through the pipes 25b. In a mode of operation the organ layer is designed to enrich only certain cavities, the valve 27c and 27a could be locked and the liquid will enrich only a part of the biochip without various cavities. The term enrich means that a medical product is provided.

Claims

1. Micro-bioreactor (1) for testing the effects of at least one medical product (S1-Sn), on a plurality of human organ samples, said micro-bioreactor (1) comprising central control system (1 b) and a biochip (1a) that comprises:

a baseplate (40) to provide a support for a stack of plates of the biochip, an adaptation plate (10) comprising at least one inlet (12) adapted to introduce said at least one substance (S1-Sn),

an organ plate (20) comprising a plurality of cavities (21 , 23) for housing an organoid (01 , 02) of a human organ, at least one of said cavities (21 , 23, 25) being connected with at least one inlet (12),

wherein:

said central control system (1b) and said biochip (1a) are in fluidic and electric connection;

the micro-bioreactor (1) comprises a closed fluidic circulation system comprising at least one circulation pump (210) and a fluidic conduct (200) configured to circulate, in operation of the bioreactor (1), a liquid between at least two of said plurality of cavities (21 , 23, 25), said fluidic circulation system comprising at least one addressable valve 27a, 27b, 27c, 27d ;

said organ plate (20) comprises at least a portion (200a) of said closed fluidic circulation system so that at least two of said plurality of cavities (21 , 23, 25) are in fluidic communication and so that, in operation of the bioreactor (1), a fluid is circulated from at least one of said plurality of cavities (21 , 23, 25) to at least another of said plurality of cavities (21 , 23, 25);

the micro-bioreactor (1) comprises at least one spectrometer (220, 222, 224) being in fluidic connection with at least one of said cavities (21 , 23, 25) and so that, in operation of the bioreactor (1), a spectral analysis may be performed of the portion of said fluid and/or organoid that is facing or is in contact with at least a portion of said spectrometer (220, 222, 224;) the bio-reactor (1) comprises an electronic system comprising electrical commands to open or close said at least one addressable valve and configured to control the circulation of said liquid in said fluidic circulation system, the bio-reactor (1) comprising also electronic means to operate said at least one spectrometer (220, 222, 224) and comprising a data processing system configured to process data provided by said spectrometer (220, 222, 224).

2. The micro-bioreactor (1) according to claim 1 wherein said at least one microspectrometer (220, 222, 224) is arranged at least partially onto or into said organ plate (20), and wherein at least one of said plates comprises electical contacts configured to receive and send electrical signals to a control and/or dataprocessing system situated outised said stack of further plates (10,20,30).

3. The micro-bio-reactor (1) according to claim 1 or claim 2 wherein said control and/or dataprocessing system is integrated at least partially into said stack of plates (10,20).

4. The micro-bioreactor (1) according to any one of claims 1 to 3 wherein at least one of said spectrometers (220, 222, 224) is an impedance spectrometer.

5. The micro-bio-reactor (1) according to claim 4 wherein said impedance spectrometer comprises at least two gold electrodes (223a, 223b) .

6. The micro-bio reactor (1) according to any one of claims 1 to 5 wherein at least one of said spectrometers (220, 222, 224) is an optical spectrometer.

7. The micro-bioreactor (1) according claim 6 wherein said optical spectrometer comprises an optical waveguide (300) configured to guide an optical lightbeam (302).

8. The micro-bioreactor (1) according claim 7 wherein at least a portion (20”a) of said optical waveguide (300) is arranged onto or into said organ plate (20).

9. The micro-bioreactor (1) according to claim 7 or claim 8 wherein said optical waveguide (300) is arranged so, in operation of said bio-reactor (1), at least a portion of it provides to at least one of said cavities (21 , 23), an evanescent wave so that the properties of said optical lightbeam (302) are altered by the presence and/or reaction of an organoids (01 , 02) present in said cavity (21 , 23) .

10. The micro-bioreactor (1) according to any one of claims 6 to 9 wherein at least one of said optical spectrometers (220, 222, 224) comprises an optical emitter (E) and an optical detector (D).

11. The micro-bio-reactor (1) according to claim 6 to 10 wherein at least one of said optical spectrometers (220, 222, 224) comprises at least one Mach-Zehnder interferometer waveguide structure (224).

12. The micro-bio-reactor (1) according to any one of claims 6 to 11 wherein at least one of said cavities (21 , 23) is connected to at least one impedance spectrometer and at least one optical spectrometer.

13. The micro-bio-reactor (1) according to any one of claims 1 to 12 wherein said stack of plates comprise a nutrition layer (30) arranged between said adaptation layer (10) and said organ layer (20) and so that at least one of said inlets (12’-12^” ) is connected to at least two cavities (2T-21^V).

14. The micro-bio-reactor (1) according to any one of claims 1 to 13 comprising mechanical means adapted to remove said adaptation plate (10) away from said organ plate (20) so as to allow to provide a direct acces to said cavities (21 , 23) to place or remove organoids.

15. The micro-bio-reactor (1) according to any one of claims 1 to 14 comprising an actuator layer configured to comprise a multiplicity of actuators arranged and configured to regulate pressure forces to at least one of said cavities and/or a portion of said circulation system.

16. The micro-bio-reactor (1) according to any one of claims 1 to 15 wherein said circulation system (50) comprises microchannels realized inside or in at least one of the surfaces of said organ plate.

17. The micro-bio-reactor (1) according to anyone of claims 1 to 16 comprising a micropump integrated onto or into one of said stack of plates (10, 20, 30).

18. The micro bio reactor (1) according to any one of claims 1 to 17 wherein said bio chip (1a) comprises an optical inspection system configured to detect, in operation, the color of at least a portion of the surface of one of said organoids.

19. The micro bio reactor (1) according to any one of claims 1 to 18 wherein said cavities comprise a mechanical structure for holding and keeing in place organoids.

20. The micro bio reactor (1) according to any one of claims 4 to 19 wherein at least one of said impedance spectrometers comprises a hollow electrode.

21. The micro bio reactor (1) according to claim 20 wherein said hollow electrode comprises a portion that is postionned inside a cavity when the bioreactor is in operation..

22. A method to test the effect of medical product on tissues comprising the steps of:

- providing a bioreactor (1) according to any one of claims 1 to 21 ;

- introducing in at least two of said cavities (2T-21^V ) an organoid (01);

- starting a pump of the bioreactor (1) so as tro circulate a circulation fluid into said fluidic circulation system;

- introducing a medical product into at least one of said inlets;

23. The method according to claim 22 comprising the further steps of

- leaving. said at least one organoid into a cavity until at least 10 million cells are formed.

24. The method according to claim 22 or claim 23 comprising the further step of

- starting a measurement by said at least one of said spectrometers, controlled by a control signal of said central subsystem 1b;

- providing spectral data;

- gathering said spectral data by a software.

25. The method according to any one of claim 22 to 24 compring the steps of:

- circulating the circulation fluid for at least 7 days to allow the medical product to reach the tumor cells of the targeted organoid;

- deciding that, if the contrast liquid has provoqued a different color to at least a portion of the organoid, the therapeutic agent has had a therapeutical effect worked on the organoids, within the size measured by one of said

spectrometers 200, 222, 224.

26. The method according to any one of claim 22 to 25 wherein identical but different shaped organoids and spheroids are used in said biochip 1a.

27. The method according to any one of claim 22 to 26 wherein an optical inspection is performed by using an optical inspection system configured to observe an organoid through a window in said biochip 1a.

28. The method according to any one of claim 22 to 27 wherein, during a test, a measurement is made of the composition of the circulation fluid.

29. The method according to any one of claim 22 to 27 wherein the circulation fluid comprises at least a fraction that is blood from a patient.