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WO2025073379A1 - Dispositif pour l'étude des interactions d'un échantillon biologique - Google Patents

Dispositif pour l'étude des interactions d'un échantillon biologique Download PDF

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Publication number
WO2025073379A1
WO2025073379A1 PCT/EP2023/077740 EP2023077740W WO2025073379A1 WO 2025073379 A1 WO2025073379 A1 WO 2025073379A1 EP 2023077740 W EP2023077740 W EP 2023077740W WO 2025073379 A1 WO2025073379 A1 WO 2025073379A1
Authority
WO
WIPO (PCT)
Prior art keywords
wells
well
conduit
aforementioned
biological specimen
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
PCT/EP2023/077740
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English (en)
Inventor
Jan Lichtenberg
Olivier Frey
Lisa HÖLTING
Michal RUDNIK
Özlem YAVAS
Frauke Greve
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.)
INSPHERO AG
Original Assignee
INSPHERO AG
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 INSPHERO AG filed Critical INSPHERO AG
Priority to PCT/EP2023/077740 priority Critical patent/WO2025073379A1/fr
Publication of WO2025073379A1 publication Critical patent/WO2025073379A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C12M23/12—Well or multiwell plates
    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • 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
    • 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
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls

Definitions

  • the present application relates to a device for studying interactions of biological specimen.
  • 3D tissue cultures are more and more used in the testing or screening of compounds for their toxicity, immunogenity or therapeutic effect.
  • Figure 1 gives a general overview of an embodiment of the device 10 for culturing and/or investigating biological specimen 16.
  • Figure 1A shows a cross section of such device.
  • the device comprises a plurality of wells 11, wherein at least two wells 11 arranged next to one another each comprise an open upper section 12, and a lower section 13.
  • the upper section and the lower section are in fluid communication.
  • the lower section 13 has a smaller cross-sectional area than the open upper section 12.
  • the wells comprise an intermediate section disposed between the open upper section 12, and the lower section 13, said intermediate section comprising a pipetting stage 17 having an inclination.
  • the wells comprise a chute 18 chute being provided between the pipetting stage 17 and the lower section 13.
  • a base layer 14 is provided that forms the bottom of at least one well 11 and at least one conduit 15. At least two lower sections 13 of at least two wells 11 are connected to one another by a conduit 15.
  • the conduit is formed by longitudinal grooves 24a in the basal parts of some wells, and the upper side of the base layer 14.
  • At least one set of at least two wells is arranged relative to at least one other set of at least two wells in such way that the conduits 15a, 15b are arranged in essentially rectangular fashion relative to one another.
  • Figure 2 shows two other embodiments of the device 10 for culturing and/or investigating biological specimen 16.
  • the conduit 15 is formed by longitudinal grooves 25 in the upper side of the base layer 14, which create a distance between the nasal parts of the wells and the base layer 14, thus determining the height of the conduit 15.
  • the height of the conduit 15 can be chosen to be as high or even higher than the diameter of the biological specimen 16, or can be chosen to be smaller than the diameter of the biological specimen 16.
  • Figures 1 and 2 are not necessarily drawn to scale.
  • the conduit 15 connects two lower sections 13 of two wells to one another. As described in the specification, even more wells can be connected by the conduit.
  • Figure 2B the conduit 15 four lower sections 13 of four wells to one another
  • Figure 3 shows other embodiments of the device 10 for culturing and/or investigating biological specimen 16, comprising a separation layer 22 arranged between the lower section 13 of the well and the conduit 15.
  • Said separation layer 22 may comprise a mesh or a semipermeable membrane.
  • a cell monolayer 23 is grown on the separation layer.
  • the base layer 14 is connected to the device 10 by a series of layers comprising adhesive layers 20a, 20b and 20c, as well as a spacer layer 21 that mainly determines the height of the conduit 15. This embodiment can also be put into practice without the separation layer 22.
  • the height of the conduit 15 can be chosen to be as high or even higher than the diameter of the biological specimen 16, or can be chosen to be smaller than the diameter of the biological specimen 16.
  • Figure 3 is not necessarily drawn to scale.
  • Figure 4 shows other embodiments of the device 10 for culturing and/or investigating biological specimen 16, with different well shapes.
  • the wells are essentially funnel-shaped while in Figure 4B, the intermediate section disposed between the open upper section 12 and the lower section 13 is hemispherical.
  • the height of the conduit 15 can be chosen to be as high or even higher than the diameter of the biological specimen 16, or can be chosen to be smaller than the diameter of the biological specimen 16.
  • Figure 3 is not necessarily drawn to scale.
  • Figure 5 shows other embodiments of the device 10 for culturing and/or investigating biological specimen 16. The dotted line symbolizes that the left and right half belong, optionally, to different embodiments.
  • These chamber bevels 19b and undercuts 19a facilitate the use of such device 10 in open top light sheet microscopy, wherein two microscope lenses 26 (excitation lens and collection lens) are being used in inclined fashion from underneath the device 10 to image the biological specimen.
  • Light sheet microscopy uses a fluorescence microscope e.g. for live cell imaging at subcellular resolution, while allowing to use standard sample carriers, like microtiter plate-shaped culturing devices, as e.g. provided by embodiments of the present invention.
  • Such light sheet microscopes are e.g. provided under the brand ZEISS Lattice Lightsheet 7.
  • the height of the undercuts 19a, or the chamber bevels 19b is preferably smaller that the diameter of the biological specimen, like e.g. a microtissue.
  • the height of the undercuts 19a and/or the chamber bevels 19b is preferably smaller than the diameter of the biological specimen, like e.g. a microtissue. In further embodiments, the height of the undercuts, the chamber bevels or the combination of the two, if applicable, is equal to, or smaller than, 3 /4, 2 A or /i of the diameter of the biological specimen, like e.g. a microtissue.
  • Figure 6 shows another embodiment of the device 10 for culturing and/or investigating biological specimen 16, comprising a separation layer 22 arranged between the lower section 13 of the well and the conduit 15.
  • the biological specimen 16 comprises cells or tissue with lower specific weight/density than aqueous media like water or saline (like e.g. adipocytes or an adipocyte microtissue).
  • the separation layer 22 avoids floating of the specimen 16, and moving thereof upwards to the liquid air interface (not shown) in the well. All this can be done on a microscopic stage with single cell resolution. In such way, it can for example be ensured that the specimen stays within the focal range of a microscopic lens 26.
  • a biological specimen 16 comprising cells or tissue with lower specific weight/density than aqueous media can be introduced into the device through the well that lacks such separation layer ( Figure 6A), and reaches the conduit. If the device is then tilted slightly, the biological specimen will travel through the conduit into the direction of the neighbouring well comprising the separation layer 22.
  • the height of the conduit 15 is preferably greater than the diameter of the biological specimen 16, in order to allow the latter to travel through the conduit.
  • Figure 7 shows embodiments of the device 10 for culturing and/or investigating biological specimen 16 when used in a transwell migration assay.
  • the device 10 comprises a separation layer 22 on which a monolayer of endothelial cells 23 is grown.
  • the assay can be used to test if and how immune cells 27 migrate through the endothelial monolayer 23 in response to the presence of a chemoattractant 28 (e.g., an inflammatory cytokine) symbolized by the star symbol, monolayer in response to the presence of a tumor sample or tumor microtissue tissue 16a. All this can be done on a microscopic stage with single cell resolution.
  • chemoattractant 28 e.g., an inflammatory cytokine
  • Figure 8 shows an embodiment of the device 10 for culturing and/or investigating biological specimen 16 in which the interaction between two biological specimen 16a, 16b is investigated.
  • Such interaction can be the effect a tumor sample or a tumor microtissue 16a being treated with a pharmaceutical entity 29 (being shown by the example of an antibody, a small molecular drug, a viral vector, or a recombinant immune cell) has on a non-tumor microtissue (e.g., a liver organoid) 16b.
  • a pharmaceutical entity 29 being shown by the example of an antibody, a small molecular drug, a viral vector, or a recombinant immune cell
  • a non-tumor microtissue e.g., a liver organoid
  • cytokines 30, symbolized by the star symbol released by the tumor sample or a tumor microtissue in response to the therapy may have a detrimental off-target effect on the non-tumor microtissue, e.g. caused by a cytokine storm.
  • Figure 9 shows an embodiment of the device 10 for culturing and/or investigating biological specimen 16 similar to the one shown in Figure 3C, with adhesive layers 20a, 20b and 20c, as well as exemplary thicknesses of the spacer layer 21, the separation layer 22 and the base layer 14.
  • Figure 10 shows an embodiment of the device 10 for culturing and/or investigating biological specimen 16, which comprises different sets of wells, some of which are connected to one another by a conduit 15a which is arranged relative to other sets of at least two wells in such way that the conduits 15a, 15b are arranged in essentially rectangular fashion relative to one another.
  • this embodiment allows to study the interaction between biological specimen that are placed within a set of wells connected to one another by conduits 15b that are arranged rectangular to the tilting axis 31, as the tilting movement facilitates the liquid flow 32 in the conduits between the wells, and the components comprised therein.
  • conduits 15a that are arranged in parallel to the tilting axis will experience less or even no exchange of liquids, and of the components comprised therein. Biological specimen placed therein will hence experience less or no interaction, and can thus serve as a negative control.
  • Figure 11 shows an embodiment of the device 10 for culturing and/or investigating biological specimen 16, which shows different numbers of wells connected to one another by the same conduit.
  • the device comprises rows with 16 wells. In the first four rows, 8 x 2 wells are connected by a conduit each (doublets). In rows 5 - 8, 4 x 4 wells are connected by a conduit each (quadruplets). In rows 9 - 12, 2 x 8 wells are connected by a conduit each (octets), in rows 17 - 20, 5 x 3 wells are connected by a conduit each (triplets), and in rows 21 - 24, l x 14 wells are connected by a conduit. It is important to mention that never all wells of one row or column are connected by the same conduit.
  • a device according to the invention can e.g. have doublets, triplets, quadruplets, octets or other combinations only, or, as exemplarily shown in Figure 11, can comprise a mixture of e.g. doublets, triplets, quadruplets, octets and 14ets, e.g., in different rows or columns, or even in the same rows or columns.
  • the device comprises e.g. n wells in a row or column, a maximum of n-2 wells are connected to one another by the same conduit.
  • a device (10) for culturing and/or investigating biological specimen (16) comprises a plurality of wells wherein at least two wells (11) arranged next to one another each comprise an open upper section (12), a lower section (13), wherein the upper section and the lower section are in fluid communication. Further at least two lower sections (13) of the at least two wells (11) are connected to one another by a conduit (15).
  • the device culturing and/or investigating biological specimen adopts the format of a microwell plate or microtiter plate, with different wells arranged in rows and columns.
  • two wells doublets
  • three wells triplets
  • four wells quadraturelets
  • five wells six wells
  • seven wells eight wells
  • octets nine wells
  • ten wells eleven wells or twelve wells
  • twelve wells are connected to one another by a conduit.
  • the device comprises e.g. n wells in a row or column, a maximum of n-2 wells are connected to one another by the same conduit.
  • the device comprises rows with 16 wells, 8 x 2 wells can be connected by a conduit each, 4 x 4 wells can be connected by a conduit each, 2 x 8 wells can be connected by a conduit each, 5 x 3 wells can be connected by a conduit each, or 1 x 14 wells can be connected by a conduit, yet never all 16 wells are connected by the same conduit.
  • the maximum number of wells connected to one another by the same conduit equals half of the number of wells in a given row or column of a given device for culturing and/or investigating biological specimen.
  • the maximum number of wells connected to one another by the same conduit equals n-2, wherein n is the number of wells in a given row or column of a given device for culturing and/or investigating biological specimen.
  • table 1 some exemplary dimensions of devices for culturing and/or investigating biological specimen are shown, and the maximum number of doublets, triplets, quadruplets, or octets connected by one conduit each is shown.
  • the device according to the present invention does not provide reservoirs comprising culture media, washing media or test media, which reservoirs would be fluidically connected to one or more wells by means of a conduit, as e.g. disclosed in W02019008189A1.
  • Table 1 some exemplary dimensions of devices according to the present invention
  • said lower section (13) has a smaller cross-sectional area than the open upper section (12).
  • the device according to the invention is a one-part structure, i.e., without a separate base layer.
  • Such one-part structure can e.g. be produced by 3D printing.
  • the device according to the invention comprises a base layer (14) that forms the bottom of at least one well and at least one conduit, hence forming a two- part structure (not counting adhesive layers or separation layers in).
  • a base layer comprises a transparent foil that is attached from underneath to the wells by means of an adhesive or an adhesive layer.
  • the wells have no bottom per se, as the bottom is formed by the base layer (14) attached thereto.
  • the base layer (14) can be releasably or irreleasably attached from underneath to the wells.
  • Such two-part structure can be produced, e.g., by die-casting, molding, embossing or 3D printing methods of suitable material, e.g., thermoplasts including the materials discussed hereinbelow.
  • At least one conduit consists (15), essentially, of a longitudinal groove defined by two lateral walls.
  • these grooves can be formed in the basal parts of some wells (with the base layer (14) forming the bottom thereof) , or in the upper side of the base layer (14), with the basal parts of the wells forming the ceiling thereof.
  • the base layer (14) comprises an optically transparent material.
  • said base layer comprises a cyclo-olefin polymer (COP).
  • COP cyclo-olefin polymer
  • said base layer comprises polystyrene. According to another embodiment, said base layer comprises PMMA (polymethyl methacrylate). According to another embodiment, said base layer comprises polypropylene. According to another embodiment, said base layer comprises Polydimethylsiloxane (PDMS).
  • PDMS Polydimethylsiloxane
  • the biological specimen (16) is one or more selected from the group consisting of
  • microtissue as used herein, is meant to encompass 3D cell culture models including organoids, 3D tissue spheroids, embryoid bodies, islets, precision cut tissue slides, and the like.
  • the microtissues can derive from stem cells or can be of natural origin, or can be engineered or passively or actively reassembled from isolated cells.
  • Microtissues can comprise, or even consist, of primary cells obtained from organs, as well as tumor cells obtained from tumors or cells obtained from cell culture. In other embodiments, such microtissues comprise two or more different cell types.
  • microtissues are created from isolated cells which aggregate when held under suitable conditions to form such microtissue. Details of how microtissues are produced are known to the skilled artisan e.g. from WO2015158777A1 (liver microtissues) or Keim et al., 2003 (tumor microtissues). The content of these documents is incorporated by reference herein for enablement purposes.
  • tissue sample refers to sample from liquid (blood, lymph, liquor) or solid tissues (connective tissue, organs, muscle, skin, or tumor, to name a few). Tissue samples can be provided as slices, aspirations and other suitable formats.
  • adhered cell monolayer relates to a type of cell culture in which cells are attached or adhered to a given surface in order for growth to occur. Most vertebrate derived cells (with the exception of hematopoietic cells) can be cultured and require a 2 dimensional monolayer to facilitate cell adhesion and spreading.
  • suspended cells relates to cells that do not attach, or have not attached, to a given surface, nor do they form, or have they formed, a microtissue.
  • suspended cells are for example immune cells, like PBMC, Macrophages, T cells, NK cells or the like, yet also circulating tumor cells (CTC).
  • the height of at least one conduit is smaller than the diameter of the biological specimen comprised in the well, so as to avoid that the biological specimen migrate between the wells.
  • a microtissue in the meaning of the above description has a diameter between 100 pm and 500 pm.
  • the conduit has a height smaller than such diameter.
  • the height of at least one conduit is greater than the diameter of the biological specimen comprised in the well, so as to let the biological specimen migrate into the conduit.
  • a microtissue in the meaning of the above description has a diameter between 100 pm and 500 pm. In such case the conduit has a height greater than such diameter.
  • At least one conduit has a height of between ⁇ 1 mm and > 0,03 mm, with 0,06; 0,09; 0,10; 0,12; 0,15; 0,18; 0,21; 0,24; 0,27; 0,3; 0,33; 0,36; 0,39; 0,42; 0,45; 0,48; 0,51; 0,54; 0,57; 0,6; 0,63; 0,66; 0,69; 0,72; 0,75; 0,78; 0,81; 0,84; 0,87; 0,9; 0,93; 0,96; or 0,99 mm as preferred heights.
  • the height of the conduit can for example be determined by the thickness of a spacer layer that separates the wells and the base layer.
  • At least one well comprises a separation layer (22) arranged between the lower section (13) of the well and the conduit (15).
  • Such separation layer can be used for different applications.
  • the biological specimen comprises cells or tissue with lower specific weight/density than aqueous media like water or saline (like e.g. adipocytes or an adipocyte microtissue)
  • the separation layer avoids floating of the specimen.
  • a cell monolayer can be grown on the separation layer, and its interaction with another biological specimen below the separation layer can then be investigated, or its effect in a transwell migration assay can be investigated.
  • said separation layer (22) comprises a mesh or a semipermeable membrane.
  • the mesh or semipermeable membrane comprises an average pore size of between > 0.1 pm and ⁇ 100 pm.
  • the pore size is oftentimes subject to a gaussian distribution.
  • the peak value of said distribution then indicates the average pore size.
  • the mesh size is a catalogue value provided by the respective manufacturers.
  • a sample can is placed under the microscope, and the pore sizes are measured directly from the magnified image. After obtaining high-resolution images of the mesh, digital image analysi s can be used to measure and average the pore sizes. Software tools can automate this process and provide distributions of pore sizes.
  • semipermeable membrane is a Permeable Polycarbonate Membrane as for example being used in CorningTM TranswellTM Multiple Well Plates with Permeable Polycarbonate Membrane Inserts, as supplied by Fisher Scientific.
  • PCTE hydrophilic polycarbonate
  • the separation layer (22) is coated with at least one of a) a hydrophilic layer, b) one or more ECM peptides or proteins, and/or c) a coating comprising poly-amino acids.
  • Extracellular matrix (ECM) peptides or proteins promote cell adhesion to a surface coated therewith. See for example Junker et al (1987), the content of which is incorporated by reference herein in its entirety for enablement purposes.
  • Such coatings can comprise e.g. collagen or collagen derivatives, or other matrix components (e.g. fibronectins or laminins), yet can also be cell-type specific, as e.g. disclosed in Zhang et al (2009), the content of which is incorporated by reference herein in its entirety for enablement purposes.
  • a coating comprising poly-amino acids may for example comprise Poly-L-Lysine or Poly-L- Omithine. Respective coating protocols are published in the scientific literature as well as by laboratory suppliers.
  • the device according to the invention comprises at least two sets of at least two wells, wherein the two lower sections of the at least two wells of each set are connected to one another by a conduit (15), wherein further at least one set of at least two wells is arranged relative to at least one other set of at least two wells in such way that the conduits (15a, 15b) are arranged in essentially rectangular fashion relative to one another.
  • this embodiment allows to study the interaction between biological specimen that are placed with a set of wells connected to one another by conduits that are arranged rectangular to the tilting axis, as the tilting movement facilitates the liquid flow in the conduits between the wells, and the components comprised therein.
  • the lower section (13) of at least one well is eccentrically arranged with respect to the longitudinal axis of the open upper (12) section thereof.
  • At least one well further comprises an intermediate section disposed between the open upper section (12), and the lower section (13), said intermediate section comprising a pipetting stage (17) having an inclination.
  • the pipetting stage is configured as a platform within the well, said platform has a slope such that any fluid applied to the platform can drain off into the lower section.
  • the slope of the pipetting platform has an angle of at least 5°.
  • the slope of the pipetting stage (17) has an angle of not more than 45°, preferably of not more than 35°, more preferably of not more than 25°, and most preferably of not more than 15°. Most preferably, the slope of the pipetting platform has an angle of 10°.
  • the angle of the slope is to be understood as the angle between the plane or the pipetting stage and the plane of the horizontal cross-sectional area, which is parallel to the plane of the multiwell plate where the pipetting stage is in contact with the wall of the upper section of the well.
  • the inclination of the pipetting stage (17) has an angle of at least 5°, and of not more than 45°, preferably of not more than 35°, more preferably of not more than 25°, and most preferably of not more than 15°.
  • the intermediate section of at least one well further comprises a chute (18), said chute being provided between the pipetting stage and the lower section.
  • the pipetting stage (17) provides a slope such that any fluid applied thereto can run off.
  • the chute (18) provides a transition between the lower edge of the pipetting stage and the lower section of the well.
  • the chute (18) has a larger inclination than the slope of the pipetting stage (17).
  • such “pipetting stage” is also called “ledge”, like e,g, in the microtiter plates produced by InSphero (CH) applying Insphero’s SureXchange technology.
  • a pipette tip is inserted into the well at the side of the pipetting stage (17) such that any fluid to be supplied to the well hits the pipetting stage first, before flowing smoothly downwards the pipetting stage and along the chute (18) into the lower section (13) of the well. Thereby, the biological specimen is not directly present within the flow path of any fluid supplied to or being aspirated from the well.
  • the pipetting stage (17) provides a slope upon which the vertical positioning of the pipette tip onto this slope forms a gap between the orifice of the pipette tip and the pipetting stage (17).
  • the chute has a slope, wherein the slope of the chute is steeper than the slope of the pipetting stage within the same well.
  • the pipetting stage and/or the chute may be present in a well on only one inner side of the well, on two adjacent inner sides of the well, on three inner sides of the well, or on all four or all inner sides of the well.
  • the device according to the present invention comprises a row of wells or an array of wells.
  • said device is configured as a multiwell plate, preferably comprising an array of 96 wells, an array of 384 wells or an array of 1536 wells.
  • the multiwell plate is a standard SBS/ANSI format multiwell plate.
  • At least a) the lower section (13) of at least one well, and/or b) at least one section of the base layer (14) is provided with an ultra-low attachment coating.
  • Providing a well of a multiwell plate with an ultra-low attachment surface can be done in various different ways.
  • An example of providing a well with an ultra-low attachment surface is covalently binding a hydrogel layer to the substrate such as polystyrene, said hydrogel being hydrophilic and neutrally charged.
  • Multiwell plates comprising such an ultra-low attachment surface are commercially available from Corning Inc.
  • Another example of an ultra-low attachment surface is a surface made of a polymer consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC).
  • MPC 2-methacryloyloxyethyl phosphorylcholine
  • Such ULA coatings are for example present in the AkuraTM Spheroid Microplates manufactured by Insphero (CH).
  • the separation layer (22) is attached to the lower section (13) of at least one well by means of an adhesive layer (20a).
  • a spacer layer (21) is attached to the lower side of the separation layer (22) by means of an adhesive layer (20b).
  • the height of the conduit is essentially determined by the thickness of the spacer layer (21).
  • the base layer (14) that forms the bottom of at least one well and at least one conduit is attached to the lower side of the spacer layer (21) by means of an adhesive layer (20c).
  • the device According to one embodiment of the device according to the present invention, the
  • the base layer (14) comprises at least one chamber bevel (19a) arranged at the edge of the conduit (15).
  • undercuts or chamber bevels facilitate the use of such device in open top light sheet microscopy, by avoiding blind angles.
  • two microscope lenses excitation lens and collection lens
  • the height of at least one undercut or chamber bevel is smaller than the diameter of the biological specimen, like e.g. a microtissue. Note that this also applies, preferably, for an embodiment where undercuts and chamber bevels are combined, as e.g. shown in Figure 5B, left part.
  • a microtissue in the meaning of the above description has a diameter between 100 pm and 500 pm.
  • the height of the undercuts, the chamber bevels or the combination of the two, if applicable, is equal to, or smaller than, 3 /4, 2 /s or /i of the diameter of the biological specimen, like e.g. a microtissue.
  • the device according to the above description is provided for use in the study of the interaction of two more biological specimen.
  • a method for studying the interaction of two more biological specimen in which method the device according to the above description is used.
  • the device can comprise a one-part structure, i.e., without a separate base layer.
  • a one-part structure can e.g. be produced by 3D printing.
  • the device can yet also comprise a two-part structure (not counting adhesive layers or separation layers in).
  • a base layer that forms the bottom of at least one well and at least one conduit is attached, releasably or irreleasably, from underneath to the wells, e.g., by means of an adhesive or an adhesive layer.
  • Such two-part structure can be produced, e.g., by die-casting, molding, embossing or 3D printing methods of suitable material, e.g., thermoplasts including the materials discussed herein..
  • Table 3 examples of biological specimen or combinations thereof that can be investigated
  • a transwell migration assay is a commonly used test to study, the migratory response of e.g. endothelial cells to angiogenic inducers or inhibitors, or of immune cells to immune cell engaging stimuli.
  • endothelial cells are grown on a separation layer comprising a permeable membrane or mesh. Then, for example, a solution containing a test agent is placed below the cell permeable membrane. Following an incubation period, the cells that have migrated through the membrane are stained and counted.
  • the membrane is usually coated with some extracellular matrix component (e.g. collagen) which facilitates both adherence and migration.
  • immune cells are added to the well, and the test agent on the other side of the separation layer is for example an immune cell engager, like e.g. an inflammatory cytokine.
  • an immune cell engager like e.g. an inflammatory cytokine.
  • the immune cells have to pass the endothelial layer and the separation layer.
  • the main advantage of this assay is its detection sensitivity. Migration through the permeable filter can be caused by very low levels of e.g. angiogenic inducers or an inflammatory cytokines.
  • the device according to the invention allows all this to be done on a microscopic stage with single cell resolution.

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Abstract

La présente invention concerne un dispositif (10) pour la culture et/ou l'étude d'échantillons biologiques. Le dispositif comporte une pluralité de puits (11), au moins deux puits agencés l'un à côté de l'autre comportant chacun une section supérieure ouverte (12) et une section inférieure (13, la section supérieure et la section inférieure étant en communication fluidique. Au moins deux sections inférieures (13) des au moins deux puits (11) sont reliées l'une à l'autre par un conduit (15).
PCT/EP2023/077740 2023-10-06 2023-10-06 Dispositif pour l'étude des interactions d'un échantillon biologique Pending WO2025073379A1 (fr)

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WO2017001680A1 (fr) 2015-07-01 2017-01-05 Insphero Ag Dispositif pour la propagation de micro-tissus
WO2019008189A1 (fr) 2017-07-07 2019-01-10 Insphero Ag Dispositif compartiment à microtissus
US20210230527A1 (en) * 2018-07-27 2021-07-29 The Trustees Of Columbia University In The City Of New York Human organ-on-chip models for predictive screening
US20220033749A1 (en) * 2018-09-28 2022-02-03 Ushio Denki Kabushiki Kaisha Cell culture chip
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
WO2022175898A1 (fr) * 2021-02-19 2022-08-25 Molecular Devices (Austria) GmbH Procédés pour le passage d'organoïdes utilisant des unités de microplaques à puits

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WO2015158777A1 (fr) 2014-04-15 2015-10-22 Insphero Ag Procédé de préparation de cellules pour une culture de tissu 3d
WO2017001680A1 (fr) 2015-07-01 2017-01-05 Insphero Ag Dispositif pour la propagation de micro-tissus
WO2019008189A1 (fr) 2017-07-07 2019-01-10 Insphero Ag Dispositif compartiment à microtissus
US20210230527A1 (en) * 2018-07-27 2021-07-29 The Trustees Of Columbia University In The City Of New York Human organ-on-chip models for predictive screening
US20220033749A1 (en) * 2018-09-28 2022-02-03 Ushio Denki Kabushiki Kaisha Cell culture chip
WO2022175898A1 (fr) * 2021-02-19 2022-08-25 Molecular Devices (Austria) GmbH Procédés pour le passage d'organoïdes utilisant des unités de microplaques à puits
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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