EP4444871A1 - Structures biocompatibles permettant la liaison et la culture de matériel biologique - Google Patents

Structures biocompatibles permettant la liaison et la culture de matériel biologique

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Publication number
EP4444871A1
EP4444871A1 EP22835228.2A EP22835228A EP4444871A1 EP 4444871 A1 EP4444871 A1 EP 4444871A1 EP 22835228 A EP22835228 A EP 22835228A EP 4444871 A1 EP4444871 A1 EP 4444871A1
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EP
European Patent Office
Prior art keywords
linker
aggregates
linkerspheres
biological material
spheres
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
EP22835228.2A
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German (de)
English (en)
Inventor
Kevin ACHBERGER
Stefan LIEBAU
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.)
Eberhard Karls Universitaet Tuebingen
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Eberhard Karls Universitaet Tuebingen
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Publication of EP4444871A1 publication Critical patent/EP4444871A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/085—Coculture with; Conditioned medium produced by cells of the nervous system eye cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/086—Coculture with; Conditioned medium produced by cells of the nervous system glial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2523/00—Culture process characterised by temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to a method for producing biocompatible structures for connecting and culturing biological material, a method for culturing aggregates of biological material, and the use of biocompatible structures for connecting and culturing biological material.
  • spheroids suspension cultures of cell aggregates, so-called spheroids or organoids
  • spheroids can be produced via self-assembly.
  • adherent individual or mixed cultures are brought together in a vessel and then form spheres independently or differentiate into organ-like, complex organoids.
  • retinal organoids They form a light-sensitive, structured retina.
  • Gut organoids may represent gut transport functions and contribute to villi in vivo.
  • Other examples are brain organoids, kidney organoids, etc.
  • organoids or spheroids remain individual organ/tissue systems and do not form complex organ systems with one another. In most cases, they also do not form any vascular or conduction pathways or structures that grow or migrate into the organ from other areas of the body during normal development.
  • assembloids represent the fusion of several organoids into a morphological and functional unit.
  • An example of this are cortico-spinal-muscle assembloids, which consist of brain areas (cortex), spinal cord spheroids (spinal) and muscle cells; see Anderson et al., Generation of Functional Human 3D Cortico-Motor Assembloids. Cell, Vol. 183, 2020, pp. 1913-1929, e1-10.
  • the assembloids like the organoids, can only be poorly oriented to one another and fuse with one another largely at random.
  • the organoids and assembloids are often of different sizes, making it difficult to bring them together in the correct spatial orientation. Frequently, one organoid or assemblage "absorbs" and overgrows another. Further, the possibilities are organoids and assembloids to give a structure, very limited. Organoids and assembloids can only be connected "in series", but not in a different orientation. After all, there is no easy way to add missing individual cells or substances or functional structures, such as immune cells, to the fused organoids or assemblages.
  • the object of the invention is to provide a method with which the disadvantages of the cultivation methods known in the prior art are avoided or at least reduced.
  • linkerspheres biocompatible structures for connecting and cultivating biological material
  • biocompatible stands for the property of being non-toxic to biological material.
  • linker spheres according to the invention and the matrix material this means in particular that the latter are suitable as a substrate for the cultivation and growth of biological material.
  • biological material includes biological cells, aggregates of biological cells, organs and parts thereof.
  • the non-polar, water-immiscible biocompatible liquid provided in step (1) is one that behaves like mineral oil, is 100% water-immiscible to form an emulsion according to the invention, and not for biological Material has toxic properties.
  • the liquid provided in step (1) is "mineral oil” (CAS number: 8042-47-5; EC number: 232-455-8), i.e. a highly viscous bioreagent having a grade and has a degree of purity that allows it to be used in molecular biology, e.g. for layering aqueous solutions and centrifugation gradients (quality level 200).
  • mineral oil from Sigma-Aldrich (M5904), Carl Roth GmbH & Co. KG (#8904), Merck (#107160; #113898) is suitable according to the invention.
  • Synonyms for the mineral oil according to the invention are "paraffin oil” and "vaseline oil”.
  • the mineral oil is preferably provided in a container, more preferably in a microreaction vessel of a suitable size, for example with a volume of 2 ml, 1.5 ml, 0.5 ml, 0.2 ml etc. (e.g. "Eppendorf '-Vessel).
  • a “matrix material” is understood to mean a mixture of molecules which is present in liquid form in step 2 and can be converted into a solid, tissue-like form by the action of physical or chemical phenomena.
  • a biocompatible, preferably network-like structure is provided by the matrix material in the solid form, which allows the cultivation of biological material.
  • matrix material suitable according to the invention include hydrogels, basement membrane-like matrices (e.g. Matrigel; contains a high, beneficial amount of laminin), and other biocompatible, gel-like substances.
  • the solution of the biocompatible matrix material is introduced into the mineral oil in step (2) using a suitable dosing device, such as a pipette. Due to the fluidity of the aqueous matrix material, it is preferably introduced into the mineral oil in the form of a liquid droplet in order to form an emulsion.
  • a suitable dosing device such as a pipette. Due to the fluidity of the aqueous matrix material, it is preferably introduced into the mineral oil in the form of a liquid droplet in order to form an emulsion.
  • the size of the linker spheres according to the invention can be easily controlled via the volume of the matrix material. A larger volume results in larger linkerspheres, a smaller volume in smaller linkerspheres.
  • step (3) an incubation takes place for a period of time which allows the formation of the linker spheres, preferably from at least 10 seconds up to 60 minutes.
  • step (4) of the method according to the invention due to the interfacial effects between the aqueous biocompatible matrix material and the surrounding mineral oil, the aqueous biocompatible matrix material in the emulsion assumes a spherical shape, which gives the linkerspheres their name.
  • biocompatible structures can be created in the form of linker spheres, which represent a valuable tool for tissue construction (engl. "Tissue Engineering”), with which the disadvantages of the prior art at least can be partially or completely remedied.
  • the linkerspheres provide a physiological environment for biological material, which also allows the formation of vascular and vascular pathways, which grow into an organ or migrate from different areas of the body during the development of an organism.
  • the linker spheres obtained according to the invention enable the reproduction of complex cell, tissue and organ interactions. They are therefore particularly suitable for examining complex biological structures such as organs, organ systems, etc.
  • the matrix material is a basement membrane-like matrix.
  • a "basal membrane-like matrix” is understood to mean a complex mixture of biomolecules that is used in 3D cell culture and in tissue construction as a basis for growth, ie as a matrix or cell substrate.
  • a basement membrane-like matrix is the purified secretion of the murine sarcoma cell line Engel-breth-Holm-Swarm (EHS cells) and is compositionally similar to the extracellular matrix of the basement membranes of animal cells. It contains laminin, entactin, collagen and heparan sulfate proteoglycans, among others. This measure has the advantage that a biocompatible matrix material that is particularly suitable according to the invention is used. At approx.
  • the basement membrane-like matrix forms a crosslinked, gel-like structure or a hydrogel through polymerization of the proteins it contains, while it is liquid at lower temperatures, for example at 4°C.
  • basal membrane-like matrices lead to the preservation of a complex, physiological, three-dimensional cell structure.
  • An overview of basement membrane-like matrices can be found, for example, in Hughes et al: A complex protein mixture required for optimal growth of cell culture. In: Proteomics. Volume 10, Number 9, 2010, ISSN 1615-9861, pp. 1886-1890.
  • Trade names for basement membrane-like matrices suitable according to the invention are e.g. B. Matrigel (Corning Life Sciences), BME, EHS matrix etc.
  • step (2) of the method a solution of a growth factor reduced (GFR) basement membrane-like matrix is introduced.
  • GFR growth factor reduced
  • GFR basement membrane matrix useful in the present invention is sold under the trade name Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, product number 354230, Corning Life Sciences.
  • step (3) of the method the emulsion is treated to solidify the matrix material, preferably by subjecting the emulsion to heat, preferably at about 37° C., more preferably for about 15 to 30 minutes.
  • This measure has the advantage that the linker spheres are brought into a solid form that is manageable and suitable for cultivation purposes by the cross-linking of the matrix material or the proteins contained therein. Solidification or cross-linking results in the formation of a solid hydrogel or gel-like structure, which allows biological material to be cultivated and grown through.
  • the heat treatment can conveniently be carried out by placing the reaction vessel containing the emulsion in a water bath.
  • the solution of the biocompatible matrix material contains cell culture medium.
  • This measure creates the physiological prerequisites for the linker spheres to be used immediately after their production for the cultivation of biological material.
  • Any type of cultivation medium is suitable according to the invention, with the specific choice by the person skilled in the art being based on which biological material is to be cultivated or connected to the linkerspheres.
  • a cell culture medium suitable for cultivating astrocytes is, for example, N2 medium and “B27-based retinal differentiation medium” (BRDM medium).
  • the solution of the matrix material contains biological material, preferably biological cells.
  • linker spheres containing biological material can be used as a separate cultivation unit or used to connect units or aggregates of biological material.
  • the solution of the biocompatible matrix material has a dye.
  • step (2) of the method the solution of the matrix material is introduced into the mineral oil as drops of fluid, preferably via a pipette tip.
  • the tip of the pipette which contains the solution of the biocompatible matrix material, is held directly over the surface of the oily liquid. If a micropipette is used, preferably up to the first pressure point pressed. In this way a drop forms at the front of the pipette. By immersing the pipette tip in the oily liquid, the drop detaches from the tip and sinks into the oily liquid to the bottom of the vessel.
  • step (4) is carried out after step (4):
  • the linker spheres are advantageously removed from the emulsion and made available for further use.
  • step (5) the following step is carried out after step (5):
  • the remaining mineral oil is removed from the linker spheres in an advantageous manner.
  • This washing step can be repeated to remove any excess oil from the linkerspheres. This prevents any excess oil from having a damaging effect in subsequent cultivation applications.
  • step (4) is carried out after step (4) and before step (5):
  • the isolated and optionally washed linker spheres are transferred to a culture vessel, preferably a culture dish.
  • the linker spheres are placed in an environment that allows them to be used directly for the cultivation and connection of biological material.
  • Commercially available culture dishes, such as Petri dishes, are suitable and their selection depends on the specific use of the linkerspheres.
  • the isolated and optionally washed and optionally transferred linkerspheres are cultivated, preferably at approx. 37° C., more preferably at approx. 20% by volume O2, more preferably at approx. 5 vol % CO2, more preferably for at least about 12 hours.
  • This measure has the advantage that particularly suitable parameters for cultivating the linker spheres are used.
  • Another subject of the present invention relates to a method for cultivating and/or connecting aggregates of biological material, which has the following steps:
  • linkerspheres biocompatible structures for connecting and culturing biological material
  • the complexes of the aggregates and the linker spheres are “cultivated” under the usual cell culture conditions, for example in an incubator in a nutrient medium at about 37° C. and optionally 5% by volume CO2.
  • the aggregates of biological material can be connected or fused to one another at a defined distance which is determined by the linker spheres.
  • more than one linker sphere or any number of them can also be interposed. Due to the distance created by the linkerspheres, there is a spatial separation of the aggregates to be fused; this means that they cannot easily grow into one another and one aggregate is prevented from overgrowing another.
  • the linkerspheres also allow a large number of aggregates to be linked to the same aggregate in order to obtain a cloverleaf-like structure, for example.
  • the linker spheres can be used to create pathway systems that link the aggregates of biological material to one another, for example conductive blood pathways, nerve pathways, or structures of the extracellular matrix (ECM). These can also be combined with each other.
  • the track systems can either be provided as further spheroid structures (e.g. by means of vascular organoids) or during the production of the linker spheres in the latter are embedded. The fusion of several linker spheres with different cell types is also possible without any problems.
  • the linkerspheres can contain any desired ECM structures and thus simulate the body/web structures.
  • linker spheres With the linker spheres according to the invention, the fusion of aggregates of different sizes is possible without any problems, since these only have to be coupled to the linker spheres and then track systems can be connected in between.
  • the aggregates of biological material are cut before being brought into contact with the linkerspheres, preferably with microscissors, wherein, more preferably, the aggregates of biological material are brought into contact with the cut surface with linkerspheres become.
  • the aggregates are brought into a shape and size which is based on the size of the linker sphere and/or the desired connection.
  • the cut surface creates a particularly good contact surface and ensures a good connection with the aggregates made of biological material.
  • the cultivation takes place at about 37° C., preferably at about 20% by volume of O 2 , more preferably at about 5% by volume of CO 2 .
  • the aggregates of biological material are organoids and/or spheroids and/or assemblages.
  • This measure has the advantage that the disadvantages described in the prior art for organoids and assemblages are reduced or even avoided.
  • the interposition of linker spheres means that there is no direct fusion of possibly different types of tissue, which is also generally not the case in the organism.
  • the interposition of the linkerspheres also allows the correct and arbitrary spatial orientation of the aggregates, which usually occurs randomly and undirectedly in the case of direct fusion, especially in the case of aggregates of different sizes.
  • the method according to the invention also allows the aggregates to be oriented “in series” for the first time.
  • the aggregates of biological material are selected from the group consisting of: retinal organoid, vascular organoid, brain organoid, neurospheroid.
  • a “retinal organoid” is a three-dimensional structure of cells of the retina differentiated from pluripotent stem cells that contains layering, cell-cell interaction, and cell-type specific diversity that mimics the human embryonic retina. Furthermore, photoreceptors can form in the retinal organoids, which are light-sensitive and have the special structure (inner and outer segment) of this type of cell.
  • a “vascular organoid” is understood to mean a three-dimensional structure which can be formed from pluripotent stem cells and contains cells of the vascular system. These include endothelial cells and pericytes.
  • a "brain ganoid” is an organoid formed from pluripotent stem cells, which can reflect the organization and cell diversity of certain areas of the brain.
  • a thalamic organoid contains neurons that occur in the thalamus.
  • a "neurospheroid” is a spheroidal arrangement of neural cells ( Neurons, glial cells and their progenitors) that can be generated from pluripotent stem cells can include organized (eg neurorosettes or cortical areas) and disorganized areas.
  • the aggregates of biological material have astrocytes, preferably those derived from induced or embryonic pluripotent stem cells (iPSC).
  • the stem cells are preferably of human or animal origin.
  • steps (2) and (3) can be repeated at least once, twice, three times, four times, ... ten times, twenty times, a hundred times, etc.
  • Another object of the present invention relates to the use of linker spheres obtained according to the production method according to the invention for the connection and cultivation of biological material.
  • Fig. 2 Astrolinker in schematic (a) and microscopic representation (b) -
  • Fig. 4 Microscopic representation of the process of connecting retinal
  • organoid and linkersphere (a), a suspension culture of the junction product (b), outgrowth of axons from the retinal organoid into a linkersphere (c), the junction product at single junction (d) and at double junction (e);
  • Fig. 7 Microscopic representation of a double connection of an astrolinker with a neuosphere and a retinal organoid
  • Fig. 8 Schematic representation of a complex optic nerve model under
  • linker spheres according to the invention Use of linker spheres according to the invention.
  • 9 Schematic representation of the support of vessel vascularization by linker spheres according to the invention.
  • Fig. 10 Schematic representation of multi-linking concepts.
  • FIG. 1 shows a schematic overview of the production of the linker spheres.
  • mineral oil is provided in a test vessel.
  • the liquid, biocompatible matrix material is drawn up in a pipette, possibly mixed with biological material, such as biological cells, and/or culture medium.
  • a drop of the solution of the biocompatible matrix material is introduced into the mineral oil.
  • the biocompatible matrix material on the underside of the pipette is brought into contact with the surface of the mineral oil.
  • the volume of the drop is then ejected from the pipette and the drop is allowed to sink to the bottom of the test tube filled with mineral oil.
  • the reaction vessel with the emulsion formed therein is subjected to a heat treatment, for example, by placing it in a water bath and incubating it at 37°C for 30 minutes.
  • the proteins of the biocompatible matrix material are cross-linked and the matrix material is solidified so that a gel-like structure or a hydrogel is formed, the "linker sphere".
  • the linker sphere is washed and transferred to culture medium. details
  • linkerspheres with cells The technical details for the production of linkerspheres with cells are described below. This is done using the example of linkerspheres that contain astrocytes, so-called "astrolinkers”.
  • DMEM/F12 with Glutamax 2% Hormone Mix, 1% Non-Essential Amino Acids (NEAA), 1% Antibiotics-Antimycotics (Anti-Anti), all Thermo Fisher Scientific)
  • the preparation of the astrocyte-containing linkerspheres is as follows:
  • AC Human iPSC-derived astrocytes
  • N2 medium at twice the amount containing TrypLE medium is added to stop the reaction.
  • the cells are transferred to a 15 ml conical tube and centrifuged at 1500 g for 2 minutes. The supernatant is discarded and the cells are resuspended in an appropriate volume of N2 medium. to count the cells (about 500 ⁇ l-1 OOOpl) in a Neubauer counting chamber diluted 1:1 with trypan blue to visualize dead cells Number of cells is transferred to a 1.5 ml Eppendorf container. 10,000 cells are required to produce 1 Astrolinker with a size of 2.5 ⁇ l per linker.
  • the collected cells are then pelleted again at 800 g for 2 minutes. The supernatant is discarded carefully and as completely as possible.
  • the cell pellet is then resuspended in cold BRDM medium to achieve 1.25 ⁇ l per astrolinker (e.g. for 10 linkers/100,000 cells, 10 ⁇ l of medium is used). It is then stored on ice. Growth factor-reduced Matrigel (thawed overnight in the refrigerator) is added to the chilled cell suspension in a 1:1 ratio and the solution is gently and thoroughly mixed, avoiding the formation of bubbles. Ink or other dyes can be added to the Matrigel to make the linkers more visible later. Here 5% of a 1:1000 dilution of ink in PBS was used.
  • reaction tubes 1.5 mL reaction tubes (1 tube per astrolinker) are filled with ⁇ 50 ⁇ L mineral oil and stored at room temperature until use.
  • 2.5 ⁇ l of the astrocyte-Matrigel mixture are then transferred into the reaction vessel filled with mineral oil using a thin 10 ⁇ l pipette tip (if possible pre-cooled).
  • the tip of the pipette which contains 2.5 ⁇ l of astrocyte-Matrigel mix, is held directly over the surface of the oily liquid and then pressed to the first pressure point. In this way a drop forms at the front of the pipette.
  • the tube containing the linker is placed on a heating block (37°C) as quickly as possible to allow rapid solidification. This is necessary to avoid the cells being distributed unevenly in the drop.
  • the tubes are then incubated for 15-30 minutes at 37°C.
  • the washed astrolinkers are transferred to a non-tissue treated 48-well plate containing 250 ⁇ l of ASC ++ prewarmed to 37 °C. Again, clipped tips are used.
  • the astrolinkers are stored at 37 °C, 20% O2 and 5% CO2 at least overnight and the medium is changed every 2-3 days until further use or fixation (half medium change).
  • the astrolinker is shown schematically in FIG. 2a.
  • 2b shows an astrolinker (linkersphere loaded with astrocytes) after one day in culture.
  • 2c shows an astrolinker with astrocytes which were previously transfected with a lentiviral construct (Lenti-GFAP-GFP) which expresses a green fluorescent protein (GFP) under a 'glial fibrillary acidic protein (GFAP) promoter.
  • Figures 2d and 2e show a three-dimensional reconstruction of part of an astrolinker in which the astrocytes, as in 2c, express GFP under a GFAP promoter.
  • FIG. 2d) shows an image of the fluorescence colored in magenta, FIG. 2e a height-coded false color image.
  • astrocytes instead of astrocytes, only medium (e.g. BRDM) is added to the GFR-Matrigel. All subsequent steps remain the same.
  • Cell-free linkerspheres can be cultured in any 37°C prewarmed medium or buffer. 2. Establishing double connections
  • RO Human iPSZ-derived retinoid organoids
  • the RO are cut in two with microscissors and transferred to a non-tissue treated 96-well V-shape plate. Half an organoid is placed in each well. An astrolinker or cell-free linkersphere with a 10OQ-1 tip is then placed in the wells containing the RO. Using a small needle or pipette tip, the RO and linker are positioned under a microscope. The RO is positioned so that the cut side touches the Astrolinker/Linkersphere directly. The plate is then placed extremely gently (without disturbing the positioning) in an incubator (37 °C, 20% O2, 5% CO2).
  • thalamic organoids differentiated according to a previously published protocol (Xiang 2019) are selected after 40-80 days of differentiation.
  • Each TO is placed in the 96-well V-shape plate containing the double link (Astrolinker/Linkersphere + RO).
  • the triple junction is made with a small needle or point, placing the thalamic organoid directly on the opposite side of the retinal organoid attached to the Astrolinker/Linkersphere. This is critical so that the cell junction must form through the astrolinker and not directly. Without moving, the plate is stored overnight in the incubator at 37 °C, 20% O2 and 5% CO2.
  • the triple compound is optionally cultivated on the following day.
  • the connection process is shown schematically in FIG.
  • the scale bar in the two micrographs shown in the figure below corresponds to a distance of 1000 pm.
  • a non-tissue-treated 48-well plate is prepared with 250 ⁇ l of BRDM medium prewarmed to 37°C.
  • the triple compounds are transferred to this 48-well plate (with 1000 ⁇ l tips cut off) and incubated again at 37°C, 20% O2, 5% CO2.
  • the linkers are checked under the microscope.
  • the RO can e.g. be stably transduced with GFP (e.g. with lentiviral vectors).
  • GFP e.g. with lentiviral vectors.
  • Medium changes for double or triple connections are performed every second to third day with warm BRDM medium (250 ⁇ l per well, half medium change).
  • FIG. 4b shows the result of the connection process between a linker sphere and a retinal organoid in a microscopic image.
  • Fig. 4b the complex is shown in a suspension culture.
  • the outgrowth of neurites from the organoid into the linker sphere can be seen in FIG. 4c.
  • FIG. 4d shows the result of a single linking of retinal organoid and linkersphere
  • FIG. 4e shows the result of a double link.
  • the astrocytes are labeled with GFP and stained accordingly.
  • FIG. 5 shows a double linkage of a linkersphere with a neuosphere on one side and a retinal organoid on the other side.
  • the neuosphere is connected to the retinal organoid by neural pathways that pass through the linkersphere.
  • Fig. 6 it is shown that GFP-labeled cells of the retinal organoid project inside the neurosphere and are positive for the ganglion cell marker NEFM.
  • FIG 7 a double linkage of an astrolinker with a neuosphere on one side and a retinal organoid on the other side is shown.
  • the Neuosphere is connected to the retinal organoid by neural pathways that pass through the linkersphere.
  • the complex optic nerve model e.g. B. to model glaucoma, consists of a retinal organoid containing retinal ganglion cells, two linkers filled with oligodendrocytes (myelinated part of the optic nerve) + astrocytes (intra-retinal part of the optic nerve)) + brain organoids (patterned e.g. for diencephalon or metathalamus).
  • FIG. 9 shows schematically how the vascularization can be supported by means of the linker body, e.g. by connecting tissue organoids to blood vessel organoids.
  • Multi-linking concepts are shown in FIGS. 10a and 10b.
  • Linkerspheres allow to combine multiple organoids and different connective concepts (e.g. neuronal connections, vascularization). Linkerspheres could potentially facilitate growth/assembly of complex multiorganoid and multicellular tissues including nerve outgrowth and vascularization.

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Abstract

La présente invention concerne un procédé de production de structures biocompatibles permettant la liaison et la culture de matériel biologique, un procédé de culture d'agrégats de matériel biologique, et l'utilisation de structures biocompatibles permettant la liaison et la culture de matériel biologique.
EP22835228.2A 2021-12-07 2022-12-07 Structures biocompatibles permettant la liaison et la culture de matériel biologique Pending EP4444871A1 (fr)

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DE102021132190.5A DE102021132190A1 (de) 2021-12-07 2021-12-07 Biokompatible Strukturen zur Verbindung und Kultivierung von biologischem Material
PCT/EP2022/084841 WO2023104909A1 (fr) 2021-12-07 2022-12-07 Structures biocompatibles permettant la liaison et la culture de matériel biologique

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