multi-organ device on chip

专利摘要:
MULTIPLE ORGAN DEVICES IN CHIP The present invention relates to a multi-organ chip device that comprises a base layer; an organ layer arranged on the base layer; a layer of dens arranged over the organ layer; and an actuator layer; wherein the base layer is configured to provide solid support for the additional layers; the organ layer is configured to comprise a multiplicity of individual organ equivalents, each organ equivalent comprising one or more sections of organ growth, each of the sections of organ growth being configured to comprise an organoid cavity to house at least one organoid of an organ and to comprise a micro-entry and a micro-exit for fluid communication between the organoid cavity of the organ growth section and an independent circulation system, wherein the organ layer comprises at least one equivalent of organ configured to represent the lung, small intestine, spleen, pancreas, liver, kidney and bone marrow, respectively, and an independent circulation system configured to be in direct fluid communication with the growth sections of (...).
公开号:BR112015006859B1
申请号:R112015006859-6
申请日:2013-08-15
公开日:2020-11-17
发明作者:Uwe Marx
申请人:Tissuse Gmbh；
IPC主号:

专利说明:

BACKGROUND OF THE INVENTION
[001] Miniaturized three-dimensional (3D) organ or organoid culture systems are of increasing academic and economic interest. These 3D culture systems aim to allow the investigation of how the organs work and behave under certain stimuli, as well as, to test the effects of compounds or chemical compositions in particular organs or groups of them and to study the pharmacokinetic behavior of such compounds or compositions. In particular, in relation to chemical safety testing, alternatives are required to replace animal experiments and generate data that can be more easily used to efficiently and reliably predict safety in humans. The quality of such a 3D in vitro culture system will depend on its ability to reflect as much as possible the physiological function and the environment of the respective organ or organoid. This objective can be achieved only if the organs are not considered as separate independent objects, but if the complexity of interaction between different organs in an organism is imitated as close as possible. In order to allow the generation of meaningful data, it is necessary that the culture system remains stable for an extended period of time. However, most of the known 3D culture systems today reflect only one cell type or model only one type of organ or organoid. 3D culture systems that take into account multiple organs and that allow dynamic culture of these multiple organs have only recently been described.
[002] In document No. W02009 / 146911 A2, an organ device on a chip was presented. This organ device on a chip is designed to be independent and sensor controlled. The device allows the establishment or maintenance of organs or organoids, as well as niches of stem cells in a miniaturized chip format. The organ device on a chip can comprise a multiplicity of sections of organ growth comprising an organ or organoid, a medium feed reservoir and a medium disposal reservoir functionally connected to each other, so that the organs or organoids of the organ growth section can be fed with the medium from the medium feed reservoir and that the degradation products and residues can be discarded through the medium disposal reservoir. Although this model allows the simultaneous culture of more than one organ on a chip, this device does not allow the interaction and cross-relationship between different organs on the chip. In addition, this device does not reflect all the functions necessary to achieve homeostasis of the culture system over an extended period of time.
[003] In document No. WO 2012/016711 A1, a 3D cell culture model is presented that comprises one or more sections of organ growth, an independent circulation system configured to supply the organs or organoids grown in the sections of organ growth with nutrients and an extracapillary fluid or waste collector to collect interstitial fluid and degradation products from the organ growth sections. This system allows simultaneous culture of more than one organ and mimics a vascular system that serves and interconnects different organs. In this way, this system allows interaction and cross-relationship between the organs or organoids of the system. Another cell culture device with a comparable composition is disclosed in US 2012/0214189 A1. However, this device does not reflect all the functions necessary to achieve the homeostasis of the culture system over an extended period of time. SUMMARY OF THE INVENTION
[004] The present invention relates to a multi-organ chip device that mimics the basic functions of an organism necessary for organ and / or organism homeostasis. The multi-organ chip device of the present invention is designed to reflect an independent circulation system that mimics the blood system of a higher organism that serves countless different organ equivalents. Organ equivalents are selected and arranged in such a way that the basic functions of food supply, waste removal and oxygen supply are represented fully functional to maintain the homeostasis of the organ equivalents throughout
[005] over a period of time An extended device. multiple organs on chip is provided
[006]
[007] base layer;
[008], wherein the device is a layer of a layer of a layer of comprises base; organ arranged over the anthers arranged over the organ layer; and
[009] - an actuator layer;
[010] where
[011] - the base layer is configured to provide solid support for additional layers;
[012] - the organ layer is configured to understand
[013] a multiplicity of individual organ equivalents, each organ equivalent comprising one or more sections of organ growth, each of the sections of organ growth being configured to comprise an organoid cavity to accommodate at least one organoid of an organ and comprise a microentry and a microsaid for fluid communication between the organoid cavity of the organ growth section and an independent circulation system, wherein the organ layer comprises at least one organ equivalent configured to represent the organs lung, small intestine, spleen, pancreas, liver, kidney and bone marrow, respectively, and
[014] an independent circulation system configured to be in direct fluid communication with the organ growth sections of the organ layer through the micro-entrances and microsaids of the organ growth sections;
[015] - the antrum layer is configured to comprise a multiplicity of cavities and tubes arranged to be in fluid communication with selected organ equivalents or sections of organ growth in order to allow the exchange of fluids between the cavities and the sections of organ growth; and
[016] - the actuator layer is configured to comprise a plurality of actuators arranged and configured to regulate a pressure force applied on a selected organ equivalent, the independent circulation system and / or part of it.
[017] Additional details and preferred embodiments of the invention are defined in the specification below and in the claims. DETAILED DESCRIPTION OF THE INVENTION
[018] In the following, the present invention will be described in more detail. Except where otherwise specified, all technical and scientific terms used in this document have the same meaning as is commonly understood by the person skilled in the relevant technique. If a first layer or object is specified to be located on top of a second layer or object, the first layer or object can be directly located on top of the second layer or object or another layer or object may be present between the first and second layer or object.
[019] The multi-organ chip device of the invention consists of numerous layers with different functionalities. The multi-organ chip device comprises a base layer, an organ layer, optionally an organ support layer, an antrum layer and an actuator layer.
[020] The base layer is configured to provide solid support for the additional layers, so that the multi-organ chip device can be easily handled and manipulated. Preferably, said base layer is made of a transparent material. This has the advantage that the organ layer is optically accessible from the bottom and thus allows the observation of organoids in the organ growth section during culture by microscopy, for example, by two-photon microscopy. Since the base layer is made of transparent material, the organ layer is accessible from the bottom side and allows the imaging of fluorescence ratio for measurement of local interstitial pH, extinction microscopy of phosphorescence of interstitial p02 and spectroscopy of infrared to detect physiological stress.
[021] Preferred materials for the base layer comprise glass and optically transparent synthetic polymers such as, for example, polystyrene (PS), polycarbonate (PC), polysiloxane and / or polydimethylsiloxane (PDMS).
[022] In order to monitor the status of the device and allow controlled culture of organoids, the base layer may comprise one or more sensors configured and arranged to measure signals emitted from and / or transmit signals to one or more of the equivalents organs, organ growth sections and / or independent circulation system. The sensors that are exhibit high sensitivity to allow accurate measurement even in small sample volumes. Preferably, the base layer comprises sensors for the main parameters of homeostasis of the human organism, such as organoid or cellular viability, temperature, pH, fluid balance, pressure, flow volume, oxygen pressure or oxygen consumption, consumption nutrients, fluid adsorption, intestinal juice secretion, albumin synthesis, bile synthesis, urea excretion, ionic balance, osmolarity and electrical coupling. The sensors that can be used include, however, are not limited to pH sensors, PO2 sensors, analyte capture sensors, conductivity sensors, plasmon resonance sensors, temperature sensors, CO2 sensors, NO sensors, chemotaxis sensors, cytokine sensors, ion sensors, pressure sensors, potentiometric sensors, amperometric sensors, through-flow sensors, load sensors, impedance sensors, electromagnetic field sensors, surface acoustic wave sensors and metabolic sensors. Preferably, the base layer comprises at least the following set of sensors:
[023] - 2 pÜ2 sensors configured and located to measure pθ2 in the fluid of the circulation system independent of the organ layer, preferably a PO2 sensor is located under the arteriolar transport channel in the vicinity of its origin from the equivalent lung and a pÜ2 sensor is located under the venular transport channel in the vicinity of its origin from the lung equivalent;
[024] - 4 transepithelial / endothelial electrical resistance sensors (TEER) to identify leakage in the independent circulation system (if the resistance between two of the TEER sensors is 0 the leak is likely), preferably, two TEER sensors are located in the system of independent circulation, for example, a TEER sensor is located close to the origin of the arteriolar transport channel from the lung equivalent and a TEER sensor is located at the end of the arteriolar transport channel farthest from the origin of the arteriolar transport channel a From the lung equivalent, two TEER sensors are configured and located in the liver equivalent, optionally, there may be two additional TEER sensors present configured and located in the skin or intestine equivalent together to monitor the functionality of cell barriers, such as epithelial barriers or endothelial between the organs and the bloodstream;
[025] - electrical sensors, which are coupled with biological neuronal ganglia, configured and located to be in contact with such ganglia in the organ equivalents.
[026] The multi-organ chip device of the invention comprises an organ layer located on top of the base layer. The organ layer is configured to comprise a multiplicity of individual organ equivalents, each organ equivalent comprising one or more sections of organ growth. Each of the organ growth sections of the organ layer is configured to comprise an organoid cavity to house an organoid of a specific organ type. Each organ growth section is configured to comprise a microentry and a microsaid for fluid communication between the organoid cavity of the organ growth section and the organ layer independent circulation system. The organ layer comprises at least one organ equivalent configured to represent the organs: lung, small intestine, spleen, pancreas, liver, kidney and bone marrow, respectively. The organ layer may comprise equivalents of additional organs, for example, equivalents of skin organs, testicles, brain and / or adipose tissue. In addition, the organ layer comprises an independent circulation system configured to be in direct fluid communication with the organ growth sections of the organ layer through the micro-entrances and microsaids of the organ growth sections of the organ equivalents.
[027] As used herein, the term "organ equivalent" refers to all sections of organ growth that comprise organoids or a particular type of organ. All the organs and systems of an organism, for example, of a human organism, are built by multiple functionally identical self-sufficient structural units, the organoid units. These organoid units are very small in size, from several cell layers to a few millimeters. The lobes of the liver, kidney nephrons, dermis and epidermis of the skin, intestinal mucosa, islets of Langerhans of the pancreas, gray and white matter of the cerebral cortex and cerebellum and niches of adult resting promoter stem cells are a small selection of examples of such human organoid structures, all with prominent functionality and highly variable conglomerate geometry. Due to the distinct functionality, a high degree of independence and multiplicity of such micro-organoids within the respective organ, their pattern of reactivity to any substances appears to be representative of the entire organ. Nature has created very small but sophisticated biological structures to perform the most prominent functions of organs and systems. The multiplication of these organoid structures within a given organ is the nature's risk management tool to prevent the total loss of functionality during partial organ damage. On the other hand, this concept allowed the easy adjustment of the size and shape of the organ to the needs of a given species - for example, liver in mice and men - still using a main plan established to build the only functional organoid unit. An exclusive and excellent chance for predictive substance tests for human exposure is the establishment of human micro-organoid equivalents in vitro. In the present invention, "organoids" means artificial, newly generated, aggregates of functional cells from different types of cells in vitro that show at least one organ or tissue function, preferably show most or essentially all of the organ or fabric. Thus, in the multi-chip device of the present invention, an organ equivalent is represented by one or more sections of organ growth, each section of organ growth comprising an organoid cavity to house an organoid of the respective type. of organ. In this way, the size of an organ equivalent can be easily adjusted by choosing the appropriate number of sections of organ growth or organoids of the respective organ type.
[028] The person skilled in the art is well aware of the structure of an organoid of a given organ and knows how to produce said organoid. The following are some examples of organ specific organanoids: lung alveolar organoids, pancreatic islet-shaped organoids, white and red spleen pulp, organoids in the form of small intestine villi, lobe-shaped organoids of the liver, nephron-shaped kidney organoids, bone marrow units, bone-shaped organoids and bone marrow cartilage, appendage-shaped units of the skin, organoids in the form of clusters of adipose tissue, organoids in the form of follicles in the testes and organoids in the form of cerebellar cortex of the brain.
[029] The liver organoid can be a liver lobe in hexagonal shape with a volume of 1.2 to 2.2 mm3.
[030] The lung organoid can be a pulmonary alveolus in spheroid shape and with a surface area of 0.15 to 0.25 mm2.
[031] The pancreatic organoid may be an islet of Langerhans surrounded by exocrine tissue, all organized in spheroidal format and with a volume of 0.2 to 0.5 mm3.
[032] The spleen organoid can be tissue of white and red pulp in spheroid shape with a volume of 0.3 to 0.6 mm3.
[033] The small intestine organoid can be a pillar-shaped villus with a surface area of 0.2 to 0.4 mm2.
[034] The kidney organoid can be a renal nephron with a spheroid capsule and a cylindrical tubule, and a filtration surface of 6 to 7.5 mm2.
[035] The bone marrow organoid can be a unit in the form of macropores formed by bone marrow, bone and cartilage with a volume of 0.006 to 0.008 mm3.
[036] The skin organoid can be a segment in hexagonal shape containing appendages, which has a surface area of 1.2 to 2 mm2.
[037] The adipose tissue organoid can be a spheroid-shaped adipose grouping with a volume of 0.0004 to 0.0006 mm3.
[038] The testicular organoid can be a spheroid-shaped testicle follicle with a volume of 0.006 to 0.008 mm3.
[039] The brain organoid can be a column of the cerebral cortex in cylindrical shape and a surface of 0.02 to 0.03 mm2.
[040] The organ layer can be designed so that:
[041] - an organ growth section of the liver equivalent is configured to provide an organoid cavity to house 5 to 15 liver organoids, where each liver organoid is a lobe of the liver, preferably the organoid cavity it is configured to house 10 liver organoids;
[042] - an organ growth section of the lung equivalent is configured to provide an organoid cavity to house 2,000 to 4,000 lung organoids, where each lung organoid is a pulmonary alveolus, preferably the organoid cavity is configured to host 3,000 lung organoids;
[043] - an organ growth section of the pancreas equivalent is configured to provide an organoid cavity to house 5 to 15 pancreatic organoids, where each pancreatic organoid is an islet of Langerhans, preferably the organoid cavity it is configured to house 10 pancreas organoids;
[044] - an organ growth section of the spleen equivalent is configured to provide an organoid cavity to house 5 to 15 spleen organoids, where each spleen organoid is a white and red pulp, preferably the cavity of organoid is configured to house 10 spleen organoids;
[045] - an organ growth section of the small intestine equivalent is configured to provide an organoid cavity to house 40 to 80 small intestine organoids, where each small intestine organoid is a villus, preferably the cavity of organoid is configured to house 60 small intestine organoids;
[046] - a kidney equivalent organ growth section is configured to provide an organoid cavity to house 10 to 30 kidney organoids, where each kidney organoid is a nephron, preferably the organoid cavity is configured to house 20 kidney organoids; and
[047] - an organ growth section of the bone marrow equivalent is configured to provide an organoid cavity to house 1,000 to 2,000 bone marrow organoids, where each bone marrow organoid is a unit made up of bone marrow, bone and cartilage, preferably the organoid cavity is configured to accommodate 1,400 bone marrow organoids.
[048] In addition, the organ layer can be designed so that:
[049] - an organ growth section of the skin equivalent is configured to provide an organoid cavity to accommodate 10 to 20 skin organoids, where each skin organoid is a skin appendage, preferably the organoid cavity it is configured to accommodate 15 skin organoids;
[050] - an organ growth section of the adipose tissue equivalent is configured to provide an organoid cavity to house 200,000 to 300,000 adipose tissue organoids, where each adipose tissue organoid is an adipose grouping, preferably the cavity organoid is configured to house 240,000 adipose tissue organoids;
[051] - a testicular equivalent organ growth section is configured to provide an organoid cavity to accommodate 10 to 20 testicular organoids, where each testicular organoid is a testicle follicle, preferably the organoid cavity it is configured to house 15 testicular organoids; and
[052] - an organ growth section of the brain equivalent is configured to provide an organoid cavity to house 100 to 300 brain organoids, where each brain organoid is a column of the cerebral cortex, preferably the organoid is configured to host 200 brain organoids.
[053] Each of the organ equivalents can be configured to accommodate a number of organoids that is proportional to the number of organoids present, on average, in the respective organ of a mammalian organism, preferably a human. In order to represent an organism, it is advantageous to select the size of all organ equivalents of the multi-chip device of the invention to reflect the relative proportionality of the organ size under physiological condition in the organism. Preferably, all of the organ equivalents of the multi-organ chip device are reduced in size by the same predetermined proportionality factor. This proportionality factor may vary depending on the desired size of the multi-organ device on chip, a preferred proportionality factor is 0.00001 (1 / 100,000). If a human organism is to be represented, the multi-organ chip device is preferably configured to understand:
[054] 1 liver organoid,
[055] 300 lung organoids,
[056] 1 pancreas organoid,
[057] 1 spleen organoid,
[058] 6 small intestine organoids,
[059] 2 kidney organoids,
[060] 140 bone marrow organoids and, optionally,
[061] 1 or 2 skin organoids,
[062] 24,000 adipose organoids,
[063] 1 or 2 testicular organoids,
[064] 20 brain organoids,
[065] or a multiple of them.
[066] In a particular preferred embodiment, the multi-organ chip device is preferably configured to comprise:
[067] 10 liver organoids,
[068] 3,000 lung organoids,
[069] 10 pancreas organoids,
[070] 10 spleen organoids,
[071] 60 small intestine organoids,
[072] 20 kidney organoids,
[073] 1. 400 bone marrow organoids, optionally
[074] 15 skin organoids,
[075] 240,000 adipose organoids,
[076] 15 testicular organoids,
[077] 200 brain organoids,
[078] or a multiple of them.
[079] Preferably, an organ growth section additionally comprises one or more niches of stem cells. In order to provide a system that can be operated under homeostatic condition for an extended period of time, it is advantageous to provide a source of cells that can facilitate cell renewal within an organoid. Each organ has a certain renewal time during which the cells of the organ are replaced by new cells. This cellular renewal of an organ ensures that the cells of an organ are vital and fully functional. Said renewal can be imitated by introducing a stem cell niche into one, some or all of the organ equivalents of the multi-organ chip device. Said stem cell niches can be part of one, some or all of the organ growth sections of an organ equivalent.
[080] The structure and method of making such sections of organ growth that include organ cavities and niches of stem cells have already been described in document No. WO 2012/016711 A1 and WO 2009/146911 A2, the disclosure of which is incorporated in this document as a reference.
[081] The organ layer can be made of a suitable material. Preferred materials comprise SiO2, glass, and synthetic polymers. Preferred synthetic polymers include polystyrene (PS), polycarbonate (PC), polyamide (PA), polyimide (PI), polyetheretherketone (PEEK), polyphenylene sulfide (PPSE), epoxy resin (EP), unsaturated polyester (UP), resin from phenol (PE), polysiloxane, for example, polydimethylsiloxane (PDMS), melamine resin (ME), cyanate ester (CA), polytetrafluoroethylene (PTFE) and mixtures thereof. Particularly preferred synthetic polymers are optically transparent and include, for example, polystyrene (PS), polycarbonate (PC), and polysiloxane, for example, polydimethylsiloxane (PDMS). A particularly preferred material comprises PDMS.
[082] The organ layer comprises an independent circulation system. The independent circulation system is designed to mimic the vascular system of an organism and thereby supply all the organ equivalents of the invention's multi-organ chip device with nutrients, O2 and allow interaction and cross-relationship between the equivalents of organs. The presence of said independent circulation system is vital for the homeostasis of the entire multi-organ device on a chip. The term "independent" refers to the fact that a fluid is circulable in the circulation system and that, preferably, there is no fluid connection to continuously supply fluid, for example, medium, blood or a blood equivalent, from an external reservoir for the circulation system. In this context, "external" means that the reservoir is not an integral part of the circulation system or the multi-organ chip device, for example, is not connected via a pipe to the circulation system. If substances, for example, nutrients and / or fluids, have to be answered during the incubation course, it is preferable that such nutrients or fluids are supplied discontinuously through an injection port which is preferably located in a transport channel. arteriolar or venular circulation system or that is located in the antrum layer.
[083] The independent circulation system is configured to be in direct fluid communication with the organ growth sections of the organ equivalent of the organ layer through the micro-entrances and microsaids of said organ growth sections. The structure and method of manufacturing such an independent circulation system have already been described in document No. WO 2012/016711 A1, the disclosure of which is incorporated by reference in this document. The internal surface of the independent circulation system can be lined with endothelial cells and, optionally, smooth muscle cells.
[084] The independent circulation system comprises:
[085] an arteriolar transport channel, which directly connects the microsaidas of the organ growth sections of the lung equivalent to the micro-entrances of the organ growth sections of the organ layer, in order to allow the transport of fluid with high pÜ2 to the said sections of organ growth; and
[086] a venular transport channel, which directly connects the micro growths of the organ growth sections to the microentries of the lung equivalent organ growth sections, in order to allow the transport of fluid with low pθ2 from the growth sections of organs to the lung equivalent.
[087] The independent circulation system can be filled with a fluid capable of transporting nutrients and O2 to organ equivalents. Preferably, said fluid is blood or a blood equivalent.
[088] The fluid in the independent circulation system is circulated in a direct way through the associated action of actuators in the actuator layer of the multi-organ chip device. In this way, it is possible to imitate not only an adequate pressure within the circulation system that corresponds to the pressure in the vasculature of an organism, but also to allow the imitation of the heartbeat. In this way, the inventive multi-organ device independent circulation system is suitable for providing shear forces and microenvironments that correspond to the situation encountered under physiological conditions.
[089] The independent circulation system can be configured, so that the microsaids of the organ growth sections of the small intestine, spleen and pancreas equivalents are connected to be in direct fluid communication with each other and with additional microentries of the sections of liver equivalent organ growth, to allow fluid communication between the spleen, pancreas, small intestine and liver equivalent, in such a way that fluid communication from the spleen, pancreas and small intestine towards the venular transport channel of the independent circulation system can occur exclusively through the passage through the liver equivalent. This architecture allows to imitate the basic functions of the digestive system of a superior organism, for example, a human being. The advantage of such an architecture is that the multi-organ chip device can be grown over an extended period of time by supplying the small intestine equivalent with nutrients from a reservoir located in the antrum layer. The organ equivalents of the multi-organ chip device of the invention will then be supplied with nutrients that have passed through a digestive system. In this way, nutrients are provided in a way and manner that are more comparable to the physiological condition in an organism. There is no longer any need for an external medium reservoir that is constantly fed into the circulation system to serve the organ equivalents.
[090] The independent circulation system and the organ equivalents are preferably configured so that the arteriolar transport channel that originates from the lung equivalent displays, in the direction of flow, bifurcations in which the arteriolar channels meet. branch, serving organ equivalents. The fluid that passes through a given organ equivalent is channeled back to the venular transport channel through venular channels that branch out from the venular transport channel at the respective bifurcations. Preferably, the independent circulation system and the organ equivalents are configured, so that the arteriolar transport channel that originates from the lung equivalent displays in the flow direction:
[091] - a first bifurcation in which a first arteriolar canal branches, serving the equivalent of the small intestine, spleen and pancreas;
[092] - a second bifurcation in which a second arteriolar canal branches, serving the liver equivalent;
[093] - a third bifurcation in which a third arteriolar channel branches, serving the kidney equivalent;
[094] - a fourth bifurcation in which a fourth arteriolar canal branches, serving the kidney equivalent;
[095] - a fifth bifurcation in which a fifth arteriolar canal branches, serving the bone marrow;
[096] - an optional sixth bifurcation in which a sixth arteriolar canal branches, serving a skin equivalent;
[097] - an optional seventh bifurcation in which a seventh arteriolar canal branches, serving an equivalent of adipose tissue;
[098] - an optional eighth bifurcation in which an eighth arteriolar canal branches, serving an equivalent of testicles; and
[099] - an optional ninth fork in which a ninth arteriolar channel branches, serving a brain equivalent.
[0100] The independent circulation system is configured, so that the diameter of the arteriolar transport channel in the flow direction is constantly reduced, so that the sum of the cross-sectional areas of all arteriolar transport channels, including all bifurcations at a certain distance from the lung equivalent, remain constant and in which in the venular transport channel said reduction in diameter is constantly reversed in the direction of flow, so that the sum of the cross-sectional areas of all the channels of venular transport, including all bifurcations at a given distance from the lung equivalent, remains constant.
[0101] The organ layer can be configured, so that the organoid cavities of the organ growth sections are opened on the opposite side to the basal layer. This allows organoids or precursor cells to be applied to the respective organoid cavities before the multi-organ chip device is fully assembled. In that case, the multi-organ chip device additionally comprises an optional organ support layer otherwise. The organ support layer is arranged between the organ layer and the den layer. The organ support layer is configured to seal and / or stabilize the organ layer, in such a way that for selected organ equivalents, communication with the antrum layer is maintained. The organ support layer can be provided as a 50 to 500 pm thick layer, preferably 100 to 300 pm thick, more preferably 200 pm thick. The organ support layer can be made of a material that comprises or consists of a synthetic polymer such as, for example, polystyrene (PS), polycarbonate (PC), polysiloxane and / or polydimethylsiloxane (PDMS). Preferably, the material comprises or consists of polycarbonate. Specifically in areas, where the organ support layer covers an organ equivalent that has an excretory function and / or produces a considerable amount of interstitial fluid, such as kidney, liver, spleen and small intestine, the organ support layer is configured to allow fluid communication between the organ layer and the dens layer. Such fluid communication can be achieved, for example, by providing pores within the organ support layer, preferably by providing pores with an average diameter of 5 to 7 pm. Alternatively, or in addition, the thickness of the organ support layer in an area that allows fluid communication between the organ layer and the organ support layer can be reduced to an average thickness of 5 to 15 pm, from preferably at 10 pm.
[0102] The multi-organ chip device of the invention comprises a layer of dens arranged on top of the organ layer. The antrum layer is configured to comprise a multiplicity of cavities and tubes arranged to be in fluid communication with selected organ equivalents or sections of organ growth, in order to allow the exchange of fluids between the cavities of the antrum layer and sections of organs. organ growth from the organ layer. Numerous organs have excretory functions and / or produce considerable amounts of interstitial fluid that need to be dissipated if culture or incubation over an extended period of time is considered. Especially, since the fluid in the independent circulation system is constantly circulated without exchange and replacement, it is vital to dissipate the degradation products from the system. In particular, the urine formed in the kidney equivalent and the feces supplied from the small intestine equivalent need to be eliminated from the system in order to allow the operation of the multi-organ chip device for an extended period of time under homeostatic conditions. In addition, since the medium is not constantly fed into the system, a reservoir to supply the small intestine equivalent with nutrients is required. Preferably, this nutrient reservoir is not disposed within the organ layer itself, but within the dens layer. This allows you to replenish the reservoir with nutrients discontinuously during operation of the multi-organ chip device without interacting directly with the organ layer.
[0103] The dens layer can be configured to understand:
[0104] a cavity that is located on top of the small intestine equivalent and is in fluid communication with the small intestine equivalent and a nutrition reservoir, so that the small intestine equivalent can be supplied with nutrients from the reservoir of nutrition;
[0105] a cavity that is located on top of the small intestine and is in fluid communication with the small intestine equivalent and a stool reservoir, so that material excreted from the small intestine equivalent can be transported to the reservoir of feces;
[0106] a cavity that is located on top of the liver equivalent and is in fluid communication with the liver equivalent and the cavity that is located on top of the small intestine equivalent, so that the material excreted from the liver equivalent can be transported to the cavity that is located on top of the small intestine; and
[0107] a cavity that is located on top of the kidney equivalent and is in fluid communication with the kidney equivalent and a urine reservoir, so that the urine reservoir receives the material excreted from the kidney equivalent.
[0108] The nutrition reservoir, the feces reservoir and the urine reservoir are integral parts of the antrum layer.
[0109] The den layer can additionally comprise a port that allows the introduction of chemical compounds, such as test compounds, into the fluid of the independent circulation system and the taking of samples from the fluid of the independent circulation system .
[0110] The multi-chip device of the invention comprises an actuator layer. The actuator layer is configured to comprise a plurality of actuators arranged and configured to regulate a pressure force applied on a selected organ equivalent, the independent circulation system and / or part thereof. In order to operate an organism under homeostatic conditions, it is necessary to ensure the movement and application of controlled force within the system. Of course, the blood in the vasculature needs to be moved in order to ensure proper functioning. However, peristaltic bowel movement is also necessary, as well as compression and decompression of the lung in order to allow air flow. In the multi-organ chip device of the present invention, said movement or introduction of force is facilitated through the actuators of the actuator layer. The configuration and arrangement of actuator elements in the actuator layer depends on the total architecture of the multi-organ device on chip, in particular, the arrangement of organ equivalents within the organ layer. The actuators can be understood as actuators based on air pressure that are configured to apply pressure force to an organ equivalent or to the independent circulation system or a part of it. These actuators can be controlled by an external device that can be programmed.
[0111] Preferably, the actuator layer comprises:
[0112] one or more actuators that act in the independent circulation system to allow the movement of directed fluid, in order to imitate the heartbeat;
[0113] one or more actuators that act on the antrum layer to allow directed movement, in order to imitate peristaltic bowel movement;
[0114] one or more actuators that act on the lung equivalent to allow the flow of air, in order to imitate breathing;
[0115] one or more actuators that act on the bone marrow equivalent to allow regulated compression, in order to imitate bone compression;
[0116] one or more actuators that act on the arteriolar transport channel of the independent circulation system, in order to imitate arteriolar constriction;
[0117] one or more actuators that act on the liver equivalent to allow the movement of directed fluid, in order to dissipate the bile from the liver equivalent; and
[0118] one or more actuators that act on the antrum layer to allow the movement of directed fluid, in order to dissipate the urine from the kidney equivalent.
[0119] In a preferred embodiment of the multi-organ chip device of the invention, the organ layer comprises or consists of polydimethylsiloxane (PDMS), the organ support comprises or consists of polycarbonate, the dens layer comprises or consists of PDMS and / or the actuator layer comprises or consists of polycarbonate.
[0120] The present invention is directed to the multi-organ chip device defined above and in the claims without organoids, cells and fluid. The present invention is also directed to the multi-organ chip device defined above, wherein the multi-organ chip device comprises the respective organoids, cells and fluids.
[0121] The multi-organ chip device of the present invention is characterized by its potential for prolonged operation in homeostatic condition and its proximity to a physiological organism. The multi-organ chip device can be applied in different configurations depending on the content and architecture of the organ equivalents present in the device. In addition to applications in systemic safety tests, immunological, infectious and / or oncological models, the following preferred uses of the multi-chip device of the invention are presented: TABLE I: PREFERRED USES OF THE MULTIPLE ORGAN DEVICES IN CHIP OF THE INVENTION






FIGURES:
[0122] Figure 1 shows a schematic overview of an embodiment of the multi-chip device of the invention with all its layer structure.
[0123] Figure 2 shows a schematic top-down view over the actuator layer of the Figure 1 modality.
[0124] Figure 3 shows a schematic top-down view of the antrum layer of the modality of Figure 1.
[0125] Figure 4 shows a schematic top-down view of the organ support layer of the embodiment of Figure 1.
[0126] Figure 5 shows a schematic top-down view of the organ layer of the modality of Figure 1.
[0127] Figure 6 shows a schematic top-down view of the base layer of the Figure 1 modality.
[0128] Figure 7 shows a multi-chip microfluidic device (MOC) at a glance, (a) The exploded view of the device comprising a CP polycarbonate, the PDMS-glass chip that accommodates two microvascular circuits (occupied area: 76 mm x 25 mm; height: 3 mm) and a MOC support that can be heated, (b) The cross section of a peristaltic micro pump on the chip operated by programmed compression and periodic decompression of three successively arranged PDMS membranes (thickness: 500 pm); the arrow indicates the direction of flow, (c) The top view of the MOC sketch illustrating the two separate microfluidic circuits (channel height: 100 pm; width: 500 pm), each, accommodating two insertion areas (compartments ) (insertion diameter: 5 mm). Points A and B of each circuit designate the position of the non-invasive fluid flow and cell analysis.
[0129] Figure 8 shows the assessment of fluid dynamics in the MOC in Figure 7. (a) The exemplary velocity profiles over the four stages of a total pumping cycle (frequency: 0.476 Hz) measured at two points of analysis of discrete fluid flow to sustain the pulsatile character of fluid flow (black circle = open valve, white circle = closed valve), (b) The average velocity magnitude (mm / s) and corresponding shear stress (dynes / cm2 ) plotted against the pumping frequencies (Hz) at both points. EXAMPLE
[0130] Example 1: Multi-chip device of the invention
[0131] As shown in Figure 1, the multi-organ device on chip 1 comprises a base layer 3, an organ layer 6, an organ support layer 5, an antrum layer 4 and an actuator layer 2.
[0132] As shown in Figure 6, base layer 3 is configured to provide solid support for additional layers. The base layer 3 is made of glass or transparent synthetic polymer such as, for example, polystyrene (PS), polycarbonate (PC), polysiloxane and / or polydimethylsiloxane (PDMS). The base layer 3 also comprises numerous sensors 32 and 33 that are designed and arranged to monitor and control the system. Some of these sensors 32 are configured to apply electrical stimuli to the organ equivalent of the organ layer, other sensors 33 are configured to measure system parameters in order to ensure proper functioning. The base layer 3 comprises ports from which the data acquired by the sensors can be extracted and used for other purposes, such as regulating the system.
[0133] Organ layer 6 is shown in Figure 5. Organ layer 6 is located on top of the base layer 3, is made of PDMS and is configured to comprise a multiplicity of individual organ equivalents, where each equivalent An organ growth comprises one or more sections of organ growth, each of the sections of organ growth being configured to comprise an organoid cavity to house at least one organoid of a given organ. The organ layer 6 comprises a lung equivalent 22, a small intestine equivalent 21, a spleen equivalent 23, a pancreas equivalent 24, a liver equivalent 25, a kidney equivalent 26, a bone marrow equivalent 27, an equivalent of adipose tissue 28, an equivalent of brain 29, an equivalent of testicles 30 and an equivalent of skin 31. Each organ growth section comprises a microentry and a microsaid for fluid communication between the organoid cavity of the growth section of organs and an independent circulation system 34. The independent circulation system 34 is configured to be in direct fluid communication with the organ growth sections of the organ layer 6 through the micro-entrances and microsaids of the organ growth sections. The independent circulation system 34 comprises an arteriolar transport channel that directly connects the microsaids of the organ growth sections of the lung equivalent 22 to the micro-entrances of all other organ growth sections of the organ layer 6, in order to allow the transport of fluid with high pC> 2 to said sections of organ growth; and a venular transport channel that directly connects the micro growths of the organ growth sections to the microentries of the lung equivalent 22 organ growth sections, to allow the transport of fluid with low pθ2 from the organ growth sections up to the lung equivalent 22. The independent circulation system 34 is configured so that the microsaids of the organ growth sections of small intestine, spleen and pancreas equivalents 21, 23, 24 are connected to stay in direct fluid communication between themselves and with the additional microentries of the organ growth sections of the liver equivalent 25, in order to allow fluid communication between the spleen, pancreas, small intestine and liver equivalent 23, 24, 21, 25, such that the fluid communication from the spleen, pancreas and small intestine equivalent 23, 24, 21 towards the venular transport channel of the independent circulation system 34 can occur exclusively by passing through the liver equivalent 25. The organ equivalents and the independent circulation system 34 are configured, so that the arteriolar transport channel that originates from the lung equivalent 22 displays in the flow direction:
[0134] - a first bifurcation in which a first arteriolar canal branches, serving the equivalent of small intestine, spleen and pancreas 21, 23, and 24;
[0135] - a second bifurcation in which a second arteriolar canal branches, serving the equivalent of liver 25;
[0136] - a third bifurcation in which a third arteriolar channel branches, serving the kidney equivalent 26;
[0137] a fourth bifurcation in which a fourth arteriolar channel branches, serving the equivalent of bone marrow 27;
[0138] - an optional fifth bifurcation in which a fifth arteriolar canal branches, serving the skin equivalent 31;
[0139] - a sixth bifurcation in which a sixth arteriolar canal branches, serving the equivalent of adipose tissue 28;
[0140] - a seventh bifurcation in which a seventh arteriolar canal branches, serving the equivalent of testicles 30; and
[0141] - an eighth bifurcation in which an eighth arteriolar canal branches, serving the brain equivalent 29.
[0142] The diameter of the arteriolar transport channel in the flow direction (from lung equivalent 22 towards other organ equivalents) is constantly reduced, so that the sum of the cross-sectional areas of all transport channels arteriolar arteries, including all the bifurcations at a certain distance from the lung equivalent 22 remain constant, and in which in the venular transport channel said reduction in diameter is constantly reversed in the direction of flow (from other organ equivalents in direction) to the lung equivalent 22), so that the sum of the cross-sectional areas of all venous transport channels, including all the bifurcations at a given distance from the lung equivalent, remains constant.
[0143] Each of the organ equivalents is configured to accommodate a number of organoids that is proportional to the number of organoids present, on average, in the respective organ of a mammalian organism, preferably of a human being, in which all the equivalents The number of organs in the multi-organ device on a chip is reduced in size by the same predetermined proportionality factor, for example, by a factor of 0.00001 (1 / 100,000).
[0144] Organ layer 6 is designed so that:
[0145] - the organ growth section of the liver equivalent 25 is configured to provide an organoid cavity to accommodate 5 to 15 liver organoids, where each liver organoid is a liver lobe, preferably the liver cavity organoid is configured to house 10 liver organoids;
[0146] - the organ growth section of lung equivalent 22 is configured to provide an organoid cavity to house 2,000 to 4,000 lung organoids, where each lung organoid is a pulmonary alveolus, preferably the organoid cavity be configured to host 3,000 lung organoids;
[0147] - the organ growth section of the pancreatic equivalent 24 is configured to provide an organoid cavity to accommodate 5 to 15 pancreatic organoids, where each pancreatic organoid is an islet of Langerhans, preferably the cavity of Langerhans organoid is configured to accommodate 10 pancreatic organoids;
[0148] - the organ growth section of the spleen equivalent 23 is configured to provide an organoid cavity to house 5 to 15 spleen organoids, where each spleen organoid is a white and red pulp, preferably the cavity organoid is configured to house 10 spleen organoids;
[0149] - the organ growth section of the small intestine equivalent 21 is configured to provide an organoid cavity to house 40 to 80 small intestine organoids, where each small intestine organoid is a villus, preferably the cavity organoid is configured to host 60 small intestine organoids;
[0150] - the organ growth section of the kidney equivalent 2 6 is configured to provide an organoid cavity to house 10 to 30 kidney organoids, where each kidney organoid is a nephron, preferably the organoid cavity be configured to house 20 kidney organoids;
[0151] - the organ growth section of the bone marrow equivalent 27 is configured to provide an organoid cavity to house 1,000 to 2,000 bone marrow organoids, where each bone marrow organoid is a unit formed by bone marrow, bone and cartilage, preferably the organoid cavity is configured to accommodate 1,400 bone marrow organoids;
[0152] - the organ growth section of the skin equivalent 31 is configured to provide an organoid cavity to accommodate 10 to 15 skin organoids, where each skin organoid is a skin appendage, preferably the skin cavity organoid is configured to accommodate 15 skin organoids;
[0153] - the organ growth section of the adipose tissue equivalent 28 is configured to provide an organoid cavity to house 200,000 to 300,000 adipose tissue organoids, where each adipose tissue organoid is an adipose grouping, preferably the organoid cavity is configured to house 240,000 adipose tissue organoids;
[0154] - the testicular equivalent 30 organ growth section is configured to provide an organoid cavity to house 10 to 20 testicular organoids, where each testicular organoid is a testicle follicle, preferably the testicular cavity organoid is configured to accommodate 15 testicular organoids; and
[0155] - the organ growth section of the brain equivalent 29 is configured to provide an organoid cavity to house 150 to 250 brain organoids, where each brain organoid is a column of the cerebral cortex, preferably the cavity organoid is configured to host 200 brain organoids. In Table 2 below, the parameters are provided for an organ layer 6 made of a layer of PDMS with a height of 3mm. TABLE 2:


[0156] The organ support layer 5 is arranged between the organ layer 6 and the anthers layer 4, see Figure 4. The organ support layer 5 is configured to seal and / or stabilize the organ layer 6 , such that for the selected organ equivalents, fluid communication with the antrum layer 4 is maintained. The organ support layer 5 is provided as a 200 µm thick layer. The organ support layer 5 is made of a material that comprises or consists of polycarbonate (PC). In areas, where the organ support layer 5 covers one of the organ equivalents 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, the organ support layer 5 is configured to allow fluid communication between the organ layer 6 and the antrum layer 4. In particular, in areas where the organ support layer 5 covers an organ equivalent that has an excretory function and / or produces a considerable amount of interstitial fluid, such as kidney 26, liver 25, spleen 23 and small intestine 21, this fluid communication can be achieved, for example, by providing pores within the organ support layer 5, preferably by providing pores with an average diameter of 5 to 7 pm. Alternatively or in addition, in an area that allows fluid communication between the organ layer 6 and the antrum layer 4, the thickness of the organ support layer 5 can be reduced to an average thickness of 5 to 15 pm preferably at 10 pm.
[0157] The antrum layer 4 is represented in Figure 3 and is configured to comprise a multiplicity of cavities and tubes arranged to be in fluid communication with selected organ equivalents or organ growth sections of the organ layer 6, in order to allow fluid exchange between cavities and organ growth sections. The antrum layer 4 comprises or consists of PDMS. The dens layer 4 is configured to comprise:
[0158] a cavity that is located on top of the small intestine equivalent 21 and is in fluid communication with the small intestine equivalent 21 and a nutrition reservoir 18, so that the small intestine equivalent 21 can be supplied with nutrients a from the nutrition reservoir 18;
[0159] a cavity that is located on top of the small intestine equivalent 21 and is in fluid communication with the small intestine equivalent 21 and a stool reservoir 19, so that the material excreted from the small intestine equivalent 21 can be transported to the stool reservoir 19;
[0160] a cavity that is located on top of the liver equivalent 25 and is in fluid communication with the liver equivalent 25 and the cavity that is located on top of the small intestine equivalent 21, so that the material excreted from the liver equivalent 25 can be transported to the cavity which is located on top of the small intestine equivalent 21; and
[0161] a cavity that is located on top of the kidney equivalent 2 6 and is in fluid communication with the kidney equivalent 26 and a urine reservoir 20, so that the urine reservoir 20 receives the excreted from the equivalent of kidney 26. The nutrition reservoir 18, the stool reservoir 19 and the urine reservoir 20 are integral parts of the antrum layer 4 and are preferably configured to be externally accessible.
[0162] Actuator layer 2 is configured to comprise a plurality of actuators arranged and configured to regulate a pressure force applicable in selected organ equivalents, in the independent circulation system and / or part thereof, see Figure 2. A actuator layer is made of polycarbonate.
[0163] The actuator layer 2 comprises:
[0164] 3 pressure-based actuators 10 that act on the independent circulation system 34 to allow the movement of directed fluid, in order to imitate the heartbeat;
[0165] 3 peristaltic based actuators 11 that act on the antrum layer 4, in such a way as to allow the directed movement, in order to imitate the peristaltic bowel movement;
[0166] an actuator 12 acting on the lung equivalent 22 to allow air to flow, in order to imitate the breathing of air;
[0167] an actuator 17 that acts on the bone marrow equivalent 27 to allow regulated compression, in order to imitate bone compression;
[0168] 8 actuators 14 that act on the arteriolar transport channel of the independent circulation system 34, in order to imitate arteriolar constriction;
[0169] 1 actuator 13 acting on the equivalent of 25 to allow the movement of directed fluid, in order to dissipate the bile from the equivalent of liver 25;
[0170] 1 actuator 13 acting on the kidney equivalent 26 to allow the movement of directed fluid, in order to dissipate the urine from the kidney equivalent 26 to the kidney reservoir 20; and
[0171] 1 actuator 13 that acts on the spleen equivalent 23.
[0172] In addition, the actuator layer comprises a port 16 to access the nutrition reservoir 18, a port 16 to access the stool reservoir, a port 16 to access the urine reservoir 20 and a port 16 to access the channel venular transport system of the independent circulation system 34.
[0173] Example 2: Integration of biological vasculature in a multi-organ device on chip of the invention
[0174] The aim is to emulate the transport part of the human vasculature - heart and vessels - on a chip, in order to demonstrate the feasibility of establishing an equivalent functional vasculature on a multi-organ device on a chip or human on a chip of the invention. A micro pump on the chip to support constant fluid flow over the long term through a microchannel system fully covered by primary human dermal microvascular endothelial cells (HDMECs) has been established. In contrast to the majority of existing microsystems to investigate the effects of shear stress in ECs that apply constant shear stress in the range of 1 to 40 dynes / cm2, we aim at pulsatile shear stress with reversal patterns that were previously used in different experimental configurations. The microvascular transport system presented in this work interconnects two separate compartments that are designed for the integration of individual organ equivalents with a biomass capacity of up to 100 mg each. Special inserts have been manufactured that support vessel branching and diameter reduction in the areas of individual organ culture compartments to support posterior organ vascularization. The rapid prototyping that applies soft lithography and replicates the PDMS molding allows for flexible adjustment of the design in relation to the number of organs and their specific arrangement, always adhering to the same standard chip base format. In addition, two important resources were implemented to overcome the technical manipulation restrictions of most existing microfluidic systems: i) the microsystem's incubator independent operation was ensured by a tempered chip support, and ii) microscopic access to any and all areas of the circuit channels was guaranteed, allowing video microscopy in real time. MATERIALS AND METHODS DEVICE DESIGN AND MANUFACTURE
[0175] A multi-organ microfluidic device (MOC) was designed and manufactured that accommodates two separate microvascular circuits, each operated by a separate peristaltic micro pump on the chip. Figure 7 illustrates the system at a glance. The cover plate accommodates six air pressure accessories and four inserts that form compartments of 300 pl, each, for changing media and later integrating organ equivalents. The MOC support supports the constant tempering of the MOC at 37 ° C (Figure 7a). Peristaltic micro pumps were installed in (Figure 7b). Micropump software control facilitates both fluid flow clockwise and counterclockwise. The flow rate (Q) can be varied by adjusting the pumping frequency. Each microchannel circuit (Figure 7c) comprises a total volume of 10 pl, while the two individual insertion-based compartments for additional organ equivalent culture each have a volumetric capacity of up to 300 pl. Standard soft lithography and replica molding of PDMS (Sylgard 184, Dow Corning, Midland, MI, USA) was applied for the manufacture of MOC. In summary, a master mold was made by connecting a silicon sheet to a glass sheet. Photosensitive resin was applied to the silicon sheet and standardized using a photomask and UV light. Subsequently, the unprotected silicon regions were etched and the photosensitive resin was removed. To manufacture the microsystem, the cover plate (CP) was treated with a silicon rubber additive (WACKER® PRIMER G 790; Wacker Chemie, Munich, Germany) at 80 ° C for 20 min. The prepared cover plate was connected to the master mold (channel height 100 pm, width 500 pm) and PDMS (v / v 10: 1 ratio between PDMS and curing agent) was injected into this casting station. The preparation was incubated at 80 ° C for at least 60 min. Teflon threads were used to generate the four PDMS-free culture compartments and the six 500 pm thick PDMS membranes that make up the two micro pumps on the chip (three membranes per micro pump). The fused PDMS portion is impermeable to fluids in the CP. Subsequently, the PDMS portion attached to the CP is irreversibly linked by low pressure plasma oxidation treatment (Femto; Diener, Ebhausen, Germany) to a microscope slide. Sterile medium is immediately injected into the two microvascular circuits to avoid surface neutralization. CHARACTERIZATION OF FLUIDS DYNAMICS
[0176] Non-invasive microparticulate image velocimetry (pPIV) was applied to characterize the fluid flow at points A and B (see Figure 7c) of the microfluidic circuit. In summary, an inverted Zeiss Primovert microscope (Zeiss, Jena, Germany) with a standard halogen lamp as a continuous light source coupled to a CMOS camera (Baumer Optronic HXC40, resolution: 2048 x 2048 pixel, interface: CameraLink; Baumer Optronic , Radeberg, Germany) was used to track the movement of 15 pm polystyrene beads (4 * 104 g / ml; Life Technologies, Darmstadt, Germany) over an exposure time of 4 ps per single image. A low magnification (4x) was chosen to limit the shift between two frames to approximately 50 pixels (1 pixel = 3.2 pm). Focus z was defined in the center of the fluidic channel at the respective point (50 pm above the glass slide) to detect the peak speed. A interrogation window in the center of the fluidic channel (1024 pixel x 100 pixel, 3.28 mm x 0.32 mm) was observed reaching frame rates of up to 3,200 fps. Finally, the correlation was performed with a software program (Fraunhofer IWS, Dresden, Germany) that analyzes a stack of images of 15,000 frames, calculating the maximum correlation for the x component of the displacement in a specified area. The calculated values from the following five tables are measured to minimize artifacts. The following pump configuration was used for all experiments: pressure - 0.05 mPa (500 mbar); vacuum - 0.052 mPa (520 mbar); and air flow - 1.5 1 / min. at 0.035 mPa (350 mbar). Time dependence was measured at two different locations (A + B) on the chip, as shown in Figure 7c.
[0177] As the laminar flow has its maximum speed (vmax) at the center of the microchannel, the average shear stress (x) can be calculated using the following equation:

[0178] where Vm, ^ is the magnitude of the velocity measured at the center of the channel, p is the dynamic viscosity (calculated as 1 mPa / s) and h is the channel height (100 pm). INSULATION AND CELL CULTURE
[0179] HDMECs were isolated from the human foreskin obtained with informed consent and ethical approval for pediatric surgery after routine circumcisions of young donors. All skin samples used for cell isolation were processed one day after removal. Before isolation, the foreskins were cleaned in 80% ethanol for 30 s and washed with phosphate buffered saline (PBS; PAA, Coelbe, Germany). The skin ring was opened and subcutaneous tissue was removed. In order to separate the thin epidermal layer from the dermis, the prepared foreskin was incubated in 5 mg / ml of dispase II solution (Sigma-Aldrich, Schnelldorf, Germany) at 4 ° C for 15 to 18 h. The dermis was cut into small pieces and then incubated with 4 mg / ml of Collagenase NB 4 solution (Serva, Heidelberg, Germany) at 37 ° C for 75 min. The mixture was passed through a 70 pm nylon filter and centrifuged at 300 g for 5 min. The resulting cell pellet was resuspended in Endothelial Cell Growth Medium -Endothelial Cell Growth Medium MV2 (ECGM-MV2; PromoCell, Heidelberg, Germany) supplemented with Supplement-Pack MV2 (PromoCell, Heidelberg, Germany), 1% PS and fungizona to 0.05%. The cells were inoculated in a T-75 flask and cultured in 5% CO2 at 37 ° C. The medium was replaced one day after inoculation. Two to five days after the initial inoculation, HDMECs were purified by associated magnetic cell separation (MACS). The cells were harvested using Trypsin / EDTA 0.05% (0.5 mg / ml) (PAA, Coelbe, Germany) and a positive selection for ECs using the CD31 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. ECGM-MV2 supplemented with Supplement-Pack MV2 and 1% P-S (complete ECGM-MV2) was used to elute the cells from the column. A purity control of the isolated cells was performed directly after each MACS by FACS analysis. Where necessary, the separation cycles were repeated until> 90% of the cells were CD31 positive. The purified HDMECs were either frozen for later use or immediately used after expansion. HDMECs were expanded in T-75 flasks with complete ECGM-MV2 up to 70 to 90% confluence in a three-day feeding regime. Cells between the 3rd and 8th pass were used in all studies to ensure that the cells maintain their primary endothelial characteristics. HDMEC CULTURE ON DIFFERENT SURFACES OF TREATED PDMS
[0180] HDMECs were inoculated at a density of 104 cells / cm2 on three types of PDMS surfaces: untreated, coated with 100 pg / ml of fibronectin (Sigma Aldrich, Schnelldorf, Germany) and treated with air plasma. Air plasma treatment was carried out in a low pressure plasma system (50W) at a frequency of 13.56 MHz for 30 s. After 48 h of culture, the growth behavior and morphology of the cells were compared by light microscopy. INOCULATION AND EC CULTURE AT MOC
[0181] Before inoculation, each MOC was washed with medium and statically incubated for 3 days in 5% CO2 at 37 ° C. HDMECs were harvested from expansion cultures using 0.05% Trypsin / EDTA (PAA, Coelbe, Germany). The cell suspension was concentrated by centrifugation and cell counts were performed using the ViCell viability counter (Beckman Coulter, Fullerton, CA, USA). Cell viability was> 90% for all experiments. The centrifuged cells were resuspended with complete ECGM-MV2 at a final concentration of 2 x 10 7 cells / ml. Subsequently, the cell suspension was transferred to a 1 ml syringe. The cells were injected through one of the two compartments of each circuit. The syringe was connected to a female x male ^ -28 Luer adapter (IDEX Health & Science, Wertheim-Mondfeld, Germany). The air was pushed out of that slot, which was then threaded to a special thread adapter (MOC) (MicCell MOC-I 1/4 "- 28 UNF x MIO Fitting (PEEK); Gesim, Dresden, Germany). empty syringe was connected in the same way to the second compartment, then the cell infusion in both circuits of the device was incubated in 5% CO2 at 37 ° C under static conditions for 3 h to allow the cells to attach to the walls A quantity of 300 pl of fresh medium was added to each compartment and then purged through the PDMS channels using the micro pump on the chip of each circuit, A frequency of 0.476 Hz was applied to each microvascular circuit of the MOCs for operation dynamic dynamics For MOC cultures under static conditions, the channels were washed with fresh medium for 5 min, using a difference in hydrostatic pressure between the inlet and outlet compartments.
[0182] An amount of 150 pl of medium per compartment was replaced every 1 to 2 days in both dynamic and static MOC systems, and cell growth and viability were monitored by light microscopy inspection at points A and B of each circulation (Figure 7c). In addition, cell viability was determined with a Calcein AM assay. A 5 pg / ml solution of CellTrace calcein red-orange AM (Life Technologies, Darmstadt, Germany) was added to both compartments of each circuit of an MOC at a volume of 100 pl. The MOC was pumped for 2 min. and then incubated under static conditions in 5% CO2 at 37 ° C for 30 min. Subsequently, the microchannels were washed twice with medium, replacing the medium in the compartment inserts with fresh medium. The images were obtained using fluorescence microscopy (BZ9000; Keyence, Neu-Isenburg, Germany). The regular MOC experiments were completed after 4 days (10 dynamic MOCs and 12 static MOCs). Individual MOCs were operated in the same mode for 7, 14 and 32 days to obtain the first indications about the long-term performance of microvascular circuits. In order to evaluate the possibility of replacing the CO2 incubator for MOC operation with the MOC support shown in Figure 7a, 9 MOC experiments (7 dynamic MOCs and 2 static MOCs) were performed using the support exclusively for operating times of up to 7 days. CHARACTERIZATION OF EC METABOLISM IN MOC
[0183] The glucose concentration of the medium was measured, according to the manufacturer's instructions, using the Stanbio Glucose Procedure LiquiColor® (Oxidase) nt 1070 (Stanbio Laboratory, Boerne, TX, USA). Briefly, 99 µl of the reagent was added to a 96-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) preheated to 37 ° C and 1 µl of sample medium was added. After another 5 min. After incubation at 37 ° C, the glucose concentration was quantified in a microplate reader (FLUOstar Omega; BMG Labtech, Ortenberg, Germany) at 500 nm, using water as a reference.
[0184] The lactate concentration of the medium was measured, according to the manufacturer's instructions, using the LOD-PAP method (Diaglobal, Berlin, Germany). Briefly, 99 µl of the reagent was mixed with 1 µl of the medium sample in a 96-well multi-well plate and absorbance was measured at 520 nm in a microplate reader, using water as a reference. ECS IMMUNOFLUORESCENCE COLORING INSIDE THE MOC
[0185] After 4 days in culture, the ECs were fixed inside the microvascular circuit with cold acetone at -20 ° C for 10 min., Washed twice with PBS for 5 min., Incubated with 10% goat serum in PBS for another 20 min. and then incubated with the primary mouse anti-human CD31 antibody (1: 500; 7.1 mg / ml; DRFZ, Berlin, Germany), at room temperature (RT) for 2 h. Subsequently, the circuits were washed twice with PBS followed by incubation with Alexa Fluor 594 secondary goat anti-mouse antibody (1: 200, 2 mg / ml; Life Technologies, Darmstadt, Germany), in the dark at RT for 40 min . After washing, vWF-FITC sheep anti-human antibody (1:50, 10 mg / ml; Abeam, Cambridge, UK) was added and incubated at RT for 2 h. The cores were contrasted with Hoechst 33342 (1: 1,000, 10 mg / ml; Life Technologies, Darmstadt, Germany). Another immunofluorescence staining with the primary anti-human mouse antibody VE-Cadherin (1: 100, 0.2 mg / ml; Santa Cruz Biotechnology, Heidelberg, Germany) was performed: ECs were fixed with 4% PFA for 10 min ., washed twice with PBS for 5 min. and permeabilized with 0.2% Triton X-100 for 5 min. After washing twice with PBS, staining for primary and secondary antibodies was performed, as described above. MOC cultures were stained for filamentous actin with phalloidin Oregon Green® 488 (Life Technologies, Darmstadt, Germany), according to the manufacturer's instructions, in combination with VE-Cadherin.
[0186] Each solvent was added to the MOC compartment inserts and pumped for 1 to 2 min. for even distribution. The images were taken either by standard fluorescence microscopy or by two-photon microscopy (TriMScope II; LaVision BioTec, Bielefeld, Germany). All microvascular channels were imaged through their microscope slide wall. The 3D images were reconstructed from the collected image stack, using the Imaris software (Bitplane, Zurich, Switzerland). CHARACTERIZATION OF SHEAR STRESS EFFECTS
[0187] HDMECs images colored by immunofluorescence were taken at points A and B of each microvascular circuit (Figure 7c) to monitor flow-induced morphological changes using a standard fluorescence microscope. The HDMEC membranes in the images were manually retracted for automatic EC recognition. A connected area recognition algorithm was used to identify the CEs and calculate the corresponding perimeter, cell size, center of gravity and orientation (main geometric axis of the second unweighted spatial moment) of each CE. A non-dimensional shape index (SI) parameter was used to quantify cell elongation which is defined as:

[0188] where A is the cell area and P is the cell perimeter. SI ranges from 0 to 1, where 0 is a straight line and 1 is a perfect circle. Additionally, the orientation angle was measured to quantify the alignment of HDMECs in the flow direction, where 0o is a geometric axis of the cell perfectly aligned with the flow direction and 90 ° is a cell aligned orthogonal to the flow direction. The source code was implemented in Matlab (Math Works, Ismaning, Germany). SI and cell orientation angle for at least 200 cells per image were used for analysis. GENERATION OF MICROChannels STRUCTURED BY FEMTHOSECOND LASER ABLATION
[0189] A femtosecond laser guided by CAM (Tecidosurgeon; Rowiak, Hannover, Germany) with a wavelength of 1,030 nm (pulse energy = 120 nJ), a pulse duration of 400 fs and a repetition rate of 10 MHz was used by Rowiak to generate microchannels as low as 40 x 40 pm2 within the PDMS material. The channel design was chosen to reveal minimum achievable diameters and to allow the flow of continuous medium through each of the branched channels. HDMECs were inoculated into pre-structured microchannels inside a PDMS mold and colored with the Calcein AM assay (Life Technologies, Darmstadt, Germany) after 1 day of culture. Subsequently, the PDMS mold was placed in the tissue compartment of the MOC. The images were acquired by standard fluorescence microscopy. RESULTS AND DISCUSSION FLUID DYNAMICS ASSESSMENT
[0190] pPIV has been successfully applied to exemplify the fluid flow profiles at different points of the MOC circuits in an exemplary manner (Figure 8a). Full microscopic access to each and every area of the MOC facilitates detailed analyzes of several other regions of the MOC and variable MOC designs in the future. The potential for optimal microscopic analysis and the operational mode of the peristaltic micro-pump membranes of a microfluidic MOC circuit filled with human red blood cells from a bottom-up angle view could be demonstrated. A robust peristaltic micro pump on the chip was integrated into a microvascular circuit capable of circulating flawless media in sterile conditions for weeks and months at a flow rate ranging from 7 pl / min. (lowest frequency) at 70 pl / min. (highest frequency). The frequency of pulsatile operation can be increased to 2.4 Hz, which corresponds to a high, however, still a physiological cardiac activity of 144 beats per minute in humans. At this frequency, the shear stress measured at points A and B of the microvascular circuit reaches approximately 25 dyn / cm2 (Figure 8b), which is a physiological shear stress at the upper end of the microvasculature scale. The average speed increased almost linearly with the pumping frequency. The pumping frequency used in the present experiments (0.476 Hz) corresponds to less than 30 "heartbeats" per minute (approximately half the physiological value of an adult at rest) to prevent loss of EC during the initial phases of surface coverage. This phase somehow resembles elements of wound healing in vivo. As illustrated in Figure 8a, the oscillatory shear stress - another desired physiological characteristic - could be implemented in MOC operation through the design of a micro pump. The waveform of such an oscillation at a given local position in the microvasculature depends on the frequency of pumping and the particular design of an MOC. Certain waveforms in humans have been associated with a certain susceptibility to the disease. This implies an additional assessment of the MOC platform for research into such pathological processes of the human cardiovascular system. EC ORIGIN, ISOLATION AND CULTURE
[0191] As of this date, most of the shear stress testing of human EC in microfluidic systems is performed on human umbilical vein endothelial cells (HUVECs) due to easy access to large numbers of cells and their high phenotypic elasticity. The hypothesis is that HDMECs have at least the same phenotype elasticity, but with a greater potential for rapid in vitro adaptation to change the local environment. Kamm and colleagues, for example, were able to grow HDMECs in a vertical microchannel plane and monitor capillary morphogenesis in collagen gels in the lateral plane. Unlike all other organs in the body, the skin in vertebrates needs to adapt quickly to changes in external temperatures due to immediate contraction and relaxation of blood vessels. In addition, the skin of carnivores is the organ with the most evident exposure to repeated injuries, due to its aggressive lifestyle. These two factors adopted in conjunction with human longevity must have HDMECs selected for unmatched elasticity of their phenotype and an exclusive potential for neoangiogenesis. Both factors are extremely important for the establishment of a functional in vitro equivalent to human vasculature. The capacity for neoangiogenesis, in particular, is crucial for the establishment of the second part of human vasculature - the capillary network of organ equivalents - in MOCs. The latest developments in molecular mechanisms of angiogenesis support the essential function of the local environment that includes shear stress. Several techniques have been described to isolate human ECs from different tissues. The isolation of magnetic beads from ECs after tissue digestion with MicroBead CD31 (PECAM-1) was applied, as it is constitutively expressed on the surface of virtually all types of ECs and is not present in any other cell type except of the leukocyte population. In particular, it is not expressed in dermal fibroblasts and smooth muscle cells. Morphology and several specific endothelial markers were examined to confirm the endothelial origin. The isolated HDMECs showed a morphology similar to a round stone in phase contrast and were positive for the specific endothelial marker CD31, VE-Caderina and Von Willebrand Factor (vWF). Staining for 5B5, a specific fibroblast marker, and smooth muscle a-actin, a specific smooth muscle cell marker, did not show growth of other cell types. In addition, HDMECs showed an absorption of Alexa594-labeled ac-LDL after 4 h of exposure. A mixture of dermal fibroblasts and smooth muscle cells served as the control for all stains (data not shown). HDMECs could be grown for up to eight passages without significant changes in morphology and marker expression. The present data indicate that this method is a robust and reproducible way to isolate CD-31 positive HDMECs from human foreskin. The average number of HDMECs that fully covers two microvascular circuits of an MOC was calculated to be in the range of 2 * 105 cells. On average, 1 * 107 primary HDMECs after separation can be prepared from a single human foreskin. A cell amplification factor of ~ 3,000 occurs between the initial inoculation and the 7-8 passage of HDMEC culture, thereby allowing the provision of 3 * 1010 cells from a single foreskin. Theoretically, this is equivalent to MOCs loaded with 5000 cells (two circuits per MOC). Optimization of preparation and propagation should be envisaged to further increase the yield of HDMEC. ESTABLISHMENT OF STABLE MICROVASCULAR CIRCUITS IN THE MOC
[0192] A pilot comparison study between the attachment of EC to surfaces of PDMS coated with fibronectin and treated with air plasma revealed at least an equal adhesion of HDMECs to PDMS in static cultures. In addition, plasma treatment has long been recognized as a viable technique for increasing the hydrophilicity of PDMS microchannels. Therefore, air plasma treatment was finally selected for surface activation during the manufacture of MOCs. Fibronectin is widely used as a coating material for the fixation and cultivation of EC in PDMS-based microfluidic devices. Although it is easy to handle on a laboratory research scale, a fibronectin coating procedure can hinder the speed and sterility of a large-scale, later industrial scale at high efficiency. The PDMS-based treatment with air plasma is a reproducible, fast and scalable method for preparing PDMS-based microdevices for efficient EC fixation.
[0193] Subsequently, a microvascular circuit comprising a peristaltic micro pump, two compartments for posterior organ equivalent cultures and connecting microchannels, fully covered with a functional HDMEC monolayer, was established in a pulsatile medium flow within 4 days of culture. Total circuit coverage with a human EC line was previously demonstrated elsewhere. Here, attention is directed to the rapid establishment of such a miniaturized human cardiovascular transport system based on primary HDMECs. In addition, daily monitoring of the metabolic activity of ECs was performed. The increased metabolic activity within the first days of fixation and surface coverage can be explained by the increased motility and proliferation of cells. A system friction rate of 50% in the early stages of experiments, caused mainly by contamination, was effectively reduced to about 20% during routine use of MOC in the laboratory. The total quality management systems installed in any and all industrial in vitro test laboratories must completely eliminate this attrition rate in a "research laboratory".
[0194] The ECs maintained adherence to the channel walls and remained viable, as observed by staining with Calcein AM red orange. In addition, the cells were tested for the absorption of Alexa594-ac-LDL. Since no further changes in endothelial morphology were observed after 4 days of culture, the experiments were stopped for analysis. Detailed immunofluorescence analyzes of the leakproof EC layer on the 4th day revealed remarkable vascular viability and functionality. The HDMECs that form the microvascular circuit were positive for CD31, vWF and VE-Caderina. In addition, HDMECs were able to cover all channel walls, forming a fluid-tight layer. Such stable microvascular circuits, on the one hand, can act as biological membranes preventing the transfer of molecules to the surrounding PDMS slides recently described. On the other hand, these can serve as networks of hemocompatible vessels for the circulation of whole blood, preventing blood clotting. IMPACT OF SHEAR STRESS
[0195] When exposed to laminar shear stress, ECs align and align their microfilaments in the flow direction. In vivo ECs in different locations are exposed to different types of flow, such as laminar, pulsatile and turbulent; the latter, for example, has been described to increase renewal. The elongation and flow alignment induced by physiological shear stress were evidenced in the present MOC cultures by plotting the SI and orientation angle of HDMECs in the microvascular circuits generated in pulsatile flow (Q = 40.56 pl / min., T = 5.17 dyn / cm2), against those generated under static culture conditions. A change in the distribution of filamentous actin (F-actin) was observed between static and dynamic cultivation. ECs under static conditions are polygonal and F-actin is organized as a dense band on the periphery of the cell; meanwhile at a shear stress of about 5 dyn / cm2, F-actin creates bundles of stress fibers. SI and the orientation angle differ significantly between the static and dynamic cultivation of ECs in the MOC, and are in the range of previous discoveries for HDMECs in microfluidic devices.
[0196] Finally, excellent cell viability was also observed at points of analysis in a limited number of long-term indicative experiments with microvascular MOCs during 14 days (n = 4) of culture and in a first single microvascular MOC during 32 days (data not shown). CONCLUSION
[0197] The present hypothesis is that the circulation of blood through EC line microcircuits that connect the organ equivalents to each other in a physiological order is the first and the fundamental requirement to fully emulate the human homeostasis of the organism on a microscale. Therefore, soft lithography, replica molding and two-photon laser ablation techniques are successfully applied here to establish an incubator-independent microvascular circulation system that simulates the transport function of the human cardiovascular system on a microscale. This is arranged in a two-layer glass-PDMS chip, the area of a standard microscopic slide, with channel heights of 100 pm and a total height of 3 mm. Two separate cylindrical tissue culture inserts, each area of a standard 96-well plate well, are positioned in the microvascular circuit. A robust 4-day procedure that applies pulsatile shear stress has been established to cover all fluid contact surfaces of the system with a functional hermetically sealed layer of HDMECs. Contrary to the growth of HDMEC in a vertical plane described in the literature, the total coverage of the present microvascular system with human ECs for the first time makes possible the biological hemocompatibility of such a microvascular system. The chip layout reduces the circulation fluid volume in the microvascular transport system to up to 10 pl, at least two magnitudes lower than the circulation volume applied to any of the systems operated with external pumps and reservoirs. The most important tissue culture inserts, each with a maximum volume of 300 pl, will allow the exact adjustment of fluid to physiological tissue ratios since individual organ equivalents are established in the next stage of development. The manufacturing technique is convenient and versatile, and design changes can be implemented in design response times for devices of only 2 to 3 months. The alignment and elongation of ECs in the flow direction, meticulously demonstrated in vitro, were monitored in greater detail through time series video microscopy. Other drawings of fluidic microchannel were also efficiently covered with HDMECs in the laboratories by the described technique. The first indications were generated that once a microvascular circulation system is established, it eventually has a useful life of at least 32 days. NUMERICAL REFERENCE LIST: 1 multi-organ device on chip 2 layer of actuator 3 base layer 4 layer of holes 5 6 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 organ support layer pressure-based actuator (heart) actuator peristaltic based actuator ** airflow actuator actuators arteriolar constriction actuator bone compression actuator nutrition reservoir stool reservoir equivalent small intestine equivalent urine reservoir equivalent spleen equivalent pancreas equivalent liver equivalent kidney equivalent bone marrow equivalent fat equivalent brain equivalent testicle equivalent skin equivalent electrical sensor sensor independent circulation system

权利要求:
Claims (15)
[0001]
1. MULTIPLE ORGANS IN CHIP DEVICE (1), characterized by comprising a base layer (3); an organ layer (6) arranged on the base layer; a layer of holes (4) disposed on the organ layer; and an actuator layer (2); wherein the base layer (3) is configured to provide solid support for the additional layers; the organ layer (6) is configured to comprise a multiplicity of individual organ equivalents, each organ equivalent comprising one or more sections of organ growth, each of the sections of organ growth being configured to comprise a organoid cavity to house at least one organoid of an organ and comprise a microentry and a microsaid for fluid communication between the organoid cavity of the organ growth section and an independent circulation system (34), in which the organ layer ( 6) comprises at least one organ equivalent configured to represent the lung, small intestine, spleen, pancreas, liver, kidney and bone marrow, respectively, and an independent circulation system (34) configured to be in direct fluid communication with the organ growth sections of the organ layer (6) through the microentry and microsaids of the organ growth sections; the antrum layer (4) is configured to comprise a multiplicity of cavities and tubes arranged to be in fluid communication with selected organ equivalents or organ growth sections to allow fluid exchange between the cavities and growth sections of organs; and the actuator layer (2) is configured to comprise a plurality of actuators arranged and configured to regulate a pressure force applicable to a selected organ equivalent, in the independent circulation system (34) and / or part thereof.
[0002]
2. MULTIPLE CHIP ORGAN DEVICE, according to claim 1, characterized in that the base layer (3) is made of a transparent material, preferably the base layer is made of a material that comprises or consists of glass or a transparent synthetic polymer.
[0003]
3. MULTIPLE ORGAN DEVICES IN CHIP, according to claim 1 or 2, characterized in that the base layer (3) comprises one or more sensors (33) configured to measure signals emitted from and / or transmit signals to one or more of the organ equivalents, sections of organ growth and / or from the independent circulation system (34).
[0004]
4. MULTIPLE ORGAN DEVICES IN CHIP, according to any one of the preceding claims, characterized by the independent circulation system (34) comprising an arteriolar transport channel that directly connects the microsaids of the organ growth sections of the lung equivalents (22 ) to the micro-entrances of all organ growth sections of the organ layer (6) in order to allow the transport of fluid with high pθ2 to organ growth sections; and a venular transport channel that directly connects the microsaids of the organ growth sections to the microentries of the lung equivalent organ growth sections (22) to allow the transport of fluid with low pCh from the growth sections of organs for the lung equivalent (22).
[0005]
5. MULTIPLE ORGAN DEVICES IN CHIP, according to claim 4, characterized in that the independent circulation system (34) is configured, so that the microsaids of the organ growth sections equivalent to small intestine, spleen and pancreas (21 , 23, 24) are connected to be in direct fluid communication with each other and to the additional micro-entrances of the organ growth sections of the liver equivalent (25) in order to allow fluid communication between the spleen, pancreas equivalent, small intestine and liver (23, 24, 21, 25), in such a way that fluid communication from the spleen, pancreas and small intestine towards the venular transport channel of the independent circulation system (34) can occur only through the pass through the liver equivalent (25).
[0006]
6. MULTIPLE ORGAN DEVICES IN CHIP, according to any one of the preceding claims, characterized in that the organ layer (6) additionally comprises organ equivalents, preferably the organ layer comprises equivalents of skin organs, testicles, brain and / or adipose tissue (31, 30, 29, 28).
[0007]
7. MULTIPLE CHIP ORGAN DEVICE, according to any one of the preceding claims, characterized in that it additionally comprises an organ support layer (5) disposed between the organ layer (6) and the dens layer (4), in that the organ support layer (5) is configured to seal and / or stabilize the organ layer (6), such that for the selected organ equivalents, fluid communication with the antrum layer (4) is maintained .
[0008]
8. MULTIPLE ORGAN DEVICES IN CHIP, according to any one of the preceding claims, characterized by the layer of dens (4) being configured to understand: a cavity that is located on top of the small intestine equivalent (21) and is in communication fluid with the small intestine equivalent (21) and a nutrition reservoir (18), so that the small intestine equivalent (21) can be supplied with nutrients from the nutrition reservoir (18); a cavity that is located on top of the small intestine equivalent (21) and is in fluid communication with the small intestine equivalent (21) and a stool reservoir (19), so that the material excreted from the intestine equivalent slender (21) can be transported to the stool reservoir (19); a cavity that is located on top of the liver equivalent (25) and is in fluid communication with the liver equivalent (25) and the cavity that is located on top of the small intestine equivalent (21), so that the material excreted from the liver equivalent (25) it can be transported to the cavity that is located on top of the small intestine equivalent (21); and a cavity that is located on top of the kidney equivalent (26) and is in fluid communication with the kidney equivalent and a urine reservoir (20), so that the urine reservoir receives the excreted material from the equivalent kidney.
[0009]
9. MULTIPLE CHIP ORGAN DEVICES, according to any of the preceding claims, characterized by the actuator layer (2) comprising: one or more actuators (10) acting on the independent circulation system (34) to allow movement directed fluid in order to imitate the heartbeat; one or more actuators (11) acting on the antrum layer (4) to allow directed movement, in order to imitate the peristaltic bowel movement; one or more actuators (12) that act on the lung equivalent (22) to allow air to flow in order to mimic breathing; one or more actuators (17) that act on the bone marrow equivalent (27) to allow regulated compression, in order to imitate bone compression; one or more actuators (14) acting on the arteriolar transport channel of the independent circulation system (34), in order to imitate arteriolar constriction; one or more actuators (13) acting on the liver equivalent (25) to allow the movement of directed fluid, in order to dissipate the bile from the liver equivalent (25); and one or more actuators (13) that act on the antrum layer (4) to allow the movement of directed fluid, in order to dissipate the urine from the kidney equivalent (26).
[0010]
10. MULTIPLE ORGAN DEVICES IN CHIP, according to any one of the preceding claims, characterized in that the organ layer (6) is designed so that: a section of organ growth of the liver equivalent (25) is configured to provide an organoid cavity to house 5 to 15 liver organoids, wherein each liver organoid is a liver lobe, preferably the organoid cavity is configured to accommodate 10 liver organoids; an organ growth section of the lung equivalent (22) is configured to provide an organoid cavity to house 2,000 to 4,000 lung organoids, where each lung organoid is a pulmonary alveolus, preferably the organoid cavity is configured to house 3,000 lung organoids; an organ growth section of the pancreas equivalent (24) is configured to provide an organoid cavity to house 5 to 15 pancreatic organoids, where each pancreatic organoid is an islet of Langerhans, preferably the organoid cavity is configured to house 10 pancreas organoids; an organ growth section of the spleen equivalent (23) is configured to provide an organoid cavity to house 5 to 15 spleen organoids, where each spleen organoid is a white and red pulp, preferably the organoid cavity be configured to accommodate 10 spleen organoids; an organ growth section of the small intestine equivalent (21) is configured to provide an organoid cavity to accommodate 40 to 80 small intestine organoids, where each small intestine organoid is a villus, preferably the organoid cavity be configured to house 60 small intestine organoids; a kidney equivalent organ growth section (26) is configured to provide an organoid cavity to house 10 to 30 kidney organoids, where each kidney organoid is a nephron, preferably the organoid cavity is configured to house 20 kidney organoids; and an organ growth section of the bone marrow equivalent (27) is configured to provide an organoid cavity to house 1,000 to 2,000 bone marrow organoids, where each bone marrow organoid is a unit made up of bone marrow, bone and cartilage, preferably the organoid cavity is configured to accommodate 1,400 bone marrow organoids.
[0011]
11. MULTIPLE ORGAN DEVICES IN CHIP, according to any one of the preceding claims, characterized in that the organ layer (6) is designed so that: an organ growth section of the skin equivalent (31) is configured to provide an organoid cavity to house 10 to 15 skin organoids, where each skin organoid is a skin appendage, preferably the organoid cavity is configured to accommodate 15 skin organoids; an organ growth section of the adipose tissue equivalent (28) is configured to provide an organoid cavity to house 200,000 to 300,000 adipose tissue organoids, where each adipose tissue organoid is an adipose grouping, preferably the organoid is configured to accommodate 240,000 fat tissue organoids; a testicular equivalent organ growth section (30) is configured to provide an organoid cavity to accommodate 10 to 20 testicular organoids, where each testicular organoid is a testicle follicle, preferably the organoid cavity is configured to house 15 testicular organoids; and an organ growth section of the brain equivalent (29) is configured to provide an organoid cavity to house 150 to 250 brain organoids, where each brain organoid is a column of the cerebral cortex, preferably the organoid is configured to host 200 brain organoids.
[0012]
12. MULTIPLE ORGAN DEVICES IN CHIP, according to any one of the preceding claims, characterized by the organ equivalents and the independent circulation system (34) being configured, so that the arteriolar transport channel that originates from the equivalent lung (22) display in the direction of flow: a first bifurcation in which a first arteriolar canal branches, serving the equivalent of small intestine, spleen and pancreas (21, 23, 24); a second bifurcation in which a second arteriolar channel branches, serving the liver equivalent (25); a third bifurcation in which a third arteriolar channel branches, serving the kidney equivalent (26); a fourth bifurcation in which a fourth arteriolar canal branches, serving the bone marrow (27); an optional fifth bifurcation in which a fifth arteriolar canal branches, serving a skin equivalent (31); an optional sixth bifurcation in which a sixth arteriolar canal branches, serving an equivalent of adipose tissue (28); an optional seventh bifurcation in which a seventh arteriolar canal branches, serving an equivalent of testicles (30); and an optional eighth bifurcation in which an eighth arteriolar canal branches, serving a brain equivalent (29).
[0013]
13. MULTIPLE CHIP ORGAN DEVICE, according to any one of claims 4 to 12, characterized in that the diameter of the arteriolar transport channel in the flow direction is constantly reduced, so that the sum of the cross-sectional areas of all channels of arteriolar transport, including all the bifurcations at a certain distance from the lung equivalent (22) remain constant, and in which in the venular transport channel said reduction in diameter is constantly reversed in the flow direction, so that the sum the cross-sectional areas of all venous transport channels, including all bifurcations at a given distance from the lung equivalent (22) remain constant.
[0014]
14. MULTIPLE CHIP ORGAN DEVICE, according to any one of the preceding claims, characterized in that the base layer (3) comprises or consists of glass, the organ layer comprises or consists of polydimethylsiloxane (PDMS), the backing layer of organ (5) comprises or consists of polycarbonate, the antrum layer (4) comprises or consists of PDMS and / or the actuator layer (2) comprises or consists of polycarbonate.
[0015]
15. MULTIPLE ORGAN DEVICES IN CHIP, according to any of the preceding claims, characterized in that each of the organ equivalents is configured to accommodate a number of organoids that is proportional to the number of organoids present, on average, in the respective organ a mammalian organism, preferably of a human being, in which all the organ equivalents of the multi-organ device on chip (1) are reduced in size by the same predetermined proportionality factor, for example, by a factor of 0.00001 (1 / 100,000).

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CA3079024A1|2017-10-31|2019-05-09|Murdoch Childrens Research Institute|Composition and method|

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US20210237057A1|2018-04-19|2021-08-05|Nanyang Technological University|Microfluidic board and method of forming the same|

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CN111971384A|2018-05-21|2020-11-20|深圳华大生命科学研究院|Bionic intestine-liver organ chip and preparation method and application thereof|

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WO2020087148A1|2018-11-02|2020-05-07|Centro Nacional De Pesquisa Em Energia E Materiais – Cnpem|Platform for multi-systems culture, bioreactors and methods for in vitro experimental assays|

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KR102019007B1|2019-01-31|2019-09-05|제주대학교 산학협력단|Organs on a chip having intergrated sensors|

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WO2021071490A1|2019-10-10|2021-04-15|Hewlett-Packard Development Company, L.P.|Dispense cassettes for microfluidic dispensers|

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WO2021071491A1|2019-10-10|2021-04-15|Hewlett-Packard Development Company, L.P.|Microfluidic dispensers for limiting dilution|

法律状态:
2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2019-07-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-03-10| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-07-14| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-11-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/08/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261706928P| true| 2012-09-28|2012-09-28|

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US61/706,928|2012-09-28|

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EP12186550.5A|EP2712918B1|2012-09-28|2012-09-28|Multi-organ-chip with improved life time and homoeostasis|

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EP12186550.5|2012-09-28|

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PCT/EP2013/067073|WO2014048637A1|2012-09-28|2013-08-15|Multi-organ-chip with improved life time and homoeostasis|

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