WO2017142410A1 - Moyens et procédés pour culture et analyse de cellules sphéroïdes - Google Patents

Moyens et procédés pour culture et analyse de cellules sphéroïdes Download PDF

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Publication number
WO2017142410A1
WO2017142410A1 PCT/NL2017/050098 NL2017050098W WO2017142410A1 WO 2017142410 A1 WO2017142410 A1 WO 2017142410A1 NL 2017050098 W NL2017050098 W NL 2017050098W WO 2017142410 A1 WO2017142410 A1 WO 2017142410A1
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WO
WIPO (PCT)
Prior art keywords
insert plate
microplate
cells
tubes
spheroid
Prior art date
Application number
PCT/NL2017/050098
Other languages
English (en)
Inventor
Rob VAN BEURDEN
Original Assignee
Perkinelmer Health Sciences B.V.
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 Perkinelmer Health Sciences B.V. filed Critical Perkinelmer Health Sciences B.V.
Publication of WO2017142410A1 publication Critical patent/WO2017142410A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/523—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for multisample carriers, e.g. used for 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • 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
    • 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
    • B01L2200/12—Specific details about manufacturing devices
    • 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
    • B01L2300/04—Closures and closing means
    • 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
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • 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
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic

Definitions

  • the invention relates to means and methods for cell culturing and cell analysis. More in particular, it relates to devices designed for growth, maintenance and/or microscopic analysis of 3D cell culture models, like cell aggregates or spheroids.
  • 3D cell culture models like cell aggregates or spheroids.
  • In vitro cellular and tissue models for various drug testing and screening experiments are often central to the development of novel therapeutics in the pharmaceutical industry.
  • Most in vitro studies are still performed under conventional two-dimensional (2D) cell culture systems, which are often not physiological models and/or hard to relate to functional tissues and tumors. Drug studies involving such models may therefore not produce accurate or realistic readouts. To obtain more meaningful results, in vivo studies involving animals are often utilized.
  • 3D in vitro models that provide more therapeutically predictive and physiologically relevant results for drug testing and screening in the pharmaceutical industry are needed.
  • One way to create 3D cell culture models is through the formation of so-called spheroids, or 3D clusters or aggregates of cells or more complex and functional organoids.
  • a primary purpose of growing 3D cell spheroids in vitro is to test
  • Scaling up of spheroid culture in a manner suitable for certain applications such as high-throughput screening and testing has several drawbacks.
  • One way of traditional spheroid formation involves cultivation of suspended cells in hanging drops on the underside of a Petri dish lid. This process requires inverting of the lid following placement of the drops. As a result, the drops are susceptible to perturbation, resulting in falling, spreading, and merging with neighboring drops.
  • this method is labor- intensive, does not permit efficient scalable production, and is not
  • this method usually requires another labor-intensive step of transferring the spheroids manually, one by one, to a multi-well culture plate for longer-term culture, treatment, analysis, and harvest.
  • An alternative is to induce the formation of spheroids under continuous agitation of cell suspension in bioreactors, such as spinner flasks and rotary culture vessels. This method requires the consumption of large quantities of culture media. It also requires specialized equipment and the size and uniformity of the spheroids are hard to control. The high variability in spheroids prohibits their use in many applications.
  • Microplates allow for a uniform, single spheroid formation across all wells and the culturing and assaying of spheroids in the same microplate, without the need for transfer to a new plate.
  • a special (e.g. Ultra-Low Attachment; ULA) coating can be applied on the well surface to avoid cellular adhesion and promote cellular aggregation by gravitational force.
  • ULA Ultra-Low Attachment
  • the microplate format also enables media exchange or spheroid treatment with test compounds, drugs or assay reagent.
  • US2014/0322806 discloses a spheroid cell culture article including a frame having a chamber including an opaque side wall surface, a top aperture, a gas-permeable, transparent bottom, and optionally a chamber annex surface and second bottom, and wherein at least a portion of the transparent bottom includes at least one concave arcuate surface.
  • Spheroid analysis by automated visualization plays an important role in drug testing and screening applications. For instance, majority of high- throughput spheroid assays involve microscopic detection of viable or dead cells, (morphological) changes of cells or cell organelles. Most automated imagers for analyzing standardized microplates are designed for "reading" the well through an optically clear ultra-flat bottom plate. Typically, this reading uses high numerical aperture (NA) water-immersion objectives and requires all well access.
  • NA numerical aperture
  • microplate standards govern various characteristics of a microplate including well dimensions (e.g. diameter, spacing and depth) as well as plate properties (e.g. dimensions and rigidity), which allows interoperability between microplates, instrumentation and equipment from different suppliers, and is particularly important in laboratory automation.
  • SBS Society for Biomolecular Sciences
  • ALA Association for Laboratory Automation
  • SLAS Society for Laboratory Automation and Screening
  • the microplate standards are also known as ANSI/SLAS standards.
  • ANSI/SLAS standards Currently known (standardized) microplates designed for spheroid handling suffer from the drawback that their application for automated visualization is not optimal.
  • the present inventors observed that the arcuate surface of round bottom well plates designed to promote spheroid formation (e.g. each well bottom of a plate according to TJS2014/0322806) significantly limits their compatibility with commonly used automated plate imagers.
  • the inventors set out to develop a new spheroid plate concept that allows not only for convenient spheroid culturing and handling, but also for automated microscopic spheroid analysis by commonly used automated plate imagers, in particular those that rely on imaging with water
  • immersion lenses like the Operetta imaging system and similar high content imaging systems.
  • the invention provides an insert plate for detachably attaching to a microplate having dimensions established by the Society of Biomolecular Sciences (SBS Standards) or the SLAS/NIST
  • the insert plate having a main body comprising a plurality of tubes for receiving a sample, each tube having a liquid impermeable optically clear bottom portion to allow for microscopic observation (visualization/imaging of the sample) and wherein at least part of said bottom portion has a concave arcuate surface, the plurality of tubes being positioned to fit into and align with a corresponding well of the microplate when the microplate and the insert plate are attached.
  • the indefinite article "a” or “an” and its corresponding definite article “the * as used herein means at least one, or one or more, unless specified otherwise.
  • An insert plate of the invention is among others characterized by a plurality of tubes for receiving a sample, each tube having a liquid impermeable optically clear bottom portion to allow for microscopic observation
  • the tube and tube bottom portion ultimately terminates, ends, or bottoms- out in a spheroid "fiiendly" rounded or curved surface, such as a dimple or a pit.
  • the concave arcuate surface of the bottom portion comprises a hemi-spherical surface or a conical surface.
  • the tubes of an insert plate comprise a low-adhesion or no-adhesion coating on the concave arcuate bottom surface to facilitate spheroid formation and/or spheroid maintenance.
  • the tubes can have any desired design as long as they fit into and align with a corresponding well of the microplate.
  • the tubes have a circular cross section or square base tapered to round bottom.
  • the plurality of tubes of the insert plate provided herein are positioned to fit into and align with a corresponding well of the microplate when the microplate and the insert plate are attached.
  • each of the tubes has a depth to position the concave arcuate surface at a desired distance from the base surface of the microplate when the microplate and the insert plate are attached.
  • said desired distance is essentially the same for each of the tubes. In one embodiment, the desired distance is 0 to 2 mm, preferably 0 to 1 mm.
  • the insert plate comprises 96 or 384 tubes to fit into an SBS-standardized or SLAS/NIST-standardized microplate comprising, respectively, 96 or 384 wells.
  • the insert plate comprises 96 tubes arranged in 8 rows and 12 columns.
  • the insert plate comprises 384 tubes arranged in 16 rows and 24 columns.
  • the tube depth is between 10 to 14 mm, preferably between 12 and 13.5 mm.
  • the tube diameter at the base of the tube is between 3 to 3.7 mm.mm.
  • the insert plate may comprise one or more additional structural features, in particular those that contribute to the handling or (automated) processing of the plate.
  • the insert plate comprises a plate orientation notch in the lower left corner.
  • the insert plate has an outer rim provided with a pressure sensitive adhesive to allow for securely attaching the insert to the microplate.
  • the insert plate includes a skirt portion about a periphery of the main body of the plate, preferably wherein said skirt portion is generally perpendicular to main body.
  • a skirt portion provides additional stability to the insert plate such that during handling (e.g. when culturing spheroids) the plate can be used as "stand alone" spheroid culturing article.
  • the skirt portion of the insert plate is suitably removed, e.g. by cutting, prior to imaging of the spheroids to allow for assembly with the microplate.
  • the insert plate is a skirtless object such that it can be assembled with the microplate without any modification.
  • the insert plate can be made from any moldable, optical transparent material, such as a plastic polymer.
  • the material is a thermoformable polymer.
  • Suitable preferred materials include plastic polymers such as polyethylene, polypropylene, polystyrol,
  • the plate is made from a material selected from polypropylene, polystyrene, polyethylene
  • the insert plate is preferably constructed from a single piece of material. There are several methods that can be used to make an insert plate having a main body comprising a plurality of tubes for receiving a sample, each tube having a liquid impermeable transparent (optically clear) bottom portion to allow for microscopic imaging and wherein at least part of said bottom portion has a concave arcuate surface.
  • the insert element may be molded, for example by injection molding, or shaped by thermoforming. Thermoforming is a production method where you force a plastic part with pressure and heat (and/or vacuum) into a certain shape.
  • the invention accordingly also relates to a method for manufacturing an insert plate as described herein above.
  • the invention provides a method for providing an insert plate as a unitary piece, comprising subjecting an optically transparent polymer sheet to (vacuum) thermoforming technology to introduce a plurality of tubes for in the main body.
  • Optically transparent polymers are known in the art.
  • the method comprises thermoforming a glycolyzed poly(ethylene terephthalate) (GPET) sheet or a polyolefin sheet.
  • GPET poly(ethylene terephthalate)
  • said polymer sheet has a thickness of between 0.3 and 1 mm, preferably between 0.5 and 0.8 mm.mm.
  • the invention provides an assembly of (i) an insert plate according to the invention, the plate being attached to (by insertion into) (ii) a standardized microplate having a plurality of wells, each well having a flat, transparent bottom portion and opaque side wall surfaces.
  • the multiwell microplate contains at least a plurality of wells in the suitable pattern to accommodate for the tubes of the insert plate.
  • the number of tubes of the insert plate corresponds to the number of wells of the microplate.
  • the dimension of the multiwell plate device, the well dimension, and/or the well spacing conform to the SBS (Society for Biomolecular Screening)standard (also known in the art as ANSI/SLAS Microplate Standards.
  • SBS Society for Biomolecular Screening
  • ANSI/SLAS Microplate Standards also known in the art as ANSI/SLAS Microplate Standards.
  • the microplate is a 96- well or 384-well SBS-standardized microplate.
  • the wells can have a circular or square cross section.
  • the microplate is 384-well SBS-standardized microplate containing 384 wells having a square cross section.
  • the microplate is 96-well SBS-standardized microplate containing 96 wells having a circular cross section.
  • the bottom portion of each well is flat and transparent to allow for accurate microscopic imaging.
  • the clear bottom portion of the microplate is ultra-flat.
  • High optical-quality film bottom microplates are ideal for performing high content cell-based assays using imaging systems.
  • the film bottom provides a flat and optically clear surface that reduces autofocus time.
  • the bottom portion of the microplate comprises cyclic olefin polymer.
  • the downward facing bottom surface of the microplates is designed to allow for all well access when using water immersion and high NA objectives and without objectives colliding with the plate skirt.
  • the side wall surfaces of the microplate wells are opaque so that light cannot be transmitted between adjacent wells through the side walls.
  • the side walls of wells be non-reflective, in which case the side walls of the wells of the microplate are preferably formed from a black or dark-colored polymer.
  • the dark polymer may be formed by the addition of carbon black in mounts ranging from about 0.5 weight % to about 15 weight %.
  • the microplate is a Low bottom height (200 ⁇ 10 pm), black cyclic olefin microplate with optically clear, cyclic olefin foil bottom (188 um).
  • Suitable plates for use in an assembly according to the invention are known in the art and commercially available. For example, it can be the CellCarrier-384 ultra plate (PN 6057300, PerkinElmer)
  • At least one tube of the insert plate optionally in assembly with the microplate, comprises a plurality of kving cells.
  • the cells are cardiomyocytes, tumor cells or cancer cell lines, hematopoietic cells, endothelial cells, insulin producing beta cells, neuronal cells, glial cells, kidney cells, hepatocytes, vascular progenitor cells or derivatives (such as hematopoietic stem cells or endothelial cells).
  • the cells may be in suspension or they form a 3D-structure, like aggregate or cluster or spheroid.
  • at least one of the tubes contains spheroids.
  • the assembly may further comprise a seal, lid or cover plate that is positioned over said plurality of tubes, for instance to protect the cells and/or spheroids during culturing, incubating, handling and/or imaging.
  • At least one of the microplate wells is provided with an optically clear matrix, like a liquid, a gel or solidifying material, to fill at least part of the void volume formed by the interior space of the well not occupied by the tube of the insert plate.
  • the optically clear matrix can reduce optical distortion of the incoming beam of light , thereby improving (auto)focussing and accurate microscopic imaging. It also avoids
  • the invention also provides a method of spheroid handling, comprising: a) inserting a plurality of cells into at least one tube of an insert plate of the invention, the insert plate preferably being comprised in an assembly with a microplate, and allowing the formation of spheroid formation and b) performing on one or more of the spheroids a handling method selected from the group consisting of spheroid culturing, spheroid maintaining, spheroid analysis, spheroid testing, and combinations thereof.
  • the formation of spheroids can be achieved by methods known in the art, typically involving gravity and/or cellular interactions.
  • one or more agents can be added to the growth medium to promote spheroid formation or maintenance. For example, it has been shown in the art that growth factors support the clustering and/or proliferation of cells.
  • a method of spheroid handling as provided herein may comprise contacting the spheroid(s) with a potential therapeutic agent.
  • the potential therapeutic agent is at least one of:
  • a a molecule selected from the group consisting of an endogenous ligand or hgands, a biological sample suspected of containing a native or endogenous ligand or ligands, a combinatorial library of small molecules, a hormone, an antibody, a polysaccharide, an anti-cancer agent, a natural product, a terrestrial product and a marine natural product;
  • siRNA silencing RNA
  • miRNA micro RNA
  • shRNA short hairpin RNA
  • the spheroid handling method further comprises assaying for a marker indicative of modulation of a cellular target of said potential therapeutic agent or screening for activity in modulating the phenotype of a spheroid.
  • the cells forming the spheroid(s) of cells are transformed with at least one heterologous nucleic acid molecule that encodes one or more biomarkers associated with a pheiiotype of interest.
  • the recombinant nucleic acid molecule(s) are chromosomally integrated into the genome of the cell.
  • the biomarkers are linked to an indicator that can be detected in situ following expression of the biomarker.
  • indicator is meant to refer to a chemical species or compound that is readily
  • indicator compounds thus include, but are not limited to, fluorogenic or fluorescent compounds, chemiluminescent compounds, calorimetric compounds, UV/VIS absorbing compounds, radionucleotides and combinations thereof.
  • Exemplary indicators for use in the screening methods of the present invention are proteins including Red Fluorescence Protein (RFP), which fluoresces when exposed to light of wavelength 558 nm, Green Fluorescent Protein (GFP) which fluoresces when exposed to light of wavelength 395 nm, and luciferase, which produces light in the conversion of luciferin and oxygen to oxyluciferin.
  • RFP Red Fluorescence Protein
  • GFP Green Fluorescent Protein
  • luciferase which produces light in the conversion of luciferin and oxygen to oxyluciferin.
  • the cells are neoplastic cells.
  • Cells may be tumor cells that have been isolated from a human.
  • the cells are human malignant tumor cells selected from the group consisting of breast cancer, lung cancer, prostate cancer, colon cancer, melanoma cancer, and cancer of the bone and connective tissues.
  • the cells are stem cells selected from the group consisting of embryonic stem cells or adult stem cells, progenitor cells, bone marrow stromal cells macrophages, fibroblast cells, endothelial cells, epithelial cells, and mesenchymal cells.
  • the invention also provides the use of an insert plate, assembly or method as described herein in a high throughput high throughput drug discovery program. For example, it provides an industrially applicable high through put screening method for assaying the non-, pro-, or anti-apoptotic or proliferative or necrotic activity of test compounds in spheroid cells.
  • Figs. 1A and IB schematically show a top view and a longitudinal cross-section of an embodiment of a microplate forming part of an
  • Figs. 2A and 2B schematically show a top view and a longitudinal cross-section of an embodiment of an insert plate according to the invention, which insert plate forms another part of an embodiment of an assembly according to the invention, respectively, and Fig. 3 schematically shows a cross-section of a part of an assembly according to the invention in which the insert plate of Figs. 2A and 2B has been detachably inserted into the microplate of Figs. 1A and IB.
  • a standardized microplate 1 is shown in top view and cross-section, respectively.
  • the microplate 1 has a plurality of wells 2, which plurality preferably is 96 or 384, although in other
  • another number of wells can be used.
  • the number of wells is 96 they preferably are arranged in 8 rows and 12 columns.
  • the microplate 1 comprises 384 wells, they are preferably arranged in 16 rows and 24 columns.
  • each well 2 has a square cross-section, although in other embodiments the wells can be circular.
  • Each well 2 has a flat, optically clear bottom portion 3 and opaque side wall surfaces 4, 4'.
  • the clear bottom portion 3 of the shown microplate 1 is formed by a clear bottom plate 3A which is attached to the bottom of the microplate 1 and extends below all the wells 2.
  • Each clear bottom portion 3 of the microplate 1 is ultra-flat. In case a clear bottom plate 3A is used, this clear bottom plate 3A is ultra-flat.
  • the shown microplate 1 has a footprint of approximately 127.76 x 85.48 mm, and as shown in Figs. 1A and IB is a Low bottom height (200 ⁇ 10 pm), black cyclic olefin microplate with optically clear, cyclic olefin foil bottom, such as a 384-well SBS-standardized microplate.
  • the micro plate 1 comprises a microplate orientation notch 5 in the lower left corner when the plate is oriented with well 1A in the upper left hand corner.
  • the wells 2 Prior to use, the wells 2 can be provided with an optically clear matrix 6 (liquid, gel, sohdifying material) such that during use (see Fig. 3) at least part of a void volume formed of the interior space of the well 2 not occupied by the tube is filled to avoid optical distortion of an incoming beam of light, thereby improving (auto)focussing and accurate microscopic imaging.
  • an optically clear matrix 6 liquid, gel, sohdifying material
  • FIGs. 2A and 2B an embodiment of an insert plate 7 according to the invention is shown in top view and cross-section, respectively.
  • the insert plate 7 is arranged to be detachably attached or inserted into the microplate 1 described above to form an assembly of microplate 1 and insert plate 7, as schematically shown in part in Fig. 3.
  • the insert plate 7 has a main body 8 comprising a plurality of tubes 9 for receiving a sample.
  • Each tube 9 has a liquid impermeable optically clear bottom portion 10 (Fig. 3) to allow for microscopic imaging.
  • Fig.3 At least part of said bottom portion 10 has a concave arcuate surface, which comprises a hemi-spherical surface or a conical surface.
  • the number of tubes 9 of the insert plate 7 corresponds to the number of wells 2 of the microplate 1, and amounts to e.g. 94 or 384, and the plurality of tubes 9 is arranged to be detachable attached or inserted, such as to fit into and align with a corresponding well 2 of the microplate 1 when the microplate 1 and the insert plate 7 are attached as shown in Fig. 3.
  • the insert plate 7 comprises a insert plate orientation notch 11 in the lower right corner.
  • the tubes 9 have a circular cross section tapered to the concave arcuate surface or round bottom.
  • the tube depth is between 10 and 13.5mm, preferably about 12 mm, and the tube diameter at the base (i.e. open end) of the tube 9 opposite of the bottom portion 10 is between 3 and 3.7 mm.
  • the tubes 9 preferably taper such that the bottom of the tubes fit into the bottom one- third of the corresponding wells 2 of the microplate 1, in other words the bottom of the tubes 9 occupy one third of the bottom of the wells 2. It will be appreciated that in other embodiments tubes can have of square base.
  • each of the tubes 9 is chosen such as to position the concave arcuate surface 10 at a desired distance between 0 to 2 mm, preferably 0 to 1 mm, from the base surface or clear bottom plate 3, 3A of the microplate 1 when the microplate 1 and the insert plate 7 are attached.
  • the desired distance can vary between various tubes, in the shown embodiment the desired distance is essentially the same for each of the tubes 9.
  • the insert plate 7 is fabricated as a unitary piece from an optically transparent polymer, preferably selected from glycolyzed poly(ethylene terephthalate) (GPET) and polyolefins.
  • GPET glycolyzed poly(ethylene terephthalate)
  • polyolefins preferably thermoforming plastic foil technology is used.
  • the at least one tube 9 comprises a plurality of living cells (schematically depicted as single item) 12
  • the at least one tube 9 comprises a low-adhesion or no-adhesion coating 13 on the inner side of the concave arcuate bottom surface 10.
  • the cells 12 can form a 3D-structure, like an aggregate or a cluster or a spheroid.
  • the insert plate or assembly may further comprise a seal, lid or cover plate (not shown) that is positioned over said plurality of tubes, for instance to protect the cells and/or spheroids during culturing/incubating, handling and/or imaging.
  • a seal, lid or cover plate (not shown) that is positioned over said plurality of tubes, for instance to protect the cells and/or spheroids during culturing/incubating, handling and/or imaging.
  • insert plate according to the invention as well as the assembly according to the invention provide a rounded-bottom
  • thermoformed, in particular injection-molded, optically clear insert plate to seed and grow spheroids which insert plate can be combined to fit into a flat microplate designed for analysis using conventional high content imaging systems.
  • the optical imaging is performed by focusing through the flat, clear-bottom of the microplate on the spheroids contained in the wells of the insert plate.
  • a convenient and cost-effective spheroid culturing and imaging system can be provided.
  • Fig. 4 shows representative
  • the insert plate is suitably manufactured by using thermoforming technology.
  • Thermoforming line includes heating, forming, cutting and stacking of selected plastic material.
  • the plastic material is based on specific customer requirements.
  • Basic raw materials in thennoforming process are polypropylene, polystyrene, polyethylene terephthalate.
  • the raw material is handled in material rolls. Conventional material roll stand can accommodate rolls with a diameter of max. 1200 mm.
  • thermoforming line that manufactures insert plate is radiation heating.
  • radiation heating the transmission is effected by electromagnetic waves in the infrared range. Infrared rays are absorbed by plastics.
  • the absorption rate is a function of material thickness and wave length of the rays. The thicker the material, the higher the absorption rate. Each material version has its own absorption curve.
  • Forming can be divided to four steps; pre-forming of heated material by pre- stretching, part forming, cooling of formed part, demolding of formed part.
  • pre-forming of heated material by pre- stretching, part forming, cooling of formed part, demolding of formed part.
  • pre-blowing formation of a bubble by pressure air
  • pre-stretching by using a pre stretching plug also called plug assist or pre-stretcher.
  • Forming possibilities include vacuum forming, forming by pressure air, forming by pressure air and vacuum and forming by embossing.
  • Colling possibilities of formed parts can be achieved by contact with forming tool, with forming tool + air, by contact with forming tool + cooled air or free cooling by air.
  • Cutting with a steel rule die is a so called knife cut.
  • Conventional terms for this type of cutting tools knife cutting tool, steel rule cutting tool.
  • thermoformed parts are broken out of the web. This can either be done manually or by forming product stacks in a stacking device.
  • the layout of the stacking station considers the admissible depths of draw. Stacking of positive or negative parts is possible.
  • HepG2 cells (ATCC HB-8065, LGC Standards GmbH, Wesel,
  • HepG2 cells were cultured for two days in thermoformed 384-tube ULA-coated insert plates to obtain spheroids as described above. HepG2 spheroids were visualized by staining the DNA of the cells. To that end, Hoechst-33342 nuclear counterstain (H3570, Life Technologies) was diluted 500-fold in culture medium, and 15 to 25ul was added to the spheroids to obtain a final concentration of 16.2 ⁇ . Spheroids were incubated with Hoechst for two hours to overnight to ensure a homogeneous distribution of dye over the spheroid. Subsequently, spheroids were imaged at the Hoechst-33342 nuclear counterstain (H3570, Life Technologies) was diluted 500-fold in culture medium, and 15 to 25ul was added to the spheroids to obtain a final concentration of 16.2 ⁇ . Spheroids were incubated with Hoechst for two hours to overnight to ensure a homogeneous distribution of dye over the s
  • Operetta imaging station PerkinElmer, Waltham, MA, USA
  • 360- 400nm excitation and 410-480nm emission filters Images were generated with 2x or lOx long working distance (LWD) objectives and captured using the Harmony 4.1 software (PerkinElmer, Waltham, MA, USA).

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Abstract

La présente invention concerne des moyens et des procédés de culture de cellules et d'analyse de cellules, en particulier des dispositifs conçus pour la croissance, la maintenance et/ou l'analyse microscopique de modèles de culture de cellules 3D, tels que des agrégats cellulaires ou des sphéroïdes. L'invention concerne une plaque d'insertion pour fixation de façon détachable à une microplaque standardisée et comportant une pluralité de puits, la plaque d'insertion ayant un corps principal comprenant une pluralité de tubes pour recevoir un échantillon, chaque tube comportant une partie de fond optiquement transparent imperméable aux liquides pour permettre l'imagerie microscopique, et au moins une partie de ladite partie de fond ayant une surface arquée concave, la pluralité de tubes étant positionnés pour être ajustés dans et alignés avec un puits correspondant de la microplaque lorsque la microplaque et la plaque d'insertion sont assemblées. L'invention concerne un ensemble de la plaque d'insertion et d'une microplaque.
PCT/NL2017/050098 2016-02-18 2017-02-17 Moyens et procédés pour culture et analyse de cellules sphéroïdes WO2017142410A1 (fr)

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WO2020234170A1 (fr) 2019-05-17 2020-11-26 Medizinische Universität Wien Production de sphéroïde cellulaire dans un système de culture cellulaire 2d
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US11441121B2 (en) 2013-04-30 2022-09-13 Corning Incorporated Spheroid cell culture article and methods thereof
US11976263B2 (en) 2014-10-29 2024-05-07 Corning Incorporated Cell culture insert
US11667874B2 (en) 2014-10-29 2023-06-06 Corning Incorporated Perfusion bioreactor platform
US11613722B2 (en) 2014-10-29 2023-03-28 Corning Incorporated Perfusion bioreactor platform
US11767499B2 (en) 2017-07-14 2023-09-26 Corning Incorporated Cell culture vessel
US11857970B2 (en) 2017-07-14 2024-01-02 Corning Incorporated Cell culture vessel
US11584906B2 (en) 2017-07-14 2023-02-21 Corning Incorporated Cell culture vessel for 3D culture and methods of culturing 3D cells
US11970682B2 (en) 2017-07-14 2024-04-30 Corning Incorporated 3D cell culture vessels for manual or automatic media exchange
US11345880B2 (en) 2017-07-14 2022-05-31 Corning Incorporated 3D cell culture vessels for manual or automatic media exchange
US11912968B2 (en) 2018-07-13 2024-02-27 Corning Incorporated Microcavity dishes with sidewall including liquid medium delivery surface
US11732227B2 (en) 2018-07-13 2023-08-22 Corning Incorporated Cell culture vessels with stabilizer devices
US11661574B2 (en) 2018-07-13 2023-05-30 Corning Incorporated Fluidic devices including microplates with interconnected wells
WO2020131658A1 (fr) * 2018-12-19 2020-06-25 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Plateforme d'analyse spatiale de pathologie à systèmes de calcul pour données d'imagerie cellulaires et infracellulaires à paramètres multiples in situ ou in vitro
US11983943B2 (en) 2018-12-19 2024-05-14 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Computational systems pathology spatial analysis platform for in situ or in vitro multi-parameter cellular and subcellular imaging data
WO2020234170A1 (fr) 2019-05-17 2020-11-26 Medizinische Universität Wien Production de sphéroïde cellulaire dans un système de culture cellulaire 2d
NL2025992B1 (en) 2020-07-03 2022-03-08 Perkinelmer Health Sciences B V Multiwell plate for 3D cell culturing and imaging, method for manufacturing and uses thereof.
WO2022093883A1 (fr) * 2020-10-30 2022-05-05 Corning Incorporated Plaque à microcavités
WO2023176949A1 (fr) * 2022-03-17 2023-09-21 日産化学株式会社 Récipient de culture cellulaire présentant une efficacité élevée d'utilisation cellulaire

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