[go: up one dir, main page]

EP4146394A1 - Dispositif microfluidique - Google Patents

Dispositif microfluidique

Info

Publication number
EP4146394A1
EP4146394A1 EP21722908.7A EP21722908A EP4146394A1 EP 4146394 A1 EP4146394 A1 EP 4146394A1 EP 21722908 A EP21722908 A EP 21722908A EP 4146394 A1 EP4146394 A1 EP 4146394A1
Authority
EP
European Patent Office
Prior art keywords
chamber
cells
hydrogel
main chamber
fluid channel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21722908.7A
Other languages
German (de)
English (en)
Inventor
Mario ROTHBAUER
Peter Ertl
Silvia Schobesberger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Wien
Original Assignee
Technische Universitaet Wien
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technische Universitaet Wien filed Critical Technische Universitaet Wien
Publication of EP4146394A1 publication Critical patent/EP4146394A1/fr
Pending legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • 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
    • C12M23/20—Material Coatings
    • 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
    • C12M23/34—Internal compartments or partitions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures

Definitions

  • the present invention relates to the field of microfluidic devices.
  • 3D cell culture techniques were combined with microfluidics, which makes this 3D cultivation method, known as organ-on-a-chip, more physiologically relevant.
  • Organ-on-a-Chips The main advantage of Organ-on-a-Chips is the precise spatially resolved control over molecules and fluids, whereby physiological molecular gradients and flows can be reflected. In addition, this control allows different cell types to be cultivated together in a controlled manner, which leads to more body-like functions of organoids or vascularization of the microchips.
  • Organ-on-a-chips as a disease model can be used on the one hand to identify new points of attack for combating disease and on the other hand to evaluate the toxicity and effectiveness of existing active ingredients.
  • Organ-on-a-chips represent an intermediate stage in the complexity between conventional 3D cell cultures and mouse models, which enables more precise drug development with increasing complexity and which could increase the number of drugs approved according to clinical studies.
  • Lungs, intestines, kidneys or placenta but also vascular and lymph models and heart and liver models, grown on chips.
  • stem cells can be differentiated into any cell type, it is possible to develop personalized microchips using individual stem cells.
  • organ-on-a-chips The application of organ-on-a-chips remains a challenge, since the 3D culture on the chip should represent all the important functions of an organ.
  • Such model systems must have a high level of reproducibility in order to be considered a valid alternative to animal models.
  • the technological framework conditions for the tool In order to make hydrogel-based organ-on-a-chip systems more reproducible, in addition to the cellular aspects (cell-cell variance and heterogeneity), the technological framework conditions for the tool must be designed as user-friendly as possible. This user-friendliness is often questionable, especially in the case of organ-on-a-chip developed in an academic research environment, since these microchips are usually developed and operated by trained scientific staff.
  • organs-on-a-chip developed in a scientific framework with regard to handling to be optimized in order to guarantee reproducible use by untrained personnel.
  • US 2013/059322 A1 discloses a microfluidic device for producing a three-dimensional cell culture which has a chamber.
  • US 2020/063081 A1 discloses a microfluidic device for producing a cell culture.
  • WO 2009/039433 A1 discloses a microfluidic system which comprises a sensor in a microfluidic device in order to determine the conditions prevailing in the device.
  • organ-on-a-chip devices generally have chambers in which an organoid can be formed by adding appropriate cells. Since the organoid formed in the chambers has a smaller volume than the chambers in which the organoid is located due to cell aggregation, the organoid formed move in the chambers depending on the flow conditions. This means that a measurement (e.g. an optical measurement) directly on the organoids is either impossible or difficult to carry out, which does not lead to a reproducible result. It is therefore an object of the present invention to provide means and methods that make it possible to position organoids within chambers of microfluidic devices at a defined location within the chambers in order to ensure the least possible mobility of the organoids.
  • a measurement e.g. an optical measurement
  • Another object of the present invention is to provide chambers of microfluidic devices that adjoin a fluid channel and are in contact with it, which allow hydrogel, which is advantageous as a matrix for the formation of organoids, in to introduce the chambers without this penetrating into the fluid channel.
  • a microfluidic device with at least one chamber and a fluid channel which at least partially traverses the chamber to provide a fluid flow preferably continuously flowing through the chamber, the chamber being connected to a charge opening and being loaded with hydrogel via the charge opening up to a target level, the chamber comprising a main chamber and a secondary chamber connected to the main chamber, the secondary chamber when loading the chamber with hydrogel up to the target level, it is at least partially filled with hydrogel backed up from the main chamber.
  • the secondary chamber is designed to be at least partially filled with hydrogel stowed back from the main chamber when the chamber is loaded with hydrogel up to the target level.
  • the microfluidic device preferably for producing a three-dimensional cell culture, in particular an organoid, comprises at least one chamber and a fluid channel which at least partially passes through the chamber for providing a fluid flow which preferably continuously passes through the chamber.
  • the chamber is connected to a charge opening and can be loaded with hydrogel via the charge opening up to a target level.
  • the chamber also comprises a main chamber and a secondary chamber connected to the main chamber, the secondary chamber being at least partially fillable with hydrogel when the chamber is loaded with hydrogel up to the target level.
  • the secondary chamber in connection with the fact that when the main chamber is filled with hydrogel up to the target level, the secondary chamber is also at least partially filled with hydrogel, has the advantage that when the three-dimensional cell culture is formed, part of the cell culture and, above all, part of itself Hydrogel compressing over time in the secondary chamber bil det.
  • this part located in the secondary chamber the entire cell culture is fixed in the chamber, which prevents the cell culture from slipping in the chamber. This improves the reproducibility of examinations and experiments carried out on the cell culture with various cell cultures which have been produced in several apparatuses according to the invention.
  • Another aspect of the present invention relates to a carrier comprising at least one microfluidic device according to the present invention.
  • microfluidic device according to the present invention
  • the Invention can be applied to a solid support.
  • the carrier comprises two or more of the devices according to the invention.
  • Yet another aspect of the present invention relates to a method for producing a three-dimensional cell culture or an organoid with a microfluidic device according to the invention, comprising a step (A) of loading a chamber of the device through a charge opening with hydrogel up to a target level .
  • three-dimensional cell cultures or organoids can be produced by introducing corresponding cells into the device.
  • hydrogel is used as the matrix.
  • Another aspect of the present invention relates to a method for determining the influence of a chemical compound and / or at least one physical parameter on cells of a three-dimensional cell culture in a microfluidic device according to the invention, comprising the steps: a) determining an initial state of the cells, bl) bringing the cells into contact with a chemical compound and / or b2) changing a physical parameter within the microfluidic device, c) determining a second state of the cells and d) determining the influence of the chemical compound and / or the at least one physical parameter the cells by finding a difference between the first state and second state of the cells.
  • the device of the present invention can be used to investigate the influence of a chemical compound and / or at least one physical parameter on cell len, in particular on a cell culture or on an orga noid which is located in the chamber of the device according to the invention.
  • a chemical compound and / or at least one physical parameter on cell len in particular on a cell culture or on an orga noid which is located in the chamber of the device according to the invention.
  • media and solutions can be reproducibly brought to the cells in the chambers via the fluid channel.
  • FIG. 1 shows a microfluidic device according to the invention
  • Figure 2 shows a side view of the microfluidic
  • FIG. 3 shows a plan view of a carrier with twelve microfluidic devices according to the invention.
  • FIG. 4 shows a side view of the carrier shown in FIG.
  • Figure 5a, Figure 5b and Figure 5c show the microfluidic device shown in Figure 2 during the loading of the device with hydrogel.
  • Figure 6a, Figure 6b and Figure 6c show a plan view of the microfluidic device during loading with hydrogel.
  • FIG. 7 shows a side view of the microfluidic
  • FIG. 8a, FIG. 8b and FIG. 8c show a side view of the microfluidic device according to the invention during the formation of a three-dimensional cell culture.
  • 9a, 9b and 9c show the microfluidic device from FIG. 8a, FIG. 8b and FIG. 8c in a side view.
  • FIG. 10 shows a top view of a microfluidic device according to the invention while primary human synovial fibroblasts remodel the hydrogel in order to, in the course of fourteen days to become a micromass with physiological structures.
  • FIG. 11 shows a change in the micromass structure of primary human synovial fibroblasts embedded in hydrogel.
  • FIG. 12 shows a course of the micromass condensation of primary human synovial fibroblasts embedded in hydrogel at different cell chamber heights.
  • FIG. 13 shows a plan view of a carrier with eight microfluidic devices according to the invention for a co-culture.
  • FIG. 14A and FIG. 14B show the condensation of micromasses in the device according to the invention as a function of the passage of the human synovial fibroblasts.
  • FIG. 15 shows the decrease in chondrocyte clumps over time with different media.
  • the device according to the invention which can be produced by known methods from the prior art (see, for example, Rothbauer M et al. (Lab Chip, 2018, 18, 249-270); Rothbauer M et al. (Biotech Adv, 2015, 33, 948-961); Rothbauer M et al. (Curr Opin Biotech, 2019, 55, 81-86)), is particularly suitable for the production of three-dimensional cell cultures and organoids and for simulating conditions similar to in vivo. Above all, the possibility of flowing a fluid around the cell cultures or the organoids is particularly advantageous with the device according to the invention.
  • Three-dimensional cell cultures have the advantage that the cells located therein, similar to organs in the body of a human or animal, adopt a spatial orientation. Such cell cultures consist of several cell layers, from which their three-dimensionality results. In order to facilitate the shaping, it is advantageous to add, for example, scaffold proteins / hydrogels such as collagen or matrigel to the cells. It has been shown that in Many cell lines form spheroids in a three-dimensional environment.
  • three-dimensional cell cultures are increasingly being used in order to be able to carry out certain investigations in an environment that is similar or modeled on nature.
  • pharmaceutical research which - in order to reduce the number of costly animal experiments in the initial phase of research projects - relies on three-dimensional cell cultures. Since three-dimensional cell cultures can exhibit organ-like behavior, meaningful results about the mode of action of an active ingredient can be achieved, which even allow conclusions to be drawn with regard to pharmacokinetics and pharmacodynamics.
  • Organoids are usually only a few millimeters in size, organ-like microstructures that can be produced with the device according to the invention. Usually organoids are grown from tissue cells, embryonic stem cells or induced pluripotent stem cells. Despite the fact that there is no stroma or vessels, they still show physiologically relevant, organ-like properties. Organoids are currently known for the heart, stomach, intestines, kidneys and brain.
  • the chamber is the one according to the invention
  • Target level means that enough hydrogel can be filled into the chamber until its main chamber and its secondary chamber are filled with hydrogel.
  • the secondary chamber must in any case also include hydrogel in order to form the anchor point of the three-dimensional cell culture or organoid that is being formed.
  • the main chamber and the secondary chamber are preferably connected by a connecting channel, the connecting channel having a smaller cross section than the main chamber and the secondary chamber.
  • the fluid channel runs at least partially through the main chamber, and the main chamber has a first chamber bottom.
  • the fluid channel also comprises a fluid inlet arranged at a distance from the first chamber floor in the main chamber, and a fluid outlet arranged at a distance from the first chamber floor in the main chamber.
  • the main chamber preferably has a first chamber floor and the secondary chamber has a second chamber floor, the first chamber floor being formed at a lower level than the second chamber floor.
  • the fluid channel expediently has a fluid channel floor, the fluid channel floor being formed at a higher level than the first chamber floor. This will prevents hydrogel from flowing while loading the chamber and fluid channel.
  • the fluid channel floor is formed at a lower level than the second chamber floor. This creates a reservoir for hydrogel in the main chamber, in which the cell culture can grow.
  • the difference in height between the fluid channel floor and the first chamber floor ensures that the hydrogel filled into the chamber does not pass into the fluid channel as long as the hydrogel volume filled into the chamber is not greater than the chamber volume.
  • the one filled hydrogel volume is preferably at least 5%, even more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30% less than the chamber volume.
  • the difference in height between the fluid channel bottom and the first chamber bottom is at least 0.1 mm, preferably at least 0.2 mm, even more preferably at least 0.25 mm, even more preferably at least 0.4 mm.
  • the ratio of the difference in height between the fluid channel base and the first chamber base and the height of the main chamber is 1: 5 to 1:15, preferably 1: 5 to 1:12, even more preferably 1: 5 to 1:10, even more preferably 1: 6 to 1:10, even more preferably 1: 7 to 1: 9, in particular 1: 8. It has been shown that with such height ratios the hydrogel introduced into the main chamber is not flushed out of the main chamber by a fluid flow in the fluid channel.
  • the height of the fluid channel extends from the fluid channel floor to the ceiling of the main chamber and is preferably in contact with it over the entire height.
  • the secondary chamber preferably has a smaller volume than the main chamber. As a result, a larger proportion of the mass of the cell culture is formed in the main chamber, and the side chamber is used to anchor the cell culture. This means that the part of the cell culture located in the main chamber can be used for experiments and investigations.
  • the charge opening is connected to the secondary chamber. This ensures that the hydrogel first falls into the secondary chamber before it passes into the main chamber. This has the advantage that it is ensured that the secondary chamber is also filled with hydrogel when the main chamber is filled.
  • the secondary chamber is preferably connected to the main chamber on a side of the main chamber opposite the fluid channel. This ensures that the hydrogel does not flow directly from the charge opening via the secondary hammer into the fluid channel when the chamber is filled.
  • a semipermeable membrane delimiting the fluid channel preferably a silicone membrane
  • the semipermeable membrane can be an additional obstacle to rinsing out the cell culture / organoid or the hydrogel arranged in the main chamber.
  • the device comprises a conduit connecting the charge opening to the chamber.
  • the main chamber preferably has a spatial diagonal which is 2 to 20 mm, preferably 2 to 15 mm, even more preferably 3 to 10 mm, even more preferably 4 to 6 mm.
  • the fluid channel base expediently has a distance of 0.1 to 10 mm, preferably 0.2 to 5 mm, even more preferably 0.2 to 2 mm, even more preferably 0.2 to 1 mm , more preferably from 0.2 to 0.4 mm, from the first chamber bottom.
  • the fluid channel base expediently has a distance of 0.1 to 10 mm, preferably 0.2 to 5 mm, even more preferably 0.2 to 2 mm, even more preferably 0.2 to 1 mm , more preferably from 0.2 to 0.4 mm, from the first chamber bottom.
  • an antifouling surface coating which is preferably selected from the group consisting of a polyethylene glycol (PEG) -based polymer, a polysaccharide, in particular agarose, a polyhydroxy polymer, in particular poly (2-hydroxyethyl methacrylate) (poly-HEMA), 2-methacryloyloxtethylphosphorylcholine (MPC), dextran or hydroxyethyl cellulose (HEC), a natural polymer, in particular an S-layer protein and combinations thereof.
  • PEG polyethylene glycol
  • polysaccharide in particular agarose
  • polyhydroxy polymer in particular poly (2-hydroxyethyl methacrylate) (poly-HEMA), 2-methacryloyloxtethylphosphorylcholine (MPC), dextran or hydroxyethyl cellulose (HEC)
  • MPC 2-methacryloyloxtethylphosphorylcholine
  • HEC hydroxyethyl cellulose
  • the carrier according to the invention comprises at least one microfluidic device according to the invention. This has the advantage that one or more devices according to the invention can be produced or provided together on a carrier.
  • FIG. 1 shows a microfluidic device according to the invention
  • Device 1 preferably for producing a three-dimensional cell culture in a top view, with at least one chamber 2 and a fluid channel 3 which at least partially passes through the chamber 2.
  • the device has a chamber 2, which is traversed by the fluid duct in an edge region of the chamber 2.
  • the fluid channel 3 is designed to provide a fluid stream preferably continuously flowing through the chamber 2.
  • the chamber 2 is connected to a charge opening 4 and can be loaded with hydrogel via the charge opening 4 up to a target level.
  • the loading with hydrogel is shown in FIGS. 5a, 5b and 5c, and is dealt with further below.
  • the chamber 2 has a main chamber 5 and a secondary chamber 6 connected to the main chamber 5.
  • the main chamber 5 and the secondary chamber 6 can be seen in FIG. 1 and in FIG. 2, FIG. 2 showing a side view of the microfluidic device 1 shown in FIG.
  • the secondary chamber 6 can be at least partially filled with hydrogel when the chamber 2 is loaded with hydrogel up to the desired fill level.
  • the entire cell culture is fixed in the chamber 2, which prevents the cell culture from slipping in the chamber 2. This improves the reproducibility of examinations and experiments carried out on the cell culture with different cell cultures which were produced in several devices 1 according to the invention.
  • the main chamber 5 and the secondary chamber 6 according to the preferred embodiment of the device 1 according to the invention are connected by a connecting channel 7, the connecting channel
  • the fluid channel 3 runs at least partially through the main chamber 5, and the main chamber 5 has a first chamber bottom 8.
  • the fluid channel 3 also comprises a fluid inlet arranged in the main chamber 5 at a distance from the first chamber bottom 8 9, as well as a fluid outlet 10 arranged at a distance from the first chamber bottom 8 in the main chamber 5.
  • the arrangement of fluid inlet 9 and fluid outlet 10 can be seen in FIG. This has the advantage that a step is formed between the main chamber 5 and the fluid inlet 9 or the fluid outlet 10, which prevents hydrogel from flowing into the fluid inlet 9 or the fluid outlet 10 when the chamber 2 is loaded.
  • the main chamber 5 preferably has the first chamber floor 8, and the secondary chamber 6 has a second chamber floor 11.
  • the first chamber floor 8 is preferably formed at a lower level than the second chamber floor 11. This has the advantage that the majority of the hydrogel accumulates in the main chamber 5, and the secondary chamber 6 provides an anchorage for a cell culture largely produced or grown in the main chamber 5.
  • the fluid channel 3 expediently has a fluid channel base 16 shown in FIG. 2, the fluid channel base 16 being formed at a higher level than the first chamber base 8. This prevents hydrogel from flowing into the fluid channel 3 when the chamber 2 is loaded.
  • the fluid channel floor 16 is formed at a lower level than the second chamber floor 11.
  • a collecting basin for hydrogel is formed in the main chamber 5, in which the cell culture can grow.
  • the preferred embodiment shown in the figures has a smaller volume than the main chamber 5.
  • a larger proportion of the mass of the cell culture in the main chamber 5 forms ge, and the secondary chamber 6 is used to anchor the cell culture.
  • the part of the cell culture located in the main chamber 5 can be used for experiments and investigations.
  • the secondary chamber 6 is preferably connected to the main chamber 5 on a side 12 of the main chamber 5 opposite the fluid channel. This ensures that the hydrogel does not flow directly from the charge opening 4 via the secondary chamber 6 into the fluid channel 3 when the chamber 2 is filled.
  • a semipermeable membrane 14 delimiting the fluid channel 3 is expediently arranged in the main chamber 5.
  • the silicone membrane 14 is never shown in Figure 2 with a dashed line.
  • the semipermeable membrane 14 forms an obstacle to the rinsing out of the cell culture arranged in the main chamber 5 or the hydrogel by the fluid flow in the fluid channel 3.
  • the device 1 comprises a line 15 connecting the charge opening 4 to the chamber 2. This can be seen in FIG. This has the advantage that the hydrogel can be filled into the chamber 2 in a reproducible manner, whereby a reproducible distribution of the hydrogel in the chamber 2 is achieved.
  • the main chamber 5 preferably has a spatial diagonal which is 2 to 20 mm, preferably 2 to 15 mm, even more preferably 3 to 10 mm, even more preferably 4 to 6 mm.
  • the fluid channel base 16 also expediently has a distance of 0.1 to 10 mm, preferably 0.2 to 5 mm, even more preferably 0.2 to 2 mm, even more preferably 0.2 to 1 mm , even more preferably from 0.2 to 0.4 mm, from the first chamber bottom 8.
  • Chamber 2 the main chamber 5, the secondary chamber 6 and / or the fluid channel 3 comprises a surface coating that prevents growth (not shown in the figures), this being the case is preferably selected from the group consisting of a polyethylene glycol (PEG) -based polymer, a poly saccharide, especially agarose, a polyhydroxy polymer, especially poly (2-hydroxyethyl methacrylate) (poly-HEMA), 2-methacryloyloxtethylphosphorylcholine (MPC), or dextran Hydroxyethyl cellulose (HEC), a natural polymer, in particular an S-layer protein and combinations thereof and / or a growth-preventing material based on fluorinated silanes or phosphatidylcholine coatings. This prevents unwanted growth of cells on these components.
  • PEG polyethylene glycol
  • poly saccharide especially agarose
  • polyhydroxy polymer especially poly (2-hydroxyethyl methacrylate) (poly-HEMA), 2-methacryloyloxteth
  • FIG. 3 shows a carrier 17 according to the invention which comprises at least one microfluidic device 1 according to the invention.
  • the carrier 17 shown in FIG. 3 comprises 12 microfluidic devices 1 according to the invention. This has the advantage that one or more devices 1 according to the invention can be produced or provided together on a carrier 17.
  • the carrier 17 shown by way of example in FIG. 3 has dimensions of 76 mm by 52 mm.
  • FIG. 4 shows the carrier 17 from FIG. 3 in a side view.
  • FIGS. 5a, 5b and 5c Spread shown in FIGS. 5a, 5b and 5c in a plan view, with a syringe which is used for introducing the hydrogel into the charge opening 4.
  • FIG. 7 shows the device 1 according to the invention in a state loaded with hydrogel, the fluid channel 3 being marked with a hatched area and being free of hydrogel.
  • Another aspect of the present invention relates to a method for producing a three-dimensional cell culture or an organoid with a microfluidic one Device according to the present invention, comprising a step (A) of loading a chamber of the device according to the invention through a loading opening with hydrogel up to a desired fill level.
  • hydrogel is introduced into the chamber. So much hydrogel is poured into the chamber until the hydrogel backs up to the side chamber. In such a case, it is ensured that the contracting hydrogel or cell / hydrogel mixture remains fixed in the secondary chamber of the main chamber.
  • cells are introduced together into the chamber (2) in and / or before step (A) through a charge opening and / or via a fluid inlet (9).
  • the cells can be applied into the chamber after and / or before step (A) (introduction of hydrogel into the chamber of the device according to the invention).
  • step (A) introduction of hydrogel into the chamber of the device according to the invention.
  • the cells are selected from stem cells and primary cells, preferably fibroblasts, cartilage cells, endothelial cells, epithelial cells, fat cells, induced pluripotent stem cells (IPS), osteoclasts, osteoblasts or osteocytes.
  • stem cells and primary cells preferably fibroblasts, cartilage cells, endothelial cells, epithelial cells, fat cells, induced pluripotent stem cells (IPS), osteoclasts, osteoblasts or osteocytes.
  • Cells in particular animal or human cells, of various origins can be introduced into the device according to the invention, provided that they are able to form a three-dimensional structure (such as an organoid). If, for example, stem cells are introduced into the device, they can be differentiated directly in the device using known methods.
  • the hydrogel comprises or consists of matrix, fibrin, collagen, PEG-based hydrogel, gelatin-based hydrogel, hyaluronic acid-based hydrogel, alginate-based hydrogel, silk-based Hydrogel and combinations thereof.
  • the hydrogel preferably has a concentration of 0.1 mg / ml to 500 mg / ml, more preferably 0.5 mg / ml to 500 mg / ml.
  • the device according to the invention is incubated after step (A) for 12 hours to 60 days, preferably for 1 to 30 days, even more preferably for 2 to 20 days.
  • the device according to the invention is incubated at a temperature of 25 to 38 ° C, preferably 37 to 38 ° C.
  • the temperature at which the device according to the invention is incubated depends on the cells to be cultivated and is preferably varied in a range from 25 to 38.degree.
  • step (A) in a step (B) the chamber is supplied with a fluid flow flowing continuously or discontinuously through a fluid channel, the fluid preferably being changed in the case of a discontinuous fluid flow.
  • the fluid flow in the fluid channel can be continuous or discontinuous (ie with interruptions or a changed flow rate). It is also possible to determine the composition of the fluid (eg nutrient medium) which flowing through the fluid channel, to change over time or during the cultivation of the cells. In this way, on the one hand, natural processes can be simulated, since the nutrient supply or a fluid flow that "flows around" tissue changes or can change in the human or animal body.
  • the change in the flow rate and composition of the fluid in the fluid channel also has the advantage that Influence of fluid flows and substances on cell growth or the condition of the cells within the device according to the invention can be examined.
  • the CO2 and / or Ck content and / or the pH value within the device and / or the fluid flow is controlled.
  • Yet another aspect of the present invention relates to a method for determining the influence of a chemical compound and / or at least one physical parameter on cells of a three-dimensional cell culture in a microfluidic device according to the invention, comprising the steps: a) determining an initial state of the cells , bl) bringing the cells into contact with a chemical compound and / or b2) changing a physical parameter within the microfluidic device, c) determining a second state of the cells and d) determining the influence of the chemical compound and / or the at least one physical rule Parameters on the cells by determining a difference between the first state and the second state of the cells.
  • the device according to the invention it is possible to influence the influence of physical parameters or substances or substance mixtures on three-dimensional cell cultures or organoids.
  • the first thing to do is to determine the state of the cells before adding a substance or a mixture of substances or before changing the physical parameters.
  • purely optical methods can also be used, in which the optical state of the culture or the organoid is observed.
  • the same examinations are carried out according to step bl) and / or b2) in order to establish a second state.
  • the influence of a physical parameter or a substance or a substance mixture on the cells can be derived from the difference between the first and second state.
  • the influence of the chemical compound and / or of the at least one physical parameter on the cells can preferably be determined by measuring the viability, morphology, secretion of substances from the cells, optical transparency and / or opacity or combinations thereof.
  • the influence of the chemical compound and / or of the at least one physical parameter on the cells is determined by measuring the release of biomolecules, in particular of antibodies, chemokines, cytokines, enzymes or microRNA
  • the physical parameter is selected from the group consisting of temperature and pH.
  • the present invention is made based on the following
  • Microfluidic device (1) preferably for producing a three-dimensional cell culture, with at least one chamber (2) and a fluid channel (3) which at least partially runs through the chamber (2) to provide a chamber (2) preferably continuously flowing fluid flow, wherein the chamber (2) is connected to a charge opening (4) and can be loaded with hydrogel via the charge opening (4) up to a target level, characterized in that the chamber (2) has a main chamber (5) and comprises a secondary chamber (6) connected to the main chamber (5), the secondary chamber (6) being at least partially fillable with hydrogel up to the target level when the chamber (2) is loaded with hydrogel.
  • Device (1) according to one of the embodiments 1 or 2, characterized in that the fluid channel (3) at least partially passes through the main chamber (5), and the main chamber (5) has a first chamber bottom (8), wherein the fluid channel (3) has a fluid inlet (9) arranged at a distance from the first chamber floor (8) in the main chamber (5) and a fluid outlet (10) arranged at a distance from the first chamber floor (8) in the main chamber (5) to summarize.
  • Device (1) according to one of the embodiments 1 to 6, characterized in that the secondary chamber (6) has a smaller volume than the main chamber (5).
  • Device (1) characterized in that the secondary chamber (6) on a side (12) of the main chamber (5) with the main chamber (5) opposite the fluid channel (3) ) connected is.
  • Device (1) according to one of the embodiments 1 to 9, characterized in that the fluid channel (3) runs along a side wall (13) of the main chamber (5).
  • Device (1) according to one of the embodiments 1 to 10, characterized in that in the main chamber
  • a semipermeable membrane (14), preferably a silicone membrane, delimiting the fluid channel (3) is arranged.
  • Device (1) according to one of the embodiments 1 to 11, characterized in that the device (1) comprises a line (15) connecting the charge opening (4) to the chamber (2).
  • Device (1) according to one of the embodiments 1 to 12, characterized in that the main chamber (5) has a spatial diagonal which is 2 to 20 mm, preferably 2 to 15 mm, even more preferably 3 to 10 mm, more preferably 4 to 6 mm.
  • the fluid channel base (16) has a distance of 0.1 to 10 mm, preferably 0.2 to 5 mm, even more preferably from 0.2 to 2 mm, even more preferably from 0.2 to 1 mm, even more preferably from 0.2 to 0.4 mm, from the first chamber bottom (8).
  • the chamber (2), the main chamber (5), the secondary chamber (6) and / or the fluid channel (3) comprises an antifouling surface coating, which is preferably selected from the group consisting of one Polyethylene glycol (PEG) -based polymer, a polysaccharide, in particular agarose, a polyhydroxy polymer, in particular poly (2-hydroxyethyl methacrylate) (poly-HEMA), 2-methacryloyloxtethylphosphorylcholine (MPC), dextran or hydroxyethyl cellulose (HEC), a natural polymer, especially an S-layer protein and combinations thereof.
  • PEG Polyethylene glycol
  • polysaccharide in particular agarose
  • polyhydroxy polymer in particular poly (2-hydroxyethyl methacrylate) (poly-HEMA), 2-methacryloyloxtethylphosphorylcholine (MPC), dextran or hydroxyethyl cellulose (HEC)
  • S-layer protein preferably selected from the group consisting of one Poly
  • Carrier (17) comprising at least one microfluidic device according to one of claims 1 to 15.
  • Cell culture or an organoid with a microfluidic device (1) comprising a step (A) of loading a chamber (2) of the device (1) through a loading opening (4) with hydrogel up to a target level .
  • the cells are selected from stem cells and primary cells, preferably fibroblasts, cartilage cells, endothelial cells, epithelial cells, fat cells, induced pluripotent stem cells (IPS), osteoclasts, osteoblasts or the Osteocytes.
  • stem cells and primary cells preferably fibroblasts, cartilage cells, endothelial cells, epithelial cells, fat cells, induced pluripotent stem cells (IPS), osteoclasts, osteoblasts or the Osteocytes.
  • the hydrogel is Matrigel, fibrin, collagen, PEG-based hydrogel, gelatin-based hydrogel, hyaluronic acid-based hydrogel, alginate-based hydrogel, silk-based hydrogel and includes or consists of combinations thereof.
  • 21 The method according to any one of embodiments 17 to 20, characterized in that the device (1) after step (A) for 12 hours to 60 days, preferably for 1 to 30 days, even more preferably for 2 to 20 days , is incubated.
  • step (A) in a step (B) the chamber (2) is supplied with a fluid flow flowing continuously or discontinuously through a fluid channel (3) is, wherein in the case of a discontinuous fluid flow, the fluid is preferably changed.
  • a method for determining the influence of a chemical compound and / or at least one physical parameter on cells of a three-dimensional cell culture in a microfluidic device comprising the steps: a) determining an initial state of the cells , bl) bringing the cells into contact with a chemical compound and / or b2) changing a physical parameter within the microfluidic device, c) determining a second state of the cells and d) determining the influence of the chemical compound and / or the at least one physical parameter on the cells by finding a difference between the first state and second state of the cells.
  • the physical parameter is selected from the group consisting of temperature and pH value.
  • a slide (76 ⁇ 26 mm) from VWR, it was first placed in a 2% Hellmanex® III solution and treated in an ultrasonic bath for 5 minutes. The solution was then discarded and the process was repeated first with isopropanol and then with water. Finally, the slides were dried with compressed air and in the oven at 80 ° C. for 1 hour.
  • Layers (76 x 26 mm) can be cut out.
  • 0.5 mm thick PDMS film was inserted into the C ⁇ MM-1 GS-24 cutter from Roland DG and the desired shape was cut out, which was removed with tweezers.
  • the individual layers were connected to one another by plasma activation, starting with the microscope slide and the first layer of PDMS film.
  • the strength of the connection was increased by heat treatment in an oven (80 ° C).
  • Synovial fibroblasts were cultivated with culture medium (DMEM, 10% FBS, 1% antibiotic, 1% MEM NEAA) in culture flasks (37 ° C., 5% CO 2) and released from the flask at 90% confluence. For this purpose, the cells were incubated for 5 minutes with IX trypsin solution at 37 ° C. and then medium was added to stop the reaction. The cell suspensions were counted using trypan blue and centrifuged (4 ° C., 1400 rpm, 5 min). The cell pellet was then resuspended in medium and the required amount of cells was separated.
  • culture medium DMEM, 10% FBS, 1% antibiotic, 1% MEM NEAA
  • the cell pellet was mixed with Matrigel (3000 cells per m2 Matrigel) and the chips were filled with 45 m2 cell / Matrigel suspension per cell chamber.
  • 3D culture medium was added (DMEM, 10% FBS, 1% antibiotic, 1% MEM NEAA, 1% ITS, 31.6 pg / ml L-ascorbic acid 2-phosphate, 2% HEPES).
  • the chips were cultivated for up to 4 weeks at 37 ° C. and 5% CO 2, the medium being changed twice a week.
  • the synovial fibroblasts enter into cell-cell connections and begin to pour out their own extracellular matrix.
  • the matrigel in which the cells are initially located, is remodeled and the so-called micromass condenses over time due to the strong cell connections (FIG. 10).
  • the condensation takes place in a controlled manner back towards the charge opening, since the remaining cells in the charge channel form a miniaturized micro-mass. This cannot enter the actual cell chamber because the inlet channel is narrowed.
  • the miniaturized micromass is connected to the large micromass in the cell chamber through cells.
  • a micromass similar to synovial tissue is created.
  • Synovial tissue is characterized by a denser cell layer in the direction of the medium, as well as a rather loose tissue inside the micromass.
  • the condensation changes the structure, because at the beginning the cell / matrigel suspension polymerizes in a cylinder-like chamber.
  • a spherical micro-mass is formed.
  • phase contrast images were recorded at regular intervals along the z-axis of the micromass.
  • the focal plane the size of the cross-section could be determined at different positions.
  • Figure 11 shows that the shape changes little after 14 days.
  • a comparison with a sphere shows that the curve should, however, be somewhat different.
  • the assumption is that the micromass is ellipsoidal in shape, since the diameter of the chamber is 2.5 times the height.
  • a slide (76 ⁇ 26 mm) from VWR, it was first placed in a 2% Hellmanex® III solution and treated in an ultrasonic bath for 5 minutes. The solution was then discarded and the process was repeated first with isopropanol and then with water. Finally, the slides were dried with compressed air and in the oven at 80 ° C. for 1 hour.
  • Layers (76 x 26 mm) can be cut out.
  • 0.5 mm thick PDMS film was inserted into the C ⁇ MM-1 GS-24 cutter from Roland DG and the desired shape was cut out, which was removed with tweezers.
  • the individual layers were connected to one another by plasma activation, starting with the microscope slide and the first layer of PDMS film.
  • the strength of the connection was increased by heat treatment in an oven (80 ° C).
  • all chambers, channels and reservoirs were rinsed with 70% ethanol.
  • the cell chambers were also coated with Lipidure®, an antifouling agent. Finally, the chip was sterilized with UV light.
  • MEM NEAA MEM NEAA cultured in culture flasks (37 ° C, 5% C02). At 90% confluence, the cells were released from the bottle. For this purpose, the cells were incubated for 5 minutes with IX trypsin solution at 37 ° C. and then medium was added in order to stop the reaction. The cell suspensions were counted using trypan blue and centrifuged (4 ° C., 1400 rpm, 5 min). The cells were resuspended in medium and some were subcultured, and another part was separated with the required amount of cells for the chip cultures. After centrifuging again, the cell pellet was mixed with Matrigel (3000 cells per m2 Matrigel) and the chips were filled with 45 m2 cell / Matrigel suspension per cell chamber.
  • Matrigel Matrigel (3000 cells per m2 Matrigel) and the chips were filled with 45 m2 cell / Matrigel suspension per cell chamber.
  • 3D culture medium was added (DMEM, 10% FBS, 1% antibiotic, 1% MEM NEAA, 1% ITS, 31.6 pg / ml L-ascorbic acid 2-phosphate, 2% HEPES ).
  • DMEM 10% FBS
  • antibiotic 1% MEM NEAA
  • ITS 31.6 pg / ml L-ascorbic acid 2-phosphate
  • HEPES 3D culture medium
  • FIG 14A shows that the higher the passage, the smaller the micro-mass, which is based on better micro-mass formation.
  • Figure 14B it can be seen that the micromass formed from passage 5 cells forms a significantly rounder and better-developed micromass, while the micromass from passage 1 cells after 4 weeks still consists of clusters and does not form a characteristic, dense outer layer in the direction of the medium.
  • the optical one The appearance of the micromass already gives essential information about the condition of the cells.
  • Example 3 Reduction in size as an indicator for the redifferentiation of chondrocytes
  • a slide (76 ⁇ 26 mm) from VWR, it was first placed in a 2% Hellmanex® III solution and treated in an ultrasonic bath for 5 minutes. The solution was then discarded and the process was repeated first with isopropanol and then with water. Finally, the slides were dried with compressed air and in the oven at 80 ° C. for 1 hour.
  • Layers (76 x 26 mm) can be cut out.
  • 0.5 mm thick PDMS film was inserted into the C ⁇ MM-1 GS-24 cutter from Roland DG and the desired shape was cut out, which was removed with tweezers.
  • the individual layers were connected to one another by plasma activation, starting with the microscope slide and the first layer of PDMS film.
  • the strength of the connection was increased by heat treatment in an oven (80 ° C).
  • the cell pellet was carefully mixed with fibrin (2000 cells per m ⁇ fibrin) and aliquoted with 30 ml each.
  • fibrin 2000 cells per m ⁇ fibrin
  • 30 ml of thrombin (4 U / ml) was mixed with fibrin and immediately loaded into the chip.
  • the chip was incubated for 15 minutes at 37 ° C so that a solid 3D network formed.
  • chip culture medium (DMEM, 1% antibiotic, 1% MEM NEAA, 1% ITS, 31.6 pg / ml L-ascorbic acid 2-phosphate, 2% HEPES) with different concentrations of FBS and TGF- ⁇ 3 or chondrocytes Sigma Aldrich differentiation medium (411D-250) added.
  • the chips were cultivated for up to 4 weeks at 37 ° C. and 5% CO 2, with the medium being exchanged every other day.
  • a medium should be found which can be used for both cell cultures on the chip.
  • the medium based on a synovial fibroblast culture on the chip was changed so that the serum concentration was varied (1 and 10%) and TGF- ⁇ 3, which is necessary for the redifferentiation of chondrocytes, was added.
  • These media were compared to a purchased differentiation medium.
  • Figure 15 shows that the size of the chondrocyte clump decreases significantly more over a time frame of 21 days for the self-defined media. While with the purchased differentiation medium the clump is still larger than 50% of the originally loaded construct after 21 days. It can be seen that the decrease is initially lower with a lower serum concentration, but after 21 days the size equals that which was cultivated with a higher serum concentration.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Sustainable Development (AREA)
  • General Engineering & Computer Science (AREA)
  • Clinical Laboratory Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Dispersion Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un dispositif microfluidique (1) de préférence destiné à la production d'une culture cellulaire tridimensionnelle, comprenant au moins une chambre (2) et un canal fluidique (3) qui parcourt au moins partiellement la chambre (2) et sert à fournir un courant de fluide qui parcourt la chambre (2) de préférence en continu, la chambre (2) étant reliée à une ouverture de chargement (4) et pouvant être chargée en hydrogel via l'ouverture de chargement (4) jusqu'à un niveau de remplissage théorique. Le dispositif selon l'invention est caractérisé en ce que la chambre (2) comprend une chambre principale (5) et une chambre secondaire (6) reliée à la chambre principale (5), la chambre secondaire (6), lors du chargement de la chambre (2) en hydrogel jusqu'au niveau de remplissage théorique, étant remplie au moins partiellement avec de l'hydrogel retenu de la chambre principale (5).
EP21722908.7A 2020-05-08 2021-05-05 Dispositif microfluidique Pending EP4146394A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP20173705.3A EP3907007A1 (fr) 2020-05-08 2020-05-08 Dispositif microfluidique
PCT/EP2021/061859 WO2021224328A1 (fr) 2020-05-08 2021-05-05 Dispositif microfluidique

Publications (1)

Publication Number Publication Date
EP4146394A1 true EP4146394A1 (fr) 2023-03-15

Family

ID=70740428

Family Applications (2)

Application Number Title Priority Date Filing Date
EP20173705.3A Withdrawn EP3907007A1 (fr) 2020-05-08 2020-05-08 Dispositif microfluidique
EP21722908.7A Pending EP4146394A1 (fr) 2020-05-08 2021-05-05 Dispositif microfluidique

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP20173705.3A Withdrawn EP3907007A1 (fr) 2020-05-08 2020-05-08 Dispositif microfluidique

Country Status (3)

Country Link
US (1) US20230203417A1 (fr)
EP (2) EP3907007A1 (fr)
WO (1) WO2021224328A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102021214281A1 (de) * 2021-12-14 2023-06-15 Robert Bosch Gesellschaft mit beschränkter Haftung Vorrichtung und Verfahren zum Splitten dreidimensionaler Agglomerate
EP4289933A1 (fr) * 2022-06-07 2023-12-13 ibidi GmbH Substrat de culture cellulaire
WO2025186340A1 (fr) * 2024-03-05 2025-09-12 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Dispositif de création de structures cellulaires tridimensionnelles dans un hydrogel, microsystème et/ou plateforme de culture cellulaire microfluidique et procédé de production du dispositif
WO2025202125A1 (fr) * 2024-03-28 2025-10-02 F. Hoffmann-La Roche Ag Dispositif de culture cellulaire

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9637715B2 (en) * 2005-07-07 2017-05-02 Emd Millipore Corporation Cell culture and invasion assay method and system
WO2009039433A1 (fr) * 2007-09-20 2009-03-26 Incept Biosystems Inc. Système de culture analytique microfluidique
CN102112593A (zh) * 2008-08-01 2011-06-29 智能生物系统公司 生物反应器平台的腔室
WO2017027838A1 (fr) * 2015-08-13 2017-02-16 President And Fellows Of Harvard College Dispositifs et systèmes microfluidiques pour culture cellulaire et/ou essai cellulaire
WO2017216113A2 (fr) * 2016-06-15 2017-12-21 Mimetas B.V. Dispositif et procédés de culture cellulaire
WO2018132831A2 (fr) * 2017-01-13 2018-07-19 Brandeis University Dispositifs pour la génération et le stockage simultanés et le stockage de gouttelettes isolées, et leurs procédés de fabrication et d'utilisation
US12104149B2 (en) * 2018-05-15 2024-10-01 The University Of North Carolina At Chapel Hill Devices, systems and apparatuses for generating self-sustaining hypoxic conditions and gaseous and non-gaseous chemical gradients for in vitro cell culture
KR102083083B1 (ko) * 2018-06-21 2020-02-28 연세대학교 산학협력단 고효율 오가노이드 배양 디바이스 및 배양 시스템

Also Published As

Publication number Publication date
US20230203417A1 (en) 2023-06-29
WO2021224328A1 (fr) 2021-11-11
EP3907007A1 (fr) 2021-11-10

Similar Documents

Publication Publication Date Title
WO2021224328A1 (fr) Dispositif microfluidique
Xie et al. Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes
TWI741980B (zh) 一種生物磚及其用途
DE60204352T2 (de) Haut/haar-äquivalent mit rekonstruierten papillen
EP2569141B1 (fr) Procédé pour réaliser une reproduction d'organe, en particulier un modèle fonctionnel
Rosellini et al. Microfluidic fabrication of natural polymer-based scaffolds for tissue engineering applications: a review
US9081003B2 (en) Systems and methods for testing drugs and drug delivery systems
EP3020480B1 (fr) Conduite de fluide destiné à l'examen de cellules
DE102019132211B3 (de) 3D-Gerüst aus biokompatiblem Polymer mit einem nach oben offenen Besiedlungsraum für biologische Zellen und mit einem den Besiedlungsraum umgebenden kanalförmigen Gefäß
Yin et al. Microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes for salivary gland tissue engineering
DE102018221838B3 (de) Mikrophysiologisches Choroidea-Modell
DE102019132214B3 (de) Mit biologischen Zellen besiedeltes 3D-Gerüst aus biokompatiblem Polymer und dessen Herstellung
US20250250522A1 (en) Recapitulating Tissue-Native Architectures in Bio-printable Hydrogels
EP1171572A2 (fr) Systemes modulaires de support cellulaire pour la croissance cellulaire en trois dimensions
DE102008056037A1 (de) Mikrofluidischer Bioreaktor
EP4274928B1 (fr) Non-tissés de nanofibres à porosité élevée en tant que structure de support pour tissu stromale
DE102019132180B3 (de) 3D-Gerüst aus biokompatiblem Polymer mit überdachtem Hohlraum zur Besiedlung mit biologischen Zellen
WO2016001442A1 (fr) Modèle tissulaire, procédé de fabrication et utilisation dudit modèle tissulaire
Wang et al. 3D bioprinting composite tissue
Mandaric Microfluidic Fabrication of Mineralized Alginate Hydrogel Fibers
HK1254994A1 (en) Organ mimic device with microchannels and methods of use and manufacturing thereof
WO2000070013A2 (fr) Microcapillaire constituant un systeme de culture
Youm et al. Concentric Multilayered Sphere Bioprinting of Tumor Assembloids with Physiologically Relevant Microenvironments
HK1206778B (en) Organ mimic device with microchannels and methods of use and manufacturing thereof
HK1206778A1 (en) Organ mimic device with microchannels and methods of use and manufacturing thereof

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20221206

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)