WO2020087148A1 - Plate-forme pour culture multi-systèmes, bioréacteurs et procédés pour essais expérimentaux in vitro - Google Patents

Plate-forme pour culture multi-systèmes, bioréacteurs et procédés pour essais expérimentaux in vitro Download PDF

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Publication number
WO2020087148A1
WO2020087148A1 PCT/BR2019/050474 BR2019050474W WO2020087148A1 WO 2020087148 A1 WO2020087148 A1 WO 2020087148A1 BR 2019050474 W BR2019050474 W BR 2019050474W WO 2020087148 A1 WO2020087148 A1 WO 2020087148A1
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Prior art keywords
media
microfluidic
mixer
cell media
bioreactor
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PCT/BR2019/050474
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English (en)
Inventor
Eduardo PAGANI
Talita Miguel MARIN
Angelo Luiz GOBBI
Rui Cesar MURER
Makoto Okuma
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Centro Nacional De Pesquisa Em Energia E Materiais – Cnpem
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Publication of WO2020087148A1 publication Critical patent/WO2020087148A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26—Conditioning fluids entering or exiting the reaction vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes

Definitions

  • the present patent application refers to devices, systems and methods which may be applied to methods for in vitro experimental assays, for example in an in vitro assessment of pharmacological profile of drugs .
  • This application discloses products, such as devices and systems, for example a cell media mixer, bioreactors and systems comprising the same, as well as methods for using the devices and systems for in vitro assays, such as assessment of pharmacological profile of drugs. All embodiments disclosed herein are linked by a single general inventive concept, forming a platform for multi-systems culture.
  • the present application refers to a device, which is a cell media mixer.
  • the cell media mixer is useful for collecting, mixing, exchanging gases and distributing a cell media circulating in a system for performing experimental assays.
  • the cell mixer device emulates the functionality of a cardiopulmonary bypass in a system for performing experimental assays.
  • the cell media mixer comprises a toroidal shaped mixer chamber having a lumen defined by bottom, internal and external walls.
  • the toroidal shape may be a polygonal toroidal shape, or a circular toroidal shape.
  • the mixer chamber comprises inlets and outlets for connecting the mixer chamber to one or more peripheral devices, and simultaneously collects, mixes, exchanges gases and distributes a cell media that circulates through the mixer chamber and said one or more peripheral devices.
  • the present patent application refers to bioreactor devices, such as bioreactors to emulate the functioning of the urinary and digestive systems and bioreactors to cultivate barrier organoids and one parenchymatous organoids, as well as two undefined groups of organoids in parallel.
  • devices which emulate the functioning of the urinary or digestive systems comprise fluidic mounting parts, whereby a cell media is allowed to circulate, as well as semipermeable membranes interfacing such fluidic mounting parts and a fluid circuit for circulating fluids collected from the cell media by one or more of said mounting parts.
  • the referred cell media mixer and the one or more peripheral devices connected to each other form a system for performing experimental assays.
  • the present patent application refers to systems for performing experimental assays.
  • Such systems comprise a media mixer for collecting, mixing, gas exchanging and distributing a cell media and one or more devices for cultivating organoids or organoid synergic groups.
  • this patent application refers to methods for in vitro assessment of pharmacological profile of drugs and method for in vitro organoid production.
  • the system for performing experimental assays may be, for example, a system for in vitro assessment of pharmacological profile of drugs.
  • the system is modular and reconfigurable, allowing, for example, connection with high degree of freedom of bioreactors.
  • bioreactors may comprise living organoids.
  • the system integrated with organoids can improve the state-of-the-art emulation of some complex body functions that depend on organs' integration by the systemic circulatory system. Besides drugs it can replace animal testing for the development of biologic drugs, cosmetics, functional foods, agrochemicals, cleaning products and others.
  • devices disclosed herein being cell mixer or cardiopulmonary bypass emulator, bioreactors or fluidic circuits, are conceived according to topologic principles, meaning that the natural topological relationships between the cultivated cells, the cell media and fluids are transposed to the systems and devices disclosed herein.
  • the aim of such devices is emulating the functioning or organs, such as heart, lungs, kidney, intestine and liver, or organic systems, such as cardiopulmonar, respiratory, urinary or digestive systems.
  • the dead volume i.e. a space within the device which is not being used for circulating the cell media is significantly reduced in the cell media mixer, or cardiopulmonary bypass emulator, due to its toroidal, or ring-shaped, topology architecture.
  • the methods disclosed herein comprise transposing the topological relationships between the human body cells and the human body fluids to organoids cultivated in bioreactors and fluids that emulate the human body fluids .
  • ADMETox an acronym that stands for Absorption, Distribution, Metabolism, Excretion and Toxicity. These pharmacological properties are usually assessed for optimizing investigative new drugs, in drugs' discovery and development. They also are part of the regulatory authorities demands for allowing clinical trials to start.
  • Barrier organoid an organoid that mimics barrier properties of some organ or anatomical structure, such as skin, intestinal epithelium, kidney, liver, blood brain barrier, placenta and others.
  • Bile emulated in this document means a fluid produced by the digestive system emulator bioreactor.
  • Bile separation in this document means a separated circuit for collecting and storing bile's emulated.
  • Bioreactor in this document means a reactor able to cultivate organoids whose fluidic circuits have been optimized for a specific organoid .
  • Cardiopulmonary Bypass a non-biologic machine that keeps the patient alive during heart surgery, emulating some functions of the circulatory and respiratory systems. It gets the venous blood from the systemic circulation at the cava veins, or the right heart atrium; performs the gases exchange functions of the lungs (receiving O2 and releasing CO2) and propels it to the aorta.
  • Cell lines cells that are cultivable and expansible, mostly derivate from cancers .
  • Cell media mixer in this document means a structure conceived to interconnect multiple peripheral devices, such as bioreactors, while emulating vital functions, as a cardiopulmonary bypass.
  • Closed loop circulation in this document means a fluid circuit that starts and finished at the same container, thus allowing the fluid to contact the same organoid several times. This must be disambiguated with the expression “closed circulatory system” that means the circulation that occurs inside vessels common to all vertebrates.
  • Culture media in this document means a nutritive fluid that circulates in the Micro Physiologic System circuits' and have contact with organoids interchanging nutrients and metabolites .
  • Emulation imitation.
  • Gas exchange is the exchange of gases between a cell media and an external environment, which may be a gas chamber or air.
  • the gas exchange is allowed or performed by a device, such as a cell media mixer or a cardiopulmonary bypass emulator.
  • GLP an acronym that stands for Good Laboratory Practices. It is a quality assurance certification demanded by most regulatory alterities to accept results of tests formally demanded for investigative new drugs.
  • Induced Pluripotent Stem Cells a kind of pluripotent stem cell that can be generated from mature cells.
  • Interstitial Fluid a fluid produced by filtration of the blood thought the capillaries into the interstitial space. It has direct contact with all cells. It is partially reabsorbed to the blood stream at the peripheral tissues and part is drained to the lymphatic system.
  • Lymph the fluid that flows through the lymphatic system (vessels and lymph nodes) .
  • the lymph circulation is an open loop, starting at the peripheral tissues with the interstitial fluid and returning to the bloodstream by the thoracic ducts, that drain to the subclavian veins. Lymph has a composition similar, but not identical to blood plasma. In the intestinal territory the lymph is rich in fat after a meal.
  • Anatomical topology in this document means the topological relationship of anatomical structures visible to the naked eye.
  • Mammals ' circulatory system macro-features in this document means having a closed loop circulation, a pulmonary and systemic circulation, and individual control of each vascular territory perfusion.
  • Media dead volume in this document means any media volume that is not in direct contact with the organoids or in mixing functions or in contact with the environment for gases exchange.
  • Micro anatomical topology In this document means the topological relationships between specific cell types and fluids that have contact with them such as: blood, lymph, urine, and bile.
  • Microphysiological System a cell culture microfluidic device integrated with living organoids.
  • Open loop circulation in this document means a fluid circuit that starts at one container and finishes at another container, thus allowing the fluid to contact the organoid just once. This must be disambiguated with the expression "open circulatory system” that means the circulation that occurs through tissues, outside vessels (eg. The hemolymph of some invertebrates) .
  • Organ on a Chip a denomination used by some groups to refer to a micro physiological system cultivating human organoids.
  • Organoid an artificial version of an organ, that mimics some functions of the organ.
  • Parenchymal organoid an organoid that mimics organs such as heart, brain, lymph node and others.
  • Peripheral irrigation territory in this document means the cells perfused by branches from a single arteriole, whose perfusion can be controlled by this arteriole.
  • Portal systems are venous irrigation territories in which a vein branches in a capillary bed that drains to another vein.
  • Primary cells (human) : cells from a human donor either alive or cadaver .
  • topology In mathematics, topology (from the Greek t ⁇ poV, place, and l ⁇ goV, study) is concerned with the properties of a geometric object that are preserved under continuous deformations, such as stretching, twisting, crumpling and bending, but not tearing or gluing.
  • Fluid in this document means excreta emulated, such as bile and urine emulated.
  • Urine emulated in this document means a fluid produced in the urinary system bioreactor.
  • One of the main challenges for achieving this goal is to create systems that are genetically and phenotypically human and allow integrated responses as promoted by the human organic systems such as digestive, urinary, circulatory, respiratory, etc. Advances in microfluidic devices, and the possibility of constructing functional organoids with genetically human cells allow envisioning a new technology for predicting human outcomes with higher accuracy. The large impact expected from it pushed the National Center for Advancing Translational Sciences (NCATS) to launch the "Micro physiological Systems Program". Its goal is to develop physiologically relevant, genetically diverse and pathologically meaningful in vitro systems on a modular, reconfigurable system 2 , 3 .
  • NCATS National Center for Advancing Translational Sciences
  • Topology is a discipline of mathematics. It deals with relationships between objects. It disregards shapes but does regard barriers. It also regards flow directions. We believe that devices conceived under topological concepts will promote disruptive advances in the emulation capability.
  • Organoids are artificial versions of an organ, made from that organ's cells, either cell lines, primary cells, iPSC and others.
  • the cells' original positioning within the organ and the relationships with neighboring cells is disassembled and reassembled in a different shape. If one complies with topology, reshaping is allowed, but breaking barriers and modifying cell's polarities is not allowed. Most currently available devices disregard these topological principles, creating relationships among cells that never existed in the human body.
  • Creating an MPS according to topological principles has specific challenges for each organic system.
  • the challenges for the circulatory system are: [059] 1-
  • the culture media emulates the interstitial fluid, but its topological relations include also the interstitial fluid, the lymph and the blood.
  • the current state-of-the art MPSs, including this patent application, use culture media to nourish the organoids.
  • Media is chemically and topologically like the interstitial fluid (IF) .
  • IF flows through the interstitial space in direct contact to all cells. Under normal heath conditions, the blood contacts only endothelial cells.
  • the circuits' directions are: IF is produced in the arterial capillaries, part returns to the bloodstream through the venous capillaries, part goes to the lymphatic system and then mixes with the blood at the subclavian veins .
  • the vascular space is a continuum with all endothelial cells in direct contact with blood.
  • the circulation is unidirectional and follows very specific pathways: the systemic and pulmonary circulations described ahead.
  • [0 62] 2 The blood circulates in a closed loop, together with IF components . Blood circulation is a closed loop. One single red cell is expected to pass through the left ventricle every 60 seconds, and then over and over for all its life of 100 days 4 . The same happens with all blood components, such as drugs and metabolites, until they are cleared from the body. A drug and its metabolites are exposed to pharmacokinetically relevant organs many times.
  • Some devices promote inter-organoid communication by getting the media from one container, passing it through one organoid and then to a second, sometimes a third, and so on. After that, the media goes to a different container and never returns to the organoids again. This does not emulate what happens with substances dissolved in the blood and in dynamic equilibrium with the interstitial fluid.
  • the blood carries the IF components through unidirectional pulmonary and systemic circulations .
  • the pulmonary the heart sends venous blood to the lungs and receives arterial blood from them.
  • the systemic the heart sends arterial blood to every single organ except the lungs and receives venous blood from the cava veins that mixes the blood from all peripheral territories.
  • the only exceptions are the portal systems, and the nephron
  • the topological blood circuit to be emulated is peripheral territory - right atrium - aeration - left ventricle - peripheral territory.
  • the perfusion of any vascular territory is variable according to homeostatic needs.
  • the muscles increase their blood demand during exercise, the digestive organs after feeding, the kidneys under hyperhydration (and decrease under dehydration) and the uterus during pregnancy.
  • Both heart and lungs increase their blood flow according to the whole-body demands, especially during physical exercise.
  • the possibility of individually controlling the perfusion each organoid is expected to increase the adherence to the very basic principle of homeostasis and therefore the physiological and pharmacological relevance of any results.
  • the in-vitro tests for developing drugs are usually run in stable pre-set drug concentrations, that don't mimic the clinical conditions.
  • the drug concentration in the plasma raises from zero, reaches a maximum (Cmax), and decreases to zero, due to metabolism and excretion.
  • Cmax a maximum
  • the drugs' concentration fluctuates between a maximum and a minimum. Therefore, in clinical conditions, the human tissues are exposed to variable concentrations of drugs and metabolites. Without true excretions, it will be unlikely to reproduce the drug fluctuations seem in patients.
  • the IF relations topologic transposal to the media circuits comprises contacting any cells but keeping the cell's polarities.
  • the IF contacts one side of the cells, while other fluids, such as urine or bile contact the opposite side. This cell's polarities must be kept by the MPSs.
  • the part of the original organ cell's that have contact with the IF, lymph or blood shall have contact with the media.
  • the part of the original organ cell's that have contact with the bile shall have contact with the bile emulated.
  • the part of the original organ cell's that have contact with the urine (or glomerular filtrate), shall have contact with the urine emulated.
  • the anatomical topology is to collect the media from the basal side of the intestinal barrier and expose it to the apical side of the hepatic cells, while the basal side will produce and have contact with the bile's emulated.
  • the most relevant anatomical topology is the relationship between the renal microvasculature and the urinary space inside the nephrons.
  • the topological emulation is to have the apical side of the glomerular cells and the basal side of tubular cells in contact with the media and the basal side of the glomerular cells and the apical side of the tubular cells basal side in contact with the nephron's lumen filled with urine's emulated.
  • the ideal micro physiological system is modular and reconfigurable, with high degree of freedom 5 . It means accepting modules that might be changed.
  • a modular system must accept modules that share certain characteristics of connection and compatibility but are optimized to the functions of each specific organ.
  • non-essential, but desirable characteristics of the micro physiologic systems are: being suitable for mass production (scalable); allowing bio-printed organoids easy integration; allowing parts pre integrated with organoids to be frozen for conservation and transport and thawed for utilization at its destiny; facilitate integration of sensors for monitoring media composition; facilitate access for collecting media for analytical purposes; organoids shall also be visible or easily recovered for morphological assessment for either physiological or pharmacological purposes.
  • Patents CN106811413 and WO201452835 A1 are about modular "generic" single organ bioreactors that can be used for several types of organoids. This king of bioreactor can be good for certain purposes but are not the best option for organoids with very specific characteristics/demands.
  • Patents US10066198 B2, doi : 10.1039/c31c50350j , and US 10087422 B2 are dedicated single organoid bioreactors, optimized for barrier organoids with physical mobility like the lung' s alveoli (which are mobile), the intestine (whenever mobility is needed for certain outcomes) .
  • the device doi : 10.1039/c31c50350j is optimized for certain electromechanical characteristics of the heart.
  • Patents US20180355298 Al, US10012640 B2, US7507579 B2, and US7407799 B2 are microfluidic multi-well assay plates. Some types emulate one single organoid in every well without integration. Other integrate two or more wells by fluidic channels allowing integrative responses. These plates are an important evolution of the conventional multi-well assay plates. They allow testing at high throughput speed for some complex outcomes. Because they don't follow topologic principles it is unlikely that they will be able to produce results that can replace animal models for the most complex outcomes.
  • the present patent application refers to a cell media mixer device.
  • a cell media mixer device may be used in a system for performing experimental assays and was designed for collecting, mixing and distributing a cell media circulating through such system, while performing and/or allowing gas exchange between the cell media and an external environment.
  • Such device comprises a toroidal-shaped mixer chamber having a lumen defined by bottom, internal and external walls.
  • the cell media mixer performs functions which are similar to the functions of a cardiopulmonary bypass. Therefore, as disclosed herein, a cell media mixer may also be understood as a cardiopulmonary emulator device in a system for performing experimental assays.
  • the mixer chamber comprises inlets and outlets for connecting the mixer chamber to one or more peripheral devices, and simultaneously collects, mixes, promotes gas exchanges and distributes a cell media which circulates through the mixer chamber and said one or more peripheral devices.
  • peripheral devices may be, for example, bioreactors.
  • Bioreactors may be devices for cultivating organoids or organoid synergic groups, for example.
  • the one or more devices for cultivating organoids or organoid synergic groups is/are fluidically connected to a device which emulates a topological cardiopulmonary bypass, such as a cell media mixer chamber as disclosed herein .
  • such cell media mixer may be useful, for example, to emulate a cardiopulmonary bypass in a system for in vitro assessment of pharmacological profile of drugs.
  • the cell media is a nutritive fluid suitable for cells that circulates in a system in which experimental assays are performed.
  • the cell media is collected from peripheral devices, circulated through and mixed in the mixer chamber, and then distributed to the peripheral devices.
  • the lumen of the toroidal shape is preferably located substantially at the center of the toroidal-shaped mixer.
  • the toroidal-shaped mixer may be made of plastic or any moldable material and may be produced by any known means, such as plastic injection molding or 3D printing.
  • the cell media mixer further comprises a mixer chamber seal, cap or the like.
  • a mixer chamber seal, cap or the like protects the mixer chamber from contamination. Therefore, the seal, cap or the like may be, for example, a plastic cap, a microfluidic mounting part, a semipermeable membrane or an external element which is not necessarily attached, connected or in touch with the cell media mixer .
  • gas exchange between the cell media and an external environment is allowed through a gas exchange interface.
  • the gas exchange interface is between the mixer chamber lumen and external environment.
  • the gas exchange interface may be formed, for example, by an opening between the mixer chamber cap and the mixer chamber lumen.
  • the opening between the mixer chamber cap and the mixer chamber lumen is formed by bars which prevent the cap from completely closing the mixer chamber lumen, allowing the exchange of gases with an external environment .
  • the seal, cap or the like is a semipermeable membrane permeable by gases, which allow the gas exchange interface path is the semipermeable membrane.
  • the gas exchange interface is such semipermeable membrane.
  • the cell media mixer chamber allows volume level variations of the cell media circulating through the system.
  • volume variations may occur due to inflow and outflow of the cell media circulating in the device.
  • the cell media mixer chamber inlet is located above the cell media outlet for preventing gas bubbles from passing to the outlet.
  • outlets are individually controlled and allow modulating the media flow between the mixer chamber and multiple peripheral devices, wherein each individually controlled outlet comprises a dedicated pump to allow individual perfusion control of each peripheral device connected thereto.
  • the cell media mixer allows simultaneous connection of several peripheral devices, such as bioreactors, and may work as a cardiopulmonary bypass, emulating some functions of the systemic circulation and some functions of pulmonary circulation.
  • the cell media mixer has means for eliminating gas bubbles and allowing buffer media level variations.
  • the toroidal or ring shape of the cell media mixer disclosed herein reduces dead volume therein, as better explained in the detailed description and embodiment examples.
  • the present patent application also refers to devices, such as bioreactors, which comprise microfluidic circuits.
  • Such devices, or bioreactors which emulate at least part of the functioning of organs, or organic systems, such as the urinary or digestive systems.
  • organs and/or systems are emulated by the interaction between fluidic mounting parts, semipermeable membranes interfacing the same, microfluid circuit and organ cells, organoids or organoid synergic groups which may be cultivated in the such bioreactor devices.
  • bioreactors and/or microfluidic circuits disclosed herein comprise
  • the first microfluidic mounting part receives media from the inlet and directs the same to the second microfluidic mounting part, which partially directs the media to the outlet.
  • the second semipermeable membrane is an interface between the first microfluidic mounting part and the third microfluidic mounting part, which receives fluids from the media circulating through the first microfluidic mounting part through the second semipermeable membrane.
  • the first semipermeable membrane is an interface between the third microfluidic mounting part and the second microfluidic mounting part, which receives fluids from the third microfluidic mounting part through the first semipermeable membrane.
  • the topology of the glomerular and tubular cells, urinary space and tubular lumen in relation to their blood perfusion, glomerular filtrate and urine are emulated.
  • the second semipermeable membrane emulates the functioning of nephron tubular cells, by absorbing fluids from the cell media.
  • the fluids may be absorbed through the second semipermeable membrane by nephron tubular cells cultivated thereon.
  • the first semipermeable membrane emulates the functioning of glomerular cells by filtrating fluids circulating through the third microfluidic mounting part.
  • the fluids may be filtrated through the first semipermeable membrane by glomerular cells cultivated thereon, forming a urine emulate.
  • the fluid carrying substances such as metabolites, is circulated from the third mounting part to the fluid circuit.
  • the fluid carrying substances such as metabolites, circulates from the third mounting part to the fluid circuit passing by the first semipermeable membrane.
  • a urinary system emulator bioreactor as disclosed herein may comprise one additional pump for sucking the media from the bioreactor's outlet.
  • a urinary system emulator bioreactor uses one single pump. Both alternatives produce urine emulate and employ the Bernoulli's principles of energy conservation .
  • the present application refers to a device, or bioreactor, which emulates the functioning of the digestive system.
  • the bioreactor which emulates the functioning of the digestive system basically comprises:
  • a microfluidic mounting of three parts a first and a second part being interfaced with a third part respectively by a first and a second semipermeable membrane;
  • a fluidic circuit, reservoir or circulation path A fluidic circuit, reservoir or circulation path.
  • a device, bioreactor, or microfluidic circuit emulating the functioning of the digestive system receives media from the inlet, which is circulated through the third microfluidic mounting part to the outlet.
  • the first semipermeable membrane is an interface between the third microfluidic mounting part and second microfluidic mounting part, which receives fluids from the media circulating through the third microfluidic mounting part through the first semipermeable membrane.
  • the second semipermeable membrane is an interface between the third microfluidic mounting part and the first microfluidic mounting part, which receives fluids from the media circulating through the third microfluidic mounting part through the second semipermeable membrane.
  • the media received from the third microfluidic mounting part through the second semipermeable membrane is circulated through the first microfluidic mounting part to a fluidic circuit, reservoir or circulation path .
  • the first semipermeable membrane emulates the functioning of the intestine wall.
  • the first semipermeable membrane intestine cells cultivated thereon.
  • the fluids are secreted though the second membrane by liver cells cultivated thereon, forming a bile emulated which is which directed into the bile emulated fluidic path, which may also be a reservoir .
  • a bile's emulate may be produced by such bioreactor, which employs the Bernoulli's principles of energy conservation.
  • a bioreactor for cultivating one barrier organoid and/or a parenchymatous organoid is disclosed herein.
  • Such barrier organoid may be, for example, an organoid emulating the blood brain barrier.
  • Such parenchymatous organoid may be, for example, an organoid emulating the Central Nervous System, .
  • a bioreactor according to such embodiment basically comprises:
  • the first part receives the media from the inlet and directs it to the outlet, both located at the first part.
  • the semipermeable membrane emulates functions of barriers, such as blood brain barrier, modulating the permeation of substances between both chambers.
  • a bioreactor for cultivating two groups of organoids in parallel is disclosed.
  • the two groups of organoids may be any type or organoid, such as adipocytes spheroids and pancreatic beta cell spheroids.
  • a bioreactor according to such embodiment comprises basically :
  • the chamber of the first part interfacing the chamber of the second part by an impermeable wall; at least one fluid inlet at one part and one fluid outlet at the other part; and
  • a fluidic circuit receives and mixes the media flow from the both chambers and drives it to the outlet.
  • the cell media and fluids are circulated according to topological principles of the cells cultivated in the semipermeable membranes.
  • the topological relationships between the cultivated cells, the cell media and fluids circulating through the system and devices e.g. cell media mixer and bioreactors, emulate the natural topological relationships occurring in an animal body.
  • transposing the topological relationships between the cultivated cells, cell media and fluids comprises maintaining a cultivated cell in contact with the fluid and/or cell media maintaining the anatomical regions of a cultivated cell in contact with a media or/or fluid which they are contacted with under natural conditions .
  • each inlet and outlet comprise individually controlled pumps for individual control or inflow and outflow or circulating media.
  • the present application refers to a system for performing experimental assays comprising:
  • a cell media mixer for collecting, mixing and distributing a cell media, as disclosed herein;
  • one or more devices for cultivating organoids or organoid synergic groups are provided.
  • the one or more devices for cultivating organoids or organoid synergic groups is/are fluidically connected to the cell media mixer for collecting, mixing and distributing a cell media.
  • the system comprises individually controlled media inlet and outlet, wherein each individually controlled inlet and/or outlet comprises a dedicated pump for allowing individual perfusion control of each bioreactor connected to the cardiopulmonar bypass emulator device.
  • peripheral devices such as bioreactors, devices for cultivating organoids or organoid synergistic groups the like may be connected to the cell media mixer in multiple reconfigurable positions.
  • the such system is a system for in vitro assessment of pharmacological profile of drugs.
  • the present application refers to a method for in vitro assessment of pharmacological profile of drugs, the method comprising the steps of:
  • cultivating cells in devices for cultivating organoids or organoid synergic groups
  • the fluids derived from the circulating cell media are formed in the devices for cultivating organoids or organoid synergic groups.
  • the fluids emulate organ excreta, such as bile and urine.
  • the topological relationships between the cultivated cells, the cell media and fluids are transposed to the system by maintaining a cultivated cell in contact with the fluid and/or cell media maintaining the anatomical regions of a cultivated cell in contact with a media or/or fluid which they are contacted natural conditions .
  • Figure 01 discloses the device with the cell media mixer and bioreactors to cultivate organoids.
  • the cell media mixer comprises a cell media mixer chamber (100), a cell media mixer cap (200) and pumps (110) .
  • the bioreactors (300, 400, 500, 600, 700, 800, 900) are attached to the cell media mixer.
  • Figure 01 A is the lateral view
  • figure 01 B is the top view
  • highlighting the pump positioning and circuits in relation to one bioreactor and the cell media mixer and figure 01c is the perspective view.
  • FIG. 02 shows the cell media mixer part of the cell media mixer (100) with the cell media mixer cap (200) in transverse sections.
  • 02B and 02C show the cell media mixer cap (200) .
  • the cell media mixer lumen mixes the media coming from all organoids. It prevents the media contamination, whereas allows media level variations and gases exchanging with the environment.
  • 02D is a schematic of the cell media mixer (100) showing its polygonal toroidal shape, the cell media mixer walls (101 and 102), and chamber (103) . All media circuits start and finish in cell media mixer chamber (103) .
  • the hole (104) is the cell media mixer outlet that connects to the pump inlet (not shown) .
  • the pump outlet feeds the bioreactors inlets .
  • the bioreactors outlets connect to the cell media mixer inlet that is the hole (105) .
  • Figure 03 shows the rational for the digestive system emulator bioreactor circuits.
  • Fig. 03 A is a schematic of the hepatic circulation showing the hepatocytes topology in relation to the blood coming from the portal vein, the biliary duct and the bile's emulated flow.
  • Figure 03 B shows the circuits for media and bile's emulated within the IL (300) bioreactor.
  • Figure 04 shows the digestive system emulator bioreactor (300) mounting with its six parts (310, 320, 330, 340, 350, and 360), two metallic parts (2000), and two semipermeable membranes (1020 and 1030) .
  • the figure 04 A is the mounted top view
  • 06 B is the mounted lateral view
  • 06 C is the exploded lateral view.
  • Figure 05 shows details of the digestive system emulator bioreactor (300) parts (310, 320, 330, 340, 350, and 360) in perspective views with the channel system highlighted.
  • igure 06 shows the rational for the urinary system emulator bioreactor (400) circuits.
  • Fig 06 A shows the nephron with the glomerulus, the tubules and their topology in relation to the blood and the urine within the urinary space.
  • Figure 06 B shows the option 01 circuits within the K bioreactor for media and urine's emulated.
  • Figure 06 C shows the option z circuits within the K bioreactor for media and urine's emulated.
  • Figure 07 shows the first option of the urinary system emulator bioreactor (400) mounting with its five parts (410, 420, 430, 440 and 450) and the two semipermeable membranes (1030 and 1040) .
  • Figure 07 A is the mounted top view
  • 07 B is the mounted lateral view
  • 07 C is the exploded lateral view.
  • Figure 08 shows details of the urinary system emulator bioreactor (400) parts (410, 420, 430, 440, and 450) in perspective views with the channel system highlighted.
  • Figure 09 shows the second option of the urinary system emulator bioreactor (400z) mounting with its five parts (410z, 420z, 430z, 440z and 450z) and the two semipermeable membranes (1030 and 1040) .
  • Figure 07 A is the mounted top view
  • 07 B is the mounted lateral view
  • 07 C is the exploded lateral view.
  • Figure 10 shows details of the second option of the urinary system emulator bioreactor (400z) parts (410, 420, 430, 440, and 450) in perspective views with the channel system highlighted.
  • Figure 11 shows the top (left column) and bottom (right column) perspective views of alternative designs of the digestive system emulator bioreactor.
  • the channels are in the edges of these parts for injection molding manufacture.
  • Figure 12 shows the top (left column) and bottom (right column) perspective views of alternative designs of urinary system emulator bioreactor first option. The channels are in edges of these parts for injection molding manufacture.
  • Figure 13 shows the top (left column) and bottom (right column) perspective views of alternative designs of urinary system emulator bioreactor second option. The channels are in edges of these parts for injection molding manufacture.
  • Figure 14 shows one bioreactor for cultivating one barrier organoid plus one parenchymatous organoid.
  • Figure 15 shows one bioreactor, able to cultivate two organoids with in parallel.
  • Figure 16 shows membranes, metallic pieces and one alternative bubble eliminator.
  • the cell media mixer has no organoids cultivated therein and works as a cardiopulmonary bypass (CPB), a non biologic machine that performs the functions of the heart and lungs in heart surgery.
  • the peripheral devices, or bioreactors cultivate organoids and emulate some functions of tissues, organs or organic systems.
  • An embodiment also includes one peripheral device, such as a bioreactor for emulating the digestive system, other for emulating the urinary system, and two multipurpose bioreactors to cultivate other organic systems or tissues.
  • the cell media mixer and all bioreactors have fluidic circuits conceived according to topologic principles.
  • the cell media comprises slots for connecting peripheral devices, such as bioreactors, which comprise media inlet and one media outlet are distributed around the cassis in a daisy shape, as shown in the Figures 01 and 02. They allow the modular and reconfigurable connection of bioreactors cultivating organoids.
  • the cell media mixer may be a cardiopulmonary bypass (CPB) emulator, in which some functions of the heart and lungs are emulated, including the pulmonary circulation by non-biologic means.
  • the cell media mixer allows surgical interventions in the heart, while keeps the patients' organs alive by performing the functions of the heart and lungs.
  • the steps made by the cell media mixer and the correspondent steps made by the cell media mixer are: [ 151 ]
  • the cell media mixer is able to emulate collection of venous blood returning from all systemic circulation territories.
  • the cell media mixer collects and mixes the media coming from all peripheral devices, such as bioreactors connected thereto.
  • the cell media circulating through the devices and systems disclosed herein emulates the interstitial fluid that passes through the systemic circulation peripheral territories.
  • the cell media mixer may also allow gas exchange, emulating lung functioning.
  • the venous blood is propelled to a machine that performs the gases exchange, enriching the blood with O2 and cleaning the CO2.
  • the cell media mixer does the same with the media. It enriches the media with O2 and cleans the CO2, while it circulates in the toroid lumen and makes direct contact with the air.
  • the cell media mixer propels the blood to the aorta, that distributes it to all systemic circulation territories.
  • the cell media mixer propels the media to all connected bioreactors .
  • one urinary system emulator bioreactor aimed at emulating the renal excretion of drugs and other substances ( Figures: 06, 07, 08, 09, 10, 12 and 13) . It cultivates organoids emulating the glomerular and tubular barriers. Its fluidic circuits transpose the micro anatomical topological relationships between the organoid's cells, blood, lymph, glomerular filtrate and urine. Its fluidic circuits have an innovative design that helps the production of urine's emulated, according to the Bernoulli's laws of conservation of energy and also use drag forces. It separates and stores urine's emulated.
  • the cell media mixer has one dedicated pump for each bioreactor. This allows individual perfusion control of each bioreactor. This function is not performed by the CPB. During extracorporeal circulation, the peripheral territories perfusion control is made by the patient's arterioles. In this patent application, this function is performed by one individual pump for each bioreactor.
  • one digestive system emulator bioreactor aims at emulating the intestinal absorption and hepatic metabolism of drugs and other substances ( Figures: 03, 04, 05 and 11) . It cultivates organoids emulating the intestinal barrier and the liver. Its fluidic circuits transpose the micro anatomical topological relationships between the organoid's cells, blood, lymph and bile. Its fluidic circuits have an innovative design that helps the production of bile's emulated according to the Bernoulli's laws of conservation of energy. It separates and stores bile's emulated.
  • All devices disclosed herein being cell media mixers, bioreactors or microfluidic circuits emulate natural topologic relations between cells, cell media, or interstitial fluid, and other body fluids, such as excreta, e.g. urine and bile.
  • excreta e.g. urine and bile.
  • the blood goes from the left ventricle to any peripheral territory and returns to the right atrium.
  • the portal systems and the nephron the blood never goes from one peripheral territory to another. Therefore, except for the portal systems and the nephron, the topological blood circuit to be emulated is peripheral tissue - cell media mixer - peripheral tissue.
  • FIG. 1 61 Another embodiment example of the subject matters disclosed herein is a device for organoid cultivation comprising: one cell media mixer (part 100 in the figures 1, and 2) and several bioreactors (parts 300, 400, 500, 600, 700, 800, X00... in the figures 3 to 15) .
  • the cell media mixer can connect several bioreactors, each cultivating one or more tissues or organoids.
  • the device is reconfigurable and has a high degree-of- freedom.
  • the cell media mixer working as a CPB has inlet and outlet ports (104 and 105 at the figure 02) and one individual pump for each cell media mixer face (six are shown at figures 01 and 02) .
  • the devices and systems disclosed herein may use many kinds of pumps. These preferably promote pulsatile flow, including but not restricted to piezoelectric, peristaltic, piston, pneumatic and others. Pumps unable to generate pulses might also be acceptable.
  • the pumps' positioning in the fluidic circuit in relation to the cell media mixer and bioreactors is an embodiment of this patent application.
  • the cell media mixer can connect to several bioreactors.
  • Each bioreactor is a module.
  • the modular concept is characterized by sharing the same connective features to the cell media mixer:
  • the cell media mixer can accept connection to many different shapes of peripheral devices.
  • the reconfigurable and high degree of freedom concepts are characterized by the possibility given to the user to choose how many and which bioreactors to connect to the cell media mixer.
  • the cell media mixer emulates by non-biologic means some functions of the following systemic circulation macro anatomical structures: left heart ventricle, aorta, arteries and arterioles (except pulmonary) .
  • the function emulated of the left heart ventricle, aorta and arteries is respectively to propel and pipeline the blood flow to each vascular territory.
  • the emulation is characterized by propelling and pipelining the media to each bioreactor.
  • the function emulated of arterioles is controlling the blood flow to each vascular territory.
  • the perfusion control of each vascular territory is performed by the dilation or constriction of the arterioles irrigating a vascular territory.
  • the perfusion is controlled by one individual pump dedicated to each bioreactor. So, the emulation is characterized by controlling the media flow to each bioreactor .
  • the cell media mixer emulates by non-biologic means the blood mixing functions of the systemic circulation anatomical structures: venules, veins excluding the porta and the pulmonary and including the cava veins and the right heart atrium. These structures collect and mix the blood coming from all body organs except the lungs. The emulation is made by the toroidal cell media mixer that collects and mixes the media coming from all bioreactors connected to it .
  • the cell media mixer emulates by non-biologic means the gases exchange function of the pulmonary circulation.
  • the cell media mixer is seen in the figures 01 and 02.
  • the culture media interfaces the environmental air (or incubator gases) in the cell media mixer chamber (103) . Whenever passing through the cell media mixer chamber, media gets 02 and delivers C02.
  • the cell media mixer has a bubble elimination capability. Air bubbles promote malfunction in fluidic circuits.
  • the cell media mixer inlets (105) are located above the outlets
  • Air bubbles arriving from the bioreactors to the cell media mixer chamber through the cell media mixer inlets (105) are likely to float up and unlikely to achieve the cell media mixer outlets/pumps inlets
  • the cell media mixer (100) has toroidal daisy shape, that reduces the media dead volume. This shape reduces the cell media mixer chamber volume, while allows connection to several bioreactors, with minimum pipelines.
  • the preferred shape is a hexagonal toroid. Other toroidal shapes like other polygons or a circle are also possible for attaching a smaller or larger number of bioreactors .
  • the cell media mixer (100) buffers media level variations between a minimum and a maximum without impacting on bioreactors perfusion.
  • One embodiment of the present patent application is the culture media circulatory system. It comprises a combination of one part communicating with the environment for gases exchange (the mixer chamber 103) and other parts not communicating with the environment (the bioreactors 300, 400, 500, 600, 700, 800) .
  • the cell media mixer works like a sink, allowing media level variations.
  • Figure 1 shows the cell media mixer composed by a mixer chamber
  • Fig 01A is the top view of the cell media mixer emulator of the circulatory and respiratory systems.
  • Fig 01B is the perspective view of the 01A.
  • Fig 01C shows the detailed of the bioreactor's connection to the cell media mixer.
  • Fig 01D shows the venous returning (120) from the bioreactor to the cell media mixer. Note the pump's (110) position.
  • Fig 01E perspective view of the cell media mixer with six bioreactors connected.
  • Figure 2 A shows the cell media mixer (100), with the mixer chamber 103 (the same of mixer lumen) and the mixer bottom (106) .
  • Figure 02 B shows the mixer cap top view (200)-.
  • Fig 02C shows the cap internal part bars ((203) and the parts (201) and (202) which are the internal and external flips of the cap that allow the gas exchange with the external environment of the device, avoiding contamination.
  • Fig 2D shows the cell media mixer external (101) and internal walls (102) which delimitates the mix chamber (103) .
  • Mix chamber outlet (104) and inlet (105) are the mix chamber.
  • Figure 02 D shows the variation range, with maximum and minimum acceptable levels.
  • the bioreactors work always full of media.
  • the system's total media volume can decrease, due to evaporation or production of emulated of urine or bile, or increase, when the operator adds more media.
  • Media volume variations within the system promote media levels variations in the cell media mixer only.
  • the bioreactors remain always full of media for proper functioning.
  • the cell media mixer outlets
  • the figure 1 shows the pumps' (110) inlets are placed at the cell media mixer outlets (104) .
  • One pump feeds each bioreactor.
  • the pumps preferably promote pulsatile flow, including but not restricted to piezoelectric, peristaltic, piston, pneumatic and others. Pumps unable to generate pulses might also be acceptable.
  • the cell media mixer (100) with the cap (200) is a gas exchanger.
  • the culture media interfaces the environmental air (or cell incubator gases) in the mixer chamber (103) . Whenever passing through the mixer chamber, media gets O2 and delivers
  • the cell media mixer additionally works as a bubble eliminator. Air bubbles can promote pumps and microfluidic circuits malfunction.
  • the cell media mixer inlets (105) are located above the outlets (104) . Air bubbles arriving from the bioreactors to the mixer chamber through the cell media mixer inlets
  • the cell media mixer cap (200) at the fig.02 closes the cell media mixer chamber (103) without sealing, because of small bars (203) . These allow gases exchange and fluid level variations, while preventing air microorganism contaminations.
  • both the cell media mixer and the bioreactors are conceived to reduce the media dead volume and optimize the media/cells proportion, to achieve more accurate and meaningful pharmacologic results.
  • the culture media emulates the extracellular fluids, comprising mainly the plasma and the intercellular fluid. Drugs are distributed in cells and extracellular fluids. MPSs with nonoptimized media/organoid proportion promote a non- optimized drug distribution. This can lead to less accurate and less meaningful pharmacologic results.
  • this patent application achieves media dead volume reduction by its toroidal daisy shape embodied in this patent application (Figs. 1 and 2) .
  • This shape reduces the mixer chamber volume, while allows connecting to several bioreactors, with minimum pipelines.
  • the preferred shape is a hexagonal toroid.
  • Other toroidal shapes like other polygons or a circle are also possible for attaching a smaller or larger number of bioreactors.
  • liver organoid metabolizes drugs from the media and delivers metabolites to the media. Some drugs and/or metabolites can be withdrawn from the media by liver organoid and excreted in the emulated of bile.
  • the kidney organoid excretes drugs, metabolites and wastes from the media to the emulated of urine; 3- Any gland organoid cultivated within a bioreactor will deliver its hormones to the media, accessing all organoids in the MPS; 4- Any cells, including but not restricted to white blood cells and cancer cells may arrive to all organoids cultivated in the MPS.
  • bioreactors integrated with the respective organoids connected to the cell media mixer give to the system the pharmacokinetic capability of absorbing, metabolizing, distributing and excreting drugs (ADME) .
  • ADME pharmacokinetic capability of absorbing, metabolizing, distributing and excreting drugs
  • This is aimed at reproducing in the media the drugs' and metabolites' fluctuations typically seen in the human plasma after taking a drug.
  • the drug administered over the intestinal barrier permeates to the media, passes through the liver organoid for metabolism, passes to the urinary system emulator, and is excreted, either in the "urine” or "bile, " while exposing the organoids to fluctuating concentrations of drugs and metabolites, as happens in human patients.
  • the organoids' morphology and some metabolites in the media can be assessed to get information of toxicological interest.
  • the system can assess the ADMETox properties of investigative new drugs, by in vitro means, in replacement of animal testing. These properties are assessed by the drug developers, to guide the drug's optimization and afterwards, in animal testing under GLP conditions for regulatory purposes. Both kinds of animal tests can be replaced by this system.
  • kidney, barrier functions such as: skin, blood brain barrier, placenta, etc. It also opens possibilities for synergistic organoid groups such the intestinal barrier and liver parenchyma.
  • organoid groups such the intestinal barrier and liver parenchyma.
  • Other examples include the skin and the lymph node for sensitization outcomes, skin and adipose tissues for cosmetic and pharmacologic outcomes, placenta or endometrium and embryos for reproductive medicine, among others.
  • the bioreactors have channels within the walls that minimize the dead volume and emulate the original organs anatomical topology regarding media, cells and urine or bile .
  • a bioreactor 300 cultivates intestine and liver organoids, emulating some functions of the digestive system.
  • the organoids cells in relation to the media are organized in the same topology that the human intestine and liver microstructures have in relation to the portal vein blood and bile.
  • a digestive system emulator bioreactor 300
  • Its fluidic routes are optimized according to the micro anatomical topological relations created by the portal vein. This is characterized by collecting the media from the basal side of the intestinal barrier and exposing it to one side of the hepatic cells, while the opposite side will produce and have contact with the bile's emulated.
  • This bioreactor separates a fluid from the media, that is called bile's emulated for the purposes of this patent application.
  • the figure 03 A shows the hepatocytes topology in relation to the blood coming from the portal vein, the biliary duct and the bile flow.
  • the venous blood drained from the intestine goes to the liver through the portal vein where a network of microscopic vessels (sinusoids) drive it to the hepatic vein after exposure to the hepatocytes .
  • the circuits for media and bile emulated are shown in the figure 03 B.
  • hepatic organoids both return metabolites to the media and excrete some in the bile's emulated, that is produced in the part (330) and stored in the part (310) .
  • the separation and storage of bile's emulated is of high utility and innovative.
  • the topological transposal from the human microanatomy to this bioreactor (300) is emulated.
  • the media flows through the part (330) chamber that cultivates the liver organoid at the semipermeable membrane (1020) of figure 04 forming its floor.
  • the part (340) above cultivates the intestinal barrier.
  • the media circulating in the part (330) nourishes the intestinal barrier at basolateral side, from below and carries the substances absorbed through the intestine organoid to the liver organoid.
  • the liver organoid secretes an emulated of bile to the part (320) bellow that drives it to the part (310) for storage and collection by the researcher/user.
  • Figure 04 shows the digestive system emulator bioreactor (300) mounting with its six parts (310, 320, 330, 340, 350, and 360) and two metallic compressive parts (2000) The mounting is bottom-up starting with the part with smaller number.
  • Figure 04 A is the top view
  • 04 B is the mounted lateral view
  • 04 C is the exploded lateral view. This view also shows the positioning of the sealing joints (1050), intestine cultivation membrane (1010) and liver cultivation membrane (1020) .
  • Figure 05 shows the parts of the digestive system emulator bioreactor (300) .
  • the part 310 is the bioreactor's base and emulates the gallbladder. Its chamber collects the emulated of bile from channel (323) of the part (320) . It has: a window to drain the emulated of bile (312), that stays closed to avoid contaminations, and a siphon (313) to allow the air outflow pushed by the fluid inflow from the part (320) and 3 bolt holes (311) .
  • the part (320) has a small chamber, whose floor (322) is impermeable or solid. It collects the emulated of bile produced by the cell cultivated in the part (330) above keeping the liver cells basal side wet.
  • the small hole (323) allows the emulated of bile to flow to the part's (310) cavity below for excretion. It has 3 bolt holes (321) .
  • the part's (330) chamber houses the liver organoid at the semipermeable membrane (1020) .
  • the liver organoid stays between the culture media (cell apical side) and the secretion produced at the basal side, the emulated of bile.
  • the emulated of bile crosses the semipermeable membrane (1020) and fills the parts' (320) cavity.
  • the channels (333) and (332) are respectively the media flow inlet and outlet that simultaneously nourishes the liver cells bellow at the apical side and at basolateral side of the intestinal cells above. It has 3 bolt holes (331) .
  • the part (340) contains a chamber for cultivates the intestinal barrier over the semipermeable membrane (1010) that is attached to the (342) . It has 3 bolt holes (341) .
  • the part (360) is a cap to close the hole (352) that provides access to the intestinal barrier preventing contaminations and is open whenever needed to deliver drugs, emulating the oral route.
  • This cap can have screw thread (361) to safely close the hole (352) avoiding leaks.
  • urinary system emulator bioreactors embody fluidic routes developed in agreement with the Bernoulli's principles of energy conservation and the Bernoulli's equation for streamlines filled with uncompressible liquids (in this patent application this liquid is the media) .
  • the streamlines embody variations in the transverse section and elevations in relation to a reference plan to produce pressure gradients through semipermeable membranes (semipermeable membranes) cultivating organoids (barrier organoids) . These gradients promote flow through the barrier organoids that generate urine's emulated.
  • Figure 06 shows the rational for the urinary system bioreactor (400) circuits.
  • the figure 06 A shows the human glomerular and tubular cells topology in relation to the nephron's irrigation, urinary space, tubular lumen and urine.
  • the figures 06 B and 06 C are two fluidic circuits options for the topological transposition made in the two urinary system emulator bioreactors. They show the circuits within the K bioreactor for media and emulated of urine.
  • Figure 06 B shows the circuits whose bioreactor is shown in the Figure 07 and parts details in the Figure 08.
  • the media comes from the arterial part of the cardiopulmonary bypass emulator, through the inlet (434) of the part (430), crosses the part (430) horizontally, crosses the part (440) vertically through the channel (443) and returns to the cell media mixer through the part (450), whose outlet is the (453) .
  • the emulated of urine is produced at the part (440) and stored in the part (410) .
  • the part (430) emulates the glomerulus. It comprises a small chamber (433) with a semipermeable membrane above (1030) that is integrated with glomerular cells (podocytes) . While the culture media passes through the circuit of the part (430), a fraction is filtered to the part's (440) cavity, emulating the glomerular filtrate.
  • the part's (440) cavity emulates the urinary space and the tubular lumen, which are topologically identical.
  • the semipermeable membrane (1040) above cultivates the nephron tubular cells. These cells reabsorb a fraction of the filtrate to the media circulating in the part (450) part above. This phenomenon is helped by one sucking pump placed at this bioreactor's outlet (453) that sucks the media against the barriers (452) at the part (450) that create turbulence and drag.
  • the fluid formed in the part's (440) cavity is the emulated of urine. It is transported to the part (410) by the (442) channel through the part (440); (432) channel through the part (430) and (422) channel through the part (420) .
  • turbulences created by a pump sucking the media through barriers create drag forces that help the glomerular fluid reabsorption in the urinary system emulator bioreactor (400) part (450) .
  • Figure 07 shows the urinary system bioreactor (400) mounting with its five parts (410, 420, 430, 440 and 450) and two metallic compressive parts (2000) .
  • the mounting is bottom-up starting with the part with smaller number.
  • Figure 07 A is the top view
  • 07 B is the mounted lateral view
  • 07 C is the exploded lateral view. This view also shows the positioning of the sealing joints (1050), glomerulus cultivation semipermeable membrane (1030) and tubules cultivation semipermeable membrane (1040) .
  • Figure 08 shows the urinary system emulator bioreactor first option (400) mounting with its five parts (410, 420, 430, 440 and 450) .
  • the part (410) is the bioreactor's base and emulates the urinary bladder. Its chamber collects the emulated of urine received from the part (440) through the circuit formed by the channels (442), (432), (422) and (413) . It has: an access hole to drain the emulated of urine (412), that stays closed to avoid contaminations, and a siphon (414) to allow the air outflow pushed by the fluid inflow from the (413) . It has 3 bolt holes (411) .
  • the part (420) locks the (430) part and comprises a channel (422) whereby the urine is conducted to the bladder (410) .
  • the part (430) emulates the glomerulus. It comprises a small chamber whose floor is a dedicated semipermeable membrane (1030) .
  • the channel (434) is the media inlet from the cardiopulmonary bypass emulator's pump and the channel (435) conducts the media to the channels (443) and (454) to the part (450) . It produces the glomerular filtrated emulate, that reaches the part's (440) cavity. It has 3 bolt holes (431) .
  • the part (440) emulates the nephron tubule together with the part (450) .
  • the floor is a semipermeable membrane (1040) where the tubular cells are cultivated.
  • the part's (440) cavity emulates the urinary space and the tubular lumen, which are topologically identical.
  • the fluid in this cavity emulates urine, after the cell's activity over the glomerular filtrated.
  • This fluid has the tubular cells above and the glomerular cells (podocytes) below in the part (430) .
  • the channel (442) allows the emulated of urine to initiate the circuit that finishes in the cavity of the (410) part.
  • the channel (443) passes through this part, driving the media from the part (430) to the part (450) . It has 3 bolt holes (441) .
  • the part (450) emulates the nephron tubule together with the (440) .
  • the culture media flows into its cavity (450) by channel (454) nourishing the basolateral side of the tubular cells seeded at the semipermeable membrane (1040) attached to the part (440) and receiving the substances reabsorbed by these cells.
  • the channel (453) is the culture media outlet.
  • This part has some specially designed barriers to create turbulence (452) and drag force to suck the liquid from the urinary space to the media. This effect depends on a sucking pump sucking the media at the venous outlet.
  • the part (450) has 3 bolt holes (451) .
  • Figure 09 shows the urinary system emulator bioreactor second option (400z) mounting with its five parts (410z, 420z, 430z, 440z and 450z) and two metallic compressive parts (2000) .
  • the mounting is bottom- up starting with the part with smaller number.
  • Figure 09 A is the top view
  • 09 B is the mounted lateral view
  • 09 C is the exploded lateral view. This view also shows the positioning of the sealing joints (1050), glomerulus cultivation semipermeable membrane (1030) and tubules cultivation semipermeable membrane (1040) .
  • Figure 10 shows the urinary system emulator bioreactor parts.
  • the part ( 410 z ) of the bioreactor ( 400 z ) is the bioreactor's base and emulates the urinary bladder. Its chamber collects the emulated of urine received from the part ( 430 z ) through the circuit formed by the channels (433z), ( 422 z ) (412z) . It has: an access to drain the emulated of urine (413z), that stays closed to avoid contaminations, and a siphon ( 414 z ) to allow the air outflow pushed by the fluid inflow from the ( 412 z ) . It has 3 bolt holes ( 411 z ) .
  • the part (420z) emulates the nephron tubule together with the (430z) .
  • the culture media flows into its cavity nourishing the basolateral side of the tubular cells at semipermeable membrane attached to the part (430z) above and receiving the substances reabsorbed by these cells.
  • the bottom of the part (420z) is nonpermeable .
  • the channel (424z) is the medium outlet and the (423z) is the tissue culture medium receiver.
  • the channel (422z) passes through it driving the emulated of urine from the part (430z) to the part (410z) . It has 3 bolt holes (421z) .
  • Figure 11 shows the top (left column) and bottom (right column) perspective views of alternative designs of the digestive system emulator bioreactor (300) .
  • the channels were moved to the edges of these parts for allowing injection molding manufacture.
  • Figure 12 shows the top (left column) and bottom (right column) perspective views of alternative designs of urinary system emulator bioreactor first option (400) .
  • the channels were moved to the edges of these parts for injection molding manufacture.
  • Figure 13 shows the top (left column) and bottom (right column) perspective views of alternative designs of urinary system emulator bioreactor second option ( 400 z ) .
  • the channels were moved to the edges of these parts for injection molding manufacture.
  • FIG 14 shows a bioreactor for cultivating one barrier organoid and one parenchymal organoid (500) with its three parts (510, 520 and 530) and two metallic compressive parts (2000) .
  • Figure 14 A is the top view
  • 14 B is the mounted lateral view
  • 14 C is the rational for fluidic circuits
  • 14 D is the exploded lateral view
  • 14 E is an alternative shape allowing production by plastic injection molding.
  • the mounting is bottom-up starting with the part with smaller number.
  • the part (510) has a chamber to cultivate parenchymal organoids such as central nervous system spheroids.
  • the part (520) is separated from the part (510) by a semipermeable membrane (1040) that is a semipermeable membrane to cultivate a barrier organoid such as the blood brain barrier.
  • the part (520) has a media inlet (525) and a media outlet (526) .
  • the part (530) is a cap for the part (520) .
  • These 3 parts have bolt holes (511) , (521) and (531) .
  • the figure 15 shows a bioreactor (600) for cultivating two organoids in parallel with its three parts (610, 620 and 630) and two metallic compressive parts (2000) .
  • Figure 15 A is the top view
  • 15 B is the mounted lateral view
  • 15 C is the rational for fluidic circuits
  • 15 D is the exploded lateral view
  • 15 E is an alternative shape allowing production by plastic injection molding.
  • the mounting is bottom-up starting with the part with smaller number.
  • the parts (610) and (620) have one chamber each to cultivate organoids that are separated by impermeable walls.
  • the part (610) has a media inlet (615), a channel (612) to split the media flow to both the 610 chamber and the channel (622) of the part (620) and a channel to direct the media flow to the channel (625) of the part (620) .
  • the part (620) receives the media flow from the part (610) and drives it to the outlet (626) that also mixes the flow from coming from the part (610) through the channel (613) .
  • the part (630) is a cap for the part (620) These 3 parts have bolt holes (611) , (621) and (631) .
  • FIG 16 shows another embodiment of the present patent application: organoids are cultivated in semipermeable membranes: (1010, 1020, 1030, 1040) for the intestine; (1020) for the liver; (1030) for the glomerulus and (1040) for the tubules; that fit in conventional assay plates (also known as microplates or micro titter plates) and are transferred to the device when they are mature to perform the experiment.
  • This shape is suitable for several kinds of organoids. This feature is important because the different organoids need different times and different culture medias to maturate. It also shows metallic pieces.
  • Figure 17a shows the relative tissue viability values obtained by colorimetric assay for assessing the mitochondrial activity as an indirect indicator of the cell viability (MTT) in an assay performed in the digestive system emulator bioreactor.
  • Figure 17b shows a comparison between the values of osmolarity measured in the culture medium and in the fluid (bile emulated) collected from the digestive system emulator bioreactor reservoir (310) .
  • Figure 18a shows the relative tissue viability values obtained by colorimetric assay for assessing the mitochondrial activity as an indirect indicator of the cell viability (MTT) in an assay performed in a urinary system emulator bioreactor.
  • Figure 18b shows a comparison between the values of osmolarity measured in the culture medium and in the fluid (urine emulated) collected from the urinary system emulator bioreactor reservoir (410) .
  • Figure 19a shows relative tissue viability values obtained by colorimetric assay for assessing the mitochondrial activity as an indirect indicator of the cell viability (MTT) in an assay performed in a second urinary system emulator bioreactor.
  • Figure 19b shows a comparison between the values of osmolarity measured in the culture medium and in the fluid (urine emulated) collected from the urinary system emulator bioreactor reservoir (410 Z) .
  • Tables 1, 2 and 3 report experimental data from assays performed in accordance with methods disclosed herein.
  • the system devices, i.e. cell media mixer and bioreactors used for performing the method are also disclosed herein as exemplificative embodiments.
  • Intestine Control samples refers to intestine tissue grown outside of the digestive system emulator bioreactor (300) .
  • Intestine refers to intestine tissue grown within the digestive system emulator bioreactor (300) .
  • Liver Control samples refers to hepatic tissue grown outside of the digestive system emulator bioreactor (300) .
  • Liver organoid cells refers to hepatic tissue grown within the digestive system emulator bioreactor (300) .
  • Renal tubule Control samples refers to tubule tissue grown outside of the urinary system emulator bioreactor (400) .
  • Renal tubular barrier refers to renal tubule tissue grown within the urinary system emulator bioreactor (400) .
  • Glomerulus Control samples refers to glomerular tissue grown outside the urinary system emulator bioreactor (400) .
  • Glomerular barrier refers to glomerular tissue grown within the urinary system emulator bioreactor (400) .
  • Renal tubule Control samples refers to tubule tissue grown outside of the urinary system emulator bioreactor (400 Z) .
  • Renal tubular barrier refers to renal tubule tissue grown within the urinary system emulator bioreactor (400 Z) .
  • Glomerulus Control samples refers to glomerular tissue grown outside the urinary system emulator bioreactor (400 Z) .
  • Glomerular barrier refers to glomerular tissue grown within the urinary system emulator bioreactor (400 Z) .

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Abstract

La présente invention concerne des dispositifs, des systèmes et des procédés qui peuvent être appliqués à des procédés pour des essais expérimentaux in vitro, par exemple dans une évaluation in vitro du profil pharmacologique de médicaments. Ces dispositifs comprennent des dispositifs mélangeurs de cellules qui émulent un pontage cardiopulmonaire, ainsi que des dispositifs bioréacteurs qui émulent des organes ou des systèmes d'organes, tels que des systèmes urinaires et digestifs.
PCT/BR2019/050474 2018-11-02 2019-10-31 Plate-forme pour culture multi-systèmes, bioréacteurs et procédés pour essais expérimentaux in vitro WO2020087148A1 (fr)

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