WO2017175236A1 - Plateforme microfluidique pour le développement de co-cultures in vitro de tissus de mammifère - Google Patents

Plateforme microfluidique pour le développement de co-cultures in vitro de tissus de mammifère Download PDF

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Publication number
WO2017175236A1
WO2017175236A1 PCT/IN2017/000071 IN2017000071W WO2017175236A1 WO 2017175236 A1 WO2017175236 A1 WO 2017175236A1 IN 2017000071 W IN2017000071 W IN 2017000071W WO 2017175236 A1 WO2017175236 A1 WO 2017175236A1
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Prior art keywords
culture
layer
organ
media
microfluidic device
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PCT/IN2017/000071
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English (en)
Inventor
Prajakta DANDEKAR JAIN
Ratnesh JAIN
Manish Ravikiran GORE
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Dandekar Jain Prajakta
Jain Ratnesh
Gore Manish Ravikiran
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Publication of WO2017175236A1 publication Critical patent/WO2017175236A1/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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • 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
    • 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
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture

Definitions

  • the present invention relates to a microfluidic device for the construction of media and scaffold guided three dimensional, tissue or organ like cellular orientation, similar to in vivo; conditions,, for preclinical or biomedical applications of pharmaceutical or cosmetic products.
  • the process of drug discovery stems from selecting few potential candidates from a library of thousands of active pharmaceutical molecules. These candidates are screened through pre -clinical and clinical trials prior to approval.
  • the pre-clinicai trials involved during the process of drug discovery consist of assessment of active, pharmaceutical molecules and/or their formulations on suitable animal models (Referred from: Drug Development Process, US-FDA Website).
  • suitable animal models Referred from: Drug Development Process, US-FDA Website.
  • these models suffer from disadvantages related to animal experimentation such as requirement of skilled manpower, rime consuming protocols and high cost.
  • a need has been realized to develop a suitable. alternati veto-animal models that may provide safe and effective models for screening pharmaceutical ingredients and/or their formulations or cosmetics and/or excipients (Doke S.K., et. al. 2015).
  • the current invention also possesses the ability to provide a realistic model to test the effects of -cosmetic ingredients and/or cosmetic products and/or excipients and actives with applications in healthcare and allied fields and/or their formulations.
  • 3D co-culture based in-vitro models of mammalian including human tissues include 3D scaffolds, hydrogeis. hanging-drop technique, magnetic levitation methods, bioreactor based platforms and microengineering. approaches, etc.
  • the microengineering-based methods usually involve use of a niicrobioreactor or microreactor or microfluidic assemblies to construct 3D environments for the growth and differentiation of cells and/or tissues.
  • microfluidic based models In contrast to the other approaches of building 3D eo-eul tures, microfluidic based models generally provide advantages such as lower consumption of media and buffers, production of considerable cell density, high surface to volume ratio and rate of mass transfer, high gas permeability, laminar flow conditions of cell culture media, provision of perfusion based physiologically relevant systems, allows local micro-environmental, control, provides flexibility for fabrication and combination with other approaches, offers real time analysis of cells being cultured, provides precise operationai control and adaptability to high throughput and scale up.
  • the potential ofmicrofluidic assemblies for developing in-vitro models thus. lies in their ability to reconstruct micro- environments at molecular, cellular, and tissue levels and flexibility for fabrication and formation of tissue co-cultures.
  • cell co-culture can be carried out in an accessible, controlled arrangement.
  • Microfluidic chip-based 3D co-culture model has also been used to. test drug sensitivity for individualized treatment of lung cancer.
  • the model involved co-culture of .lung cancer cells and stromal cells, under continuous supplementation of medium, to assay ahri-cancer drug efficacy in an in-vivo like tumor micro-environment.
  • a drug gradient generator was- integrated into this platform to collect information about responses of tumor cells to different concentrations of drugs.
  • device,, sensitive single or multidrug combination" chemotherapy schemes at appropriate doses were compared and investigated effectively.
  • this model was projected to assist researchers to study toxicities and side effects of anti-cancer drugs as well as the economic factors of a combination scheme before treatment (Xu, Zhi-yun, et al., 2013).
  • Efforts have been made to design placenta-on-chip model by co-cuhuring human trophoblasts (JEG- 3) and human -umbilical - vein endothelial ceils (HUVECs), to -mimic the placental barrier.
  • JEG- 3 co-cuhuring human trophoblasts
  • HUVECs human -umbilical - vein endothelial ceils
  • the two cell types were cultured in different compartments separated by a vitrified collagen membrane (Lee. JiSoo, et al., 201 5).
  • the present invention cultures cells in a chamber layer.
  • a microfluidic platform has been developed to allow for long-term maintenance of full thickness human skin equivalents (USE), comprising of epidermal and dermal compartments,
  • USE human skin equivalents
  • This model exhibited physiological relevance by mimicking the blood residence times in human skin tissue and lead to the establishment of an -air-epidermal interface that is essential for maturation and terminal differentiation of HSEs.
  • these -efforts put forward several attempts for developing microfluidic based in-vitro. models for testing various pharmaceutical molecules (Abaci, Hasan Erbil, et al.,.
  • the related art includes United States published application No. 2009/0234332 Al(M/s The Charles Stark Draper Laboratory, Inc,) describes a microfluidic system for developing artificial micro-vascular constructs for drug screening applications, in order to mimic the ability of the in-vivo blood vessels to stretch in response to several mechanical or biochemical stimuli, the device has reinforced the design of distensible wails aligning the cell culture channels.
  • Two co-culture channels are used for culturing vascular and endothelial cell types separately. These channels are separated by distensible walls. Distensibility of walls has been proposed to be dependent upon thickness and elastic modulus of the material used for fabricating the wall.
  • the present invention comprises of a single channel for the flow of materials.
  • micro- organ device comprising of a microscale support having one or more microfluidic channels for housing a micro-organ, particularly a micro-liver.
  • the patent claims micro-organ printing by computer-aided tissue engineering system. It also teaches that the cells are cultured on a 31) scaffold to mimic the physiological state of the in-vivo organ.
  • the present invention cultures cells and does not involve micro-organ printing, which is a more complex procedure involving sophisticated instrumentation.
  • the device consists of a microchannel layer consisting of plurality of channels, a single/multiple cell culture chamber supported by a polymeric membrane, lower gasket and metal base.
  • the device is claimed to provide more physiological relevance and applicability for drug screening applications.
  • the present invention consists of a single microchannel for providing media to the cells and includes a pump to enable accurate control over media flow.
  • a barrier-fike strueiiire that allows selective passage of nutrients, biochemical factors, gases, chemicals, etc, via passive or active mechanisms.
  • This is referred to as a diffusionsl barrier, which limits the transport of various biochemical and functional moieties and/or chemical factors. Integrity of this barrier plays a vital role in the smooth functioning of various tissues and/or organs.
  • the human brain i s guarded by the presence of blood-brain barrier achieved by the closely packed structure of brain endothelial ceils and astrocytes.
  • porous membrane between individual ceil types under co-culture may lead to the involvement of an additional diftusional. battier, if these models are to be explored former for the drug screening and drug development applications; Moreover, their ability to mimic the in-vivo spatial organization of cell types of different lineages of a particular tissue would also be limited due to the presence of porous membrane, unless it is essential as observed in the case of barrier based models [for example, blood brain barrier (BBB) model], The presence of additional barrier (usually a porous membrane] between the cellular layers, therefore, may lead to false positive or negative results with respect to drug pharmacokinetic studies.
  • BBB blood brain barrier
  • the present invention provides a more realistic approach to overcome these limitations by developing a unique 3D co-culture model, which can be practically applied to several tissues, and can develop but is not limited to the development of skin, lung and retina. Objective of the invention
  • the objectives of the present invention are a) to design a microfluidic device for constructing a physiologically relevant in-vitro model mimicking in-vivo cellular architecture and conditions of human and mammalian tissues
  • Another objective of the invention is to apply the developed platform to conduct pre-clinical studies, including hut not restricted to pharmacokinetic, pharmacodynamic, efficacy and toxicity studies, and to assess molecular mechanisms contributing to disease pathogenesis, progression and remodeling, in order to alleviate several disease conditions.
  • microchannel layer comprising a single channel
  • baffle or wedge layer optionally baffle or wedge layer
  • a riiicrofluidic device for preclinical or. biomedical applications of pharmaceutical or cosmetic- products comprising
  • microchannel layer comprising a single channel
  • baffle or wedge layer optionally baffle or wedge layer
  • the present. invention provides a novel, simple in vitro model of mammalian including human tissues that will, accurately mimic the in vivo tissues.
  • the proposed model serves as an in-vitro model for conducting preclinical as well as for disease or tissue specific molecular studies. It also provides to the scientists and clinicians a scalable platform to provide artificial tissue constructs or equivalents for biomedical applications.
  • invention is a microfiuidic device for the construction of media and scaffold guided three dimensional tissue or organ like cellular orientation, similar to in vivo conditions, comprising
  • microchannel layer comprising a single channel
  • baffle or wedge layer optionally baffle or wedge layer.
  • the scaffold may be made from biopolymers like but not limited to collagen, cfaitosan, cellulose and the like.
  • the organ in the organ like cellular orientation may be selected from skin, lungs, retina and the like.
  • the layers are fabricated using polydimethylsiloxane (PDMS), Poly methyl methacrylare (PMMA), Glass (Silica), Polystyrene, Polycarbonate. Polyurethane. Cyclic Olefin Copolymer (COO), Mixed Cellulose esters, PDMS, PMMA, Polycarbonate-, Polyester (PET), Poly vinylidene fluoride (PVDF), Poly tetrafluoroethylene (PTFE) and the like.
  • the device involves sequential arrangement of the following layers in three dimensional manner.
  • the top polymeric layer is between 40-60 mm long, 15-25 mm wide and 2-6 mm thick. It consists of a single, circular cell culture chamber at the centre of diameter ranging from 6-10 mm. It is fabricated from polymer selected from polydimethylsiloxane (PDMS). Poly methyl methacrylate (PMMA), Glass (Silica), Polystyrene, Polycarbonate, Polyurethane, Cyclic Olefin Copolymer (COC), Mixed Cellulose esters, PDMS, PMMA. Polycarbonate, Polyester (PET), Poly vinylidene fluoride (PVDF), Poly tetrafluoroethylene (PTFE) and the like.
  • PDMS polydimethylsiloxane
  • PMMA Poly methyl methacrylate
  • Glass Glass
  • Polystyrene Polycarbonate
  • Polyurethane Polyurethane
  • COC Cyclic Olefin Copolymer
  • Mixed Cellulose esters PDMS, PMMA.
  • the surface area of the cell culture chamber is in the range of 0.4-1 cm 2 .
  • This layer facilitates contact co-culture of individual cell types of mammalian including human tissues and obviates: the limitations observed in conventional contact less co-culture approaches.
  • number of cell-culture chambers may be constructed in this layer depending on the number of tissues to he cultured on the device.
  • the present embodiment will support multi-organ development by designing multiple cell-culture chambers.
  • the design of the cell-culture chamber of this type offers flexibility to directly load the cell suspension of specific seeding density as well as stains, enzymes , cell culture reagents, chemicals, pharmacceutical molecules and/or pharmaceutical formulations and/or cosmetic ingredients and/or cosmetic products and/or excipients and actives within applications in healthcare and allied fields and/or their formulations, directly into the cavity of the cell-culture chamber during culturing and while testing efficiency of the in-vitro model through -characterization studies.
  • the culture is cultivated insitu and may be mono or contact co-culture, b) Porous membrane:
  • a porous polymeric membrane of 8-12- microns in thickness and 0.1-5 microns with respect to pore size is attached using plasma treatment or a suitable adhesive at the lower side of the cell culture chamber (between upper cell culture chamber layer and middle channel layer).
  • These porous membranes may he made of polymers selected front polyethylene terephfhalate (PET), polytetrafluoroethylene (PTFE). Polycarbonate (PC), Mixed Cellulose Esters (MCE) and the like.
  • PET polyethylene terephfhalate
  • PTFE polytetrafluoroethylene
  • PC Polycarbonate
  • MCE Mixed Cellulose Esters
  • the membrane also allows passage of nutrient media and gases flowing in the middle niicrochannel layer at a controlled flow rate by capillary action.
  • the hydrophilic nature of the membrane and water absorption properties of the scaffold are also estimated to be the driving factors for enhancing the capillary forces for medium delivery,
  • the middle layer consisting of microchannel, is between 40*60 mm long, 15-25 mm wide and 2-6 mm thick, Height of niicrochannel ranges between 0.5-2 mm.
  • the inlet and outlet openings of the microftuidic device are fabricated to range between 1 -4 mm of thickness.
  • PDMS polydlmethylsiloxane
  • PMMA Poly methyl methacryiate
  • Glass Glass
  • Polystyrene Polycarbonate
  • Polyurethane Cyclic Olefin Copolymer
  • CDC Cyclic Olefin Copolymer
  • PDMS Polydlmethylsiloxane
  • PMMA Polymethyl methacryiate
  • PMMA Glass
  • COC Cyclic Olefin Copolymer
  • Mixed Cellulose esters PDMS
  • PMMA Polycarbonate
  • Polyester PET
  • PVDF Poly vinylidene fluoride
  • PVDF Poly tetrafiuoroethyiene
  • PTFE Poly tetrafiuoroethyiene
  • the device is attached by tubings made up of polyethylene or polymer of similar chemistry, with or without connectors, to the syringe pump.
  • Syringe pump of a suitable configuration supplies culture medium at a controlled flow rate and concomitantly remove waste from the device.
  • the flow rates of the medium are optimized in the range of few microliters per minute, depending on the need of perfusion for the tissue to be co-cultured in the device.
  • Bottom Layer The device is supported at the bottom with a support comprising of glass or polymer of similar chemistry,
  • the glass support is 70-80 mm long, 20-30 mm wide and 1 -3 mm thick.
  • This bottom layer provides a mechanical support to the device for ease of handling 10 facilitate cuituring and characters zation procedures.
  • Lower Baffle/Wedge Laver The dimensions of the lower layer, including positively protruding baffle or wedge -will he fabricated to lie between 15-25 mm in width, 0,3-0,6 mm in thickness and 3-6 mm in length. Height of the baffle will be between. 2-3 mm and thickness will be around 0.5-1 ,5 mm.. Suitable number of baffles or wedges will be fabricated in this layer depending on requirement of flow rate.
  • the polymeric microfluidic devices are fabricated from metal molds, comprising of aluminium or ether metals of suitable strength and operational flexibility. Molds are machined to be between 70-80 mm long and wide with thickness ranging from 6-8 mm.
  • metal molds are fabricated using machining, methods, such as, Computer Numerical Control (CNC) machining or other mold fabrication techniques that are suitable to yield the device with desired resolution and dimensions.
  • CNC Computer Numerical Control
  • Polymeric solutions comprising selected from polydimethylsifoxane (PDMS), Poly methyl methacrylate (PMMA), Glass (Silica), Polystyrene, Polycarbonate, Polyurethane, Cyclic Olefin Copolymer (COC), Mixed Cellulose esters, PDMS, PMMA.
  • Polycarbonate, Polyester (PET), Poly vinyl idene fluoride (PVDP), Poly tetrafiuoroethylene (PTFE) and the like are subject to the heat curing treatment at the temperatures ranging between 60-80"C.
  • Time duration of the curing treatment for the fabrication, of three polymeric layers will depend on the temperature utilized, such as, ranging from 4 h for 60°C to 2 h for 80°C .
  • the three polymeric layers are bonded together using plasma treatment or suitable adhesive which render the desired precision and resolution.
  • the porous polymeric membrane, beneath the top polymeric layer, is attached to the lower side of cell-culture chamber using plasma treatment or suitable adhesive or other similar technique which provides requisite degree of resolution and accuracy.
  • the components of the microfluidic device have been specifically designed to provide a system that facilitates contact co-culture of mammalian including human cells and/or tissues in a single chamber or compartment. This geometry is particularly required to retain the integrity of diffusional barriers and cellular organization observed in the in-vivo tissue.
  • the device also possesses significant potential for scale up by fabricating and operating parallelized -automation units and multi-welled assemblies. It is a novel platform to conduct high- throughput screening of pharmaceutical molecules and/or pharmaceutical formulations: and/or cosmetic ingredients and/or cosmetic products and/or excipients and actives with applications in healthcare and allied fields and/or their formulations, thereby expediting process of their transition into market.
  • microfluidic device of the present invention is used for preclinical or biomedical applications, of pharmaceutical or cosmetic products,
  • Microchannel layer comprising a Single channel
  • baffle or wedge layer optionally baffle or wedge layer.
  • the layers are fabricated using polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • PMMA Poly methyl methaerylate
  • Glass Glass
  • Polystyrene Polycarbonate. Polyurethane, Cyclie Olefin Copolymer (COC), Mixed Cellulose esters.
  • PDMS Polymethyl methaerylate
  • PMMA Polymethyl methaerylate
  • Glass Glass
  • COC Cyclie Olefin Copolymer
  • Mixed Cellulose esters PDMS, PMMA, Polycarbonate, Polyester (PET), Poly vinylidene fluoride (PVDF).
  • PVDF Poly tetrafluoroethylene
  • the device involves sequential arrangement of the following layers in three dimensional manner.
  • Upper cell culture chamber layer The top polymeric layer is .between 40-6.0 mm long, 15-25 mm wide and 2-6 mm thick.
  • the cell culture chamber consists of a single, circular cell culture chamber at the centre of diameter ranging from 6- 10 mm. It is fabricated from polymer selected from polydimethylsiloxane (PDMS), Poly methyl metbacrylate (PMMA). Glass (Silica). Polystyrene, Polycarbonate. Polyursthane, Cyclic Olefin Copolymer (COC). Mixed Cellulose esters, PDMS, PMMA, Polycarbonate, Polyester (PET), Poly vinyiidene fluoride (PVDF), Poly tetrafluoroethylene (PTFE) and the like.
  • the surface area of the cell culture chamber is in the range of 0.4-1 cm 2 .
  • This layer facilitates contact co-culture of individual cell types of mammalian including human tissues and obviates the limitations observed in conventional contactless eo-euliure approaches.
  • number of cell-culture chambers may be constructed in this layer depending on the number of tissues to be cultured on the device.
  • the present embodiment will support multi-organ development by designing multiple cell-culture chambers.
  • the design of the cell-culture chamber of this type offers flexibility to directly load the cell suspension of specific seeding density as well as stains, enzymes, cell culture reagents, chemicals, pharmaceutical molecules and/or pharmaceutical formulations and/or cosmetic ingredients and/or cosmetic products and/or excipienis and actives within applications in healthcare and allied fields and/or their formulations, directly into the cavity of the cell-culture chamber during cultunng and while testing efficiency of the in-vitro model through characterization studies.
  • the culture is .cultivated insitu and may be mono or contact co-culture, b) Porous membrane:
  • a porous polymeric membrane of 8-12 microns in thickness and 0.1 -5 microns with respect to pore size is attached using plasma treatment or a. suitable adhesive at the lower side of the cell culture chamber (between upper cell culture chamber layer and middle channel, layer).
  • These porous membranes may be made of polymers selected from polyethylene terephthalate (PET). polytetrafluoroethyl.ene (PTFE), Polycarbonate (PC), Mixed Cellulose Esters (MCE) and the like.
  • PET polyethylene terephthalate
  • PTFE polytetrafluoroethyl.ene
  • PC Polycarbonate
  • MCE Mixed Cellulose Esters
  • the membrane also allows passage of nutrient media and gases fiowing in the middle microchannel layer at a controlled flow rate by capillary action.
  • the hydrophilic nature of the membrane and water absorption properties of the scaffold are also estimated to be the driving factors for enhancing the capillary forces for medium delivery.
  • the middle layer consisting of micrpchannel, is between 40-60 mm long, 15-25 mm wide, and 2-6 mm thick. Height, of microchannel -ranges between 0.5-2 mm.
  • the inlet and outlet openings of the microrluidic device are fabricated to range between 1 -4 mm of thickness, It is fabricated from polymer selected from polydimelhylsiloxane (PDMS). Poly methyl methaqryjate (PMMA), Glass (Silica). Polystyrene.
  • This layer will render passage of nutrient media and gases to the upper cell-culture chamber layer at a controlled flow rate.
  • Inlet and outlet openings are fabricated in this layer.
  • the device is attached, by tubings made up of polyethylene or polymer of similar chemistry, with or without connectors to the syringe pump.
  • Syringe pump of a suitable configuration supplies culture medium at a controlled flow rate and concomitantly remove waste from the device.
  • the flow rates of the medium are optimized in the range of few microliters per minute, depending on the need of perfusion for the tissue to be co-cultured in the device.
  • the device is supported at the bottom with a support comprising of glass or polymer of similar chemistry.
  • the glass support is 70-80 mm long, 20-30 mm wide and 1 -3 mm thick. This bottom layer provides a mechanical support to the device for ease of handling to facilitate culturing and characterization procedures.
  • Lower Baffle/Wedge Layer :
  • the dimensions of the lower layer including positively protruding baffle or wedge will be fabricated to lie between 15-25 mm in width, 0.3-0,6 mm in thickness and 3-6 mm in length. Height of the baffle will be between 2-3 mm and thickness wil l be around 0,5- 1.5 mm. Suitable number of baffles or wedges will be fabricated in this layer depending on requirement of flow rate. Baffles or wedges arising from the lower layer will be protruding in the middle inicrochannel layer, thereby, obstructing the medium flow. Due to the obstruction created in the channel layer located beneath the circular cell culture chamber area, the medium is expected to flow over the baffles or wedges, thereby, expediting its delivery to the cells through enhancement of the rate of capillary action.
  • the polymeric micrafluidic device are fabricaied from the metal molds, comprising of aluminium orother -metals of suitable strength and operational flexibility . Molds are machined to be between 70-80 mm long and wide with thickness ranging from 6-8 mm. These metal molds are fabricaied using machining methods, such as, Computer Numerical Control (CNC) machining or other mold fabrication techniques that are suitable to yield the device with desired resolution and dimensions, Polymeric solutions comprising selected from polydimetbySsiloxane (PDMS). Poly methyl methacfylate (PMMA). Glass (Silica), Polystyrene, Polycarbonate, Polyurethane, Cyclic Olefin Copolymer (COC).
  • CNC Computer Numerical Control
  • the three polymeric layers are bonded together using plasma treatment or suitable adhesive- which render the desired precision and resolution.
  • the porous polymeric membrane, beneath the top polymeric layer, is attached to the lower side of cell-culture chamber using plasma treatment or suitable adhesive or other similar technique which provides requisite degree of resolution and accuracy.
  • the components of the microfluidic device have been specifically designed to provide a system that facilitates contact co-culture of mammalian including human cells and/or tissues in a single chamber or compartment. This geometry is particularly required to retain the integrity of difrusional barriers and cellular organization observed in the in-vivo tissue.
  • the device also possesses significant potential for scale up by fabricating and operating parallelized automation units and multi-welled assemblies, it is a novel platform to conduct high-throughput screening of pharmaceutical molecules and/or pharmaceutical formulations, and/or cosmetic ingredients and/or cosmetic products and/or excipients and actives with applications in healthcare and allied fields and/or their formulations; thereby expediting process of their transition into market.
  • micro-organ devices for various appl ications, such as experimental pharmaceutical screening for efficacy, adsorption, distribution, metabol ism, elimination, and toxicity, Thus, these devices may be used to assess the beneficial and detrimental effects of a novel drug after it passes through a given metabolic pathway. In addition, these devices may be used to evaluate the therapeutic benefits or toxicities of a drug compound or its formulations. or pharmaceutical or cosmetic excipients, Micro-organ devices as disclosed herein can address the need for in vitro niicro-organ-like structures that substantially replicate in vivo structure and function.
  • a micro fluidie device as used herein refers to a set of micro-channels etched or molded into a material (glass, silicon or polymer such as PDMS, for Polydimethylsiloxane), The micro-channels are connected together in order to achieve the desired features.
  • "Scaffold” as used herein is a. polymeric biomaterial either derived front natural source or prepared synthetically to form an in-vivo mimicking extracellular matrix structure thereby promoting cellular adhesion, differentiation, proliferation and tissue regeneration in a three dimensional orientation.
  • a "co-culture” as used herein can be defined as the growth of more than one distinct ceil type in a combined culture.
  • Contact co-culture involves direct contact between different ceil types and populations, without the presence of additional diffusional -barrier (viz. porous membrane) between them, it is also known as mixed co-culture,
  • tissue or organ like cellular orientation as used herein is defined as arrangement/organization in which cells comprising of a tissue/organ are patterned three-dimensionally to mimic the in-vivo architecture (i.e. anatomical layout), dynamics and physiological functionality (viz. barrier properties) of a tissue/organ.
  • Channel as used herein is defined as the hydraulic component of the anicrofluidic device fabricated/etched out of a specific geometry (viz. rectangular, circular, etc.) and dimension. It provides a gateway for fluid flow through it, under pressure.
  • “Baffle” as used herein is defined as a mechanical component or device specifically added to the obstruct/restrain/regulate the fluid flow.
  • “Wedge” as -used herein is. defined as a component possessing two principal fates which meet in a sharply acute angle, It is also used to regulate the fluid flow pattern (here).
  • FIG. 1 Front view of microfluidic device
  • FIG. 3 Front perspective view of microfluidic device
  • FIG. 4 Phase contrast microscopical analysis of 3D HADF culture using microfluidic device on 48 h culture. Scale bar corresponds, to 400 ⁇ m ( 10X magnification)
  • Figure 5 3D Morphology of HADF cells (Phase contrast image) showing development of processes to establish contact with hydrogel matrix and adjacent cells. Scale bar corresponds to 100 ⁇ m (40X magnification)
  • Figure 7 PI stained red fluorescent (dead) cell population observed after 48 h culture. [Scale bar corresponds to 400 ⁇ m ( 10X magnification)]
  • Figure 8 Z-stack confocal image observed after 48 h culture exhibiting 3D distribution of HADF cells [Scale bar corresponds to 200 ⁇ m (10X magnification)]
  • Scale bar corresponds to 200 ⁇ m (10X magnification)
  • HADF Human Primary Dermal Fibroblast
  • hydrogel based scaffold was used for conducting 3D culture of skin cells. HADF cells were mixed with the hydrogel based scaffold in the ratio, ranging from 1 : 1 to 1 :2 to facilitate their entrapment within the matrix (in cold, at 4°C) and were seeded in the culture chamber at the seeding density of 25000-45000 cells per ml.
  • the device Prior to the cell seeding, the device was subjected to fluid wetting by perfusing culture media (Dulbecco's Modified Eagle Medium with high-glucose comprising of 10 % Fetal Bovine Serum w/o 1 % Antibiotic Antimycotic Solution) at the infusion and/or withdrawal flow rate of 50-300 ul/min using a programmable push pull syringe pump.
  • culture media Dulbecco's Modified Eagle Medium with high-glucose comprising of 10 % Fetal Bovine Serum w/o 1 % Antibiotic Antimycotic Solution
  • K.D. Scientific Legato 272 Push-Pull Programmable Syringe Pump and Teflon tubing were used for conducting the experiments.
  • the chamber was covered with a glass cover slip and parafilin and maintained for culturing at the infusion and/or withdrawal flow rate of 0.5-1 ⁇ /mjn using a programmable push pull syringe pump.
  • the volume of the fluid in the chamber and the outlet was monitored throughout the culture operation.
  • Example 2 Test method for testing culture
  • the device has been tested for 3D monoculture studies of Human Primary Dermal Fibroblast Cells.
  • the 48h culture obtained from microfluidic device was processed and characterized by phase contrast ( Figures 4 and 5). fluorescence and confocal microscopy.
  • Live-Dead [Calcein AM-Propidium Iodide (PI)] staining was used for assessment of cell viability. Live cells emit green fluorescence ( Figure 6) while dead cells exhibited fluorescence ( Figure 7).
  • Calcein AM staining based confocal studies were carried out using laser ofexcitation wavelength of 488 nm and emission wavelength of 505-525 nm wavelength.
  • PI staining based confocal studies were carried out using laser of Excitation wavelength of 561 nm and emission wavelength of 623-665 nm wavelength.
  • 3D Z-stacking was conducted using confocal microscopy in order to assess 3D distribution of cells in the hydrogel matrix (figure 8).

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Abstract

La présente invention concerne un dispositif microfluidique pour la construction d'un tissu ou d'un organe tridimensionnel guidé par un support et un échafaudage, tel qu'une orientation cellulaire, similaire aux conditions in vivo, comprenant: a. une couche de chambre de culture; b. une membrane poreuse; c. une couche à microcanal comprenant un canal unique; et d. éventuellement une couche de chicane ou de coin.
PCT/IN2017/000071 2016-04-06 2017-04-06 Plateforme microfluidique pour le développement de co-cultures in vitro de tissus de mammifère WO2017175236A1 (fr)

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CN108034586A (zh) * 2018-01-02 2018-05-15 清华大学深圳研究生院 一种用于单细胞捕捉和培养的微流控芯片
CN109055204A (zh) * 2018-10-19 2018-12-21 杭州捷诺飞生物科技股份有限公司 药物筛选用器官芯片
CN111269833A (zh) * 2018-12-05 2020-06-12 中国科学院大连化学物理研究所 一种基于器官芯片的人胰岛类器官模型构建方法
CN111269572A (zh) * 2018-12-05 2020-06-12 中国科学院大连化学物理研究所 一种聚二甲基硅氧烷楔形多孔薄膜的制备方法
WO2020225777A1 (fr) * 2019-05-08 2020-11-12 Molecular Devices (Austria) GmbH Système et procédé pour la culture d'organoïdes
CN111996121A (zh) * 2020-09-30 2020-11-27 北京大橡科技有限公司 3d多器官共培养芯片
CN112080425A (zh) * 2020-09-07 2020-12-15 中国科学院上海微系统与信息技术研究所 一种器官芯片、上皮/内皮屏障模型器件及其制作方法
CN112574884A (zh) * 2020-11-19 2021-03-30 深圳先进技术研究院 基于微流控技术的多功能器官芯片、制备方法及其应用
EP3907277A1 (fr) * 2020-05-01 2021-11-10 The Charles Stark Draper Laboratory, Inc. Plaque de culture cellulaire microfluidique pour interface air-liquide et applications de tissus cultivés en 3d
CN114854585A (zh) * 2022-05-11 2022-08-05 中国中医科学院中药研究所 一种用于中药肝毒性评价的基于微流控体系的精密肝切片培养模型
CN114849801A (zh) * 2022-04-26 2022-08-05 复旦大学 通量化体外细胞、组织、器官培养和分析的微流控装置

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CN108034586A (zh) * 2018-01-02 2018-05-15 清华大学深圳研究生院 一种用于单细胞捕捉和培养的微流控芯片
CN108034586B (zh) * 2018-01-02 2023-09-22 清华大学深圳研究生院 一种用于单细胞捕捉和培养的微流控芯片
CN109055204A (zh) * 2018-10-19 2018-12-21 杭州捷诺飞生物科技股份有限公司 药物筛选用器官芯片
CN109055204B (zh) * 2018-10-19 2024-03-26 杭州捷诺飞生物科技股份有限公司 药物筛选用器官芯片
CN111269572B (zh) * 2018-12-05 2021-10-15 中国科学院大连化学物理研究所 一种聚二甲基硅氧烷楔形多孔薄膜的制备方法
CN111269833A (zh) * 2018-12-05 2020-06-12 中国科学院大连化学物理研究所 一种基于器官芯片的人胰岛类器官模型构建方法
CN111269572A (zh) * 2018-12-05 2020-06-12 中国科学院大连化学物理研究所 一种聚二甲基硅氧烷楔形多孔薄膜的制备方法
WO2020225777A1 (fr) * 2019-05-08 2020-11-12 Molecular Devices (Austria) GmbH Système et procédé pour la culture d'organoïdes
CN113950523A (zh) * 2019-05-08 2022-01-18 分子装置(奥地利)有限公司 用于类器官培养的系统和方法
US12024696B2 (en) 2019-05-08 2024-07-02 Molecular Devices (Austria) GmbH System and method for organoid culture
EP3907277A1 (fr) * 2020-05-01 2021-11-10 The Charles Stark Draper Laboratory, Inc. Plaque de culture cellulaire microfluidique pour interface air-liquide et applications de tissus cultivés en 3d
CN112080425A (zh) * 2020-09-07 2020-12-15 中国科学院上海微系统与信息技术研究所 一种器官芯片、上皮/内皮屏障模型器件及其制作方法
CN111996121A (zh) * 2020-09-30 2020-11-27 北京大橡科技有限公司 3d多器官共培养芯片
CN112574884A (zh) * 2020-11-19 2021-03-30 深圳先进技术研究院 基于微流控技术的多功能器官芯片、制备方法及其应用
CN114849801A (zh) * 2022-04-26 2022-08-05 复旦大学 通量化体外细胞、组织、器官培养和分析的微流控装置
CN114854585A (zh) * 2022-05-11 2022-08-05 中国中医科学院中药研究所 一种用于中药肝毒性评价的基于微流控体系的精密肝切片培养模型
CN114854585B (zh) * 2022-05-11 2022-09-30 中国中医科学院中药研究所 一种用于中药肝毒性评价的基于微流控体系的精密肝切片培养模型

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