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
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/22—Settling tanks; Sedimentation by gravity
• • 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

Definitions

• EP 3425043 describes a screening method using spheroids organized in a multi-well culture plate.
• the spheroids are formed in U-shaped wells whose walls are non-adherent for the cells.
• the effect of the tested drugs is evaluated by validating whether a spheroid is self-assembled or not.
• WO 2017-008699 entitled “cell traction force measuring device, measuring method and preparation method” describes a measuring device using a layer of nano-pillars of known Young's modulus at the top of which the cells are cultivated. Analysis of the images reveals the deformation of the nano-pillars, and thus a map of the forces applied by each cell can be established.
• This method is compatible with high-resolution imaging, allowing us to understand what happens at the cellular and subcellular level.
• this method measures the forces exerted by individual cells which are not necessarily representative of the pathology and/or the organ that we wish to study.
• manufacturing nanopillars is expensive and complex. This prevents the adaptation of these methods to multi-well plates which are standard for drug screening.
• the main disadvantage of this technology relates to the ease of use of the process.
• the rings of heart tissue must be removed from the molds while this manipulation is difficult. Additionally, imaging of individual cells is not possible because the rings are too thick, while ring imaging and force assessment cannot be performed simultaneously, leading to a time-consuming process. time-consuming and very expensive.
• the device can be made at least partially, preferably completely, in a material based on a flexible or soft biocompatible hydrogel making it possible to measure the force of contraction of the aggregate of cells forming the annular-shaped tissue;
• the cells When the cells are seeded on top of this substrate, the cells fall to the bottom of the substrate under the effect of gravity or an artificially applied force such as a centrifugal force so that they are guided along the flanks towards the groove, leading to the formation of a cellular aggregate or tissue whose shape corresponds to that of the groove.
• the structured culture substrate is preferably composed of a flat support on which the grooves have been formed (preferably molded) in which the cells spontaneously organize themselves into a tissue.
• the opening of the cavity is circular and has a diameter greater than the diameter of the annular groove.
• the device has a central pillar disposed in the center of the groove when the latter is of annular shape.
• the central pillar is completely surrounded by the annular groove and preferably delimits the internal perimeter of the annular groove.
• the central pillar comprises a local reduction of its perimeter to an intermediate height in order to allow reliable and constant maintenance of the position of the cell ring around the pillar, in particular when the cells contract or if an external force comes. Apply to the cell ring, for example if the culture medium is replaced. This local reduction in the perimeter prevents the ring from sliding along the pillar if it contracts around it.
• the local reduction in perimeter is less than 20% of the average perimeter of the central pillar to facilitate the unmolding of the structure.
• the local reduction in perimeter is less than 20% of the average perimeter of the central pillar to facilitate the unmolding of the structure.
• the device according to the invention is characterized in that the base of the central pillar is integral with the structured substrate and in that the other end of the central pillar is preferably conical in shape to guide the seeded cells towards the groove.
• the device is made partially or totally of a photo-polymerizable polymer material in order to reduce its manufacturing time.
• the walls of the cavity are made of non-adherent material for the cells in order to let the cells freely exert their own force on the aggregate.
• the lower part of the groove (bottom), to which the cells adhere can be adherent to provide support for the formation of the aggregate, which makes it possible to stabilize its shape and prevent the aggregate
• the cell contracts on itself and takes the shape of a simple spheroid.
• the culture device according to the invention, the multi-well support and the method can be used for producing cell cultures, in particular cells derived from human cells.
• the dimensional analysis shows us that the force applied by the organoid can be determined as follows:
• the structured substrate of the device according to the invention can be manufactured using any type of material which has the capacity to polymerize and which is biocompatible.
• the monomer molecules react so as to form a three-dimensional network or polymer chain by polymerization and forming polymers which may be synthetic or natural in nature.
• the polymer will be a hydrogel allowing the diffusion of nutritional molecules and the oxygenation of the organoid and therefore its well-being.
• the material constituting the structured substrate 1 (at least the walls of the cavity) will preferably be a non-adherent material to help guide the cells towards the annular groove and/or to prevent adhesion of the organoid to the substrate. .
• a material allowing cell adhesion will be used. This can be further improved by the presence on the surface of a specific extra cellular matrix such as collagen, fibronectin, etc.
• the dimensions of the annular groove can be adapted to the size of the organoids used in the process within the limits described above.
• FIG.l shows a sectional view of a device according to the invention
• FIG.4 shows the steps of molding a device according to the invention
• FIG.5 shows photographs of molds for manufacturing the device according to the invention
• the cells can be cultured in two ways: either simply suspended in a cell culture medium, or mixed with an extracellular matrix, either natural or synthetic, such as for example type I, II, III, IV and V collagen.
• an extracellular matrix either natural or synthetic, such as for example type I, II, III, IV and V collagen.
• Collagen is the main compound of the natural extracellular matrix in the human body, this protein is present between cells in many connecting tissues. It can also be a matrigel, a hydrogel or other extracellular matrices.
• FIG.4 illustrates a method of manufacturing the device according to the invention.
• a preferably flat support 23 glass plate petri dish, multi-well plate, etc.
• a layer 24 of the chosen polymer in the unpolymerized state
• the coated layer 24 takes the desired form of positive structure of the micro-structured substrate 1.
• demolding [Fig.4]
• the final 3D micro-structured substrate 1 is obtained.
• the interior cavity is produced in two stages: a first stage during which a drill bit with a diameter less than that of the central pillar is used to dig a cylindrical hole, and a second stage during which a drill with a spherical profile is used to finely adjust the profile of the walls, allowing the manufacture of "S" shaped or curved profiles and a sharp conical hole which gives the conical profile of the central pillar.
• CNC computer numerical control
• the example below uses a substrate based on polyacrylamide.
• the starting support consists of glass strips 16 mm in diameter which are cleaned and silanized to promote adhesion.
• UV radiation of appropriate wavelength for example, 365 nm
• 365 nm UV radiation of appropriate wavelength
• the following reagents are used in the formulation of these types of gels: C3H5O Acrylamide 40%; C7H10O2N2 N,N’-Methylenebisacrylamide 2%; Irgacure 2959 (1% v/v) - UV sensitive (wavelength 365 nm);
• FIG.7 is an image of the fibroblast cells after centrifugation while [Fig.8] is an image of the same cells after collagen polymerization (1 hour, 37 degrees) showing that the individual fibroblast cells form a perfect ring around of the central pillar.
• This example shows the rapid and spontaneous assembly of a 3D organoid in an annular shape using the device of the invention.
• This example also demonstrates that the organoid can be preserved in structured substrates for a long period.
• the "s" shaped mold plays an important role, as it allows the ring-shaped organoid to stay in place even when the cells contract and could possibly slide down the central pillar, or when fluid is agitated, for example when changing the environment. Maintaining the organoid in place allows stable observation over a long period while allowing the surrounding environment to be easily changed.
• Measuring the variation in the diameter of the pillar (in other words the internal diameter of the ring) is used to evaluate the variation in the contraction force applied by the cellular structure on the central pillar.
• a drug such as Latrunculin A (actin depolymerization inducer) can be used to inhibit cell contraction and thus deduce the force of contraction before application of the drug.
• Latrunculin A actin depolymerization inducer
• the diameter of the central pillar increases by approximately 1.5% in the presence of Latrunculin A compared to the control experiment without the drug. This is demonstrated in [Fig. He],

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• Health & Medical Sciences (AREA)
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• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical & Material Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Sustainable Development (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Clinical Laboratory Science (AREA)
• Molecular Biology (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 2022
  • 2022-06-13 FR FR2205692A patent/FR3136480B1/fr active Active
• 2023
  • 2023-06-05 EP EP23734337.1A patent/EP4536799A1/de active Pending
  • 2023-06-05 WO PCT/FR2023/050791 patent/WO2023242495A1/fr not_active Ceased

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