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WO2025073684A1 - Procédé et appareil pour la liaison ciblée d'une matrice de culture cellulaire - Google Patents

Procédé et appareil pour la liaison ciblée d'une matrice de culture cellulaire Download PDF

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Publication number
WO2025073684A1
WO2025073684A1 PCT/EP2024/077578 EP2024077578W WO2025073684A1 WO 2025073684 A1 WO2025073684 A1 WO 2025073684A1 EP 2024077578 W EP2024077578 W EP 2024077578W WO 2025073684 A1 WO2025073684 A1 WO 2025073684A1
Authority
WO
WIPO (PCT)
Prior art keywords
matrix
cell
component
cell culture
capture system
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/EP2024/077578
Other languages
German (de)
English (en)
Inventor
Eva WEIMER
Tabea Hein
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.)
Robert Bosch GmbH
Original Assignee
Robert Bosch GmbH
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 Robert Bosch GmbH filed Critical Robert Bosch GmbH
Publication of WO2025073684A1 publication Critical patent/WO2025073684A1/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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • Reliable binding of the cell culture matrix, especially the cell-laden matrix is a challenging task, especially when undefined carryover of matrix residues must be avoided while simultaneously preventing cell loss to maximize cell yield. This is often not sufficiently possible with the state-of-the-art methods.
  • Phase separation by centrifugation of the matrix/cell/medium mixture for cell harvesting and/or purification often results in a clean separation of the middle matrix phase and the lower cell phase; either matrix residues remain in the solution and/or cells are removed with the matrix.
  • Manual removal of the upper medium and middle matrix phases by pipette is also difficult, as well as visually and manually challenging in order to clearly and unambiguously identify and "hit" the different phases.
  • the method and device according to the invention offer a solution for the reliable and required binding of a cell culture matrix, enabling selective and specific retention of the components to be removed from the cell culture, regardless of their density, while simultaneously protecting the cells. Furthermore, this opens up the possibility of using them in an automated process, particularly in an automated miniature process, such as in a (micro)fluidic system, without the clumping known from the prior art and the associated partial or complete blockage of a device suitable for carrying out the method.
  • the invention therefore proposes a method for binding a matrix of a cell culture, which comprises the following steps: a. Providing a cell culture with at least one, preferably several, identical or different cells, wherein the cell culture comprises a matrix; and b. Contacting the cell culture with at least a first component (31) of a biological, chemical and/or physical capture system, wherein the first component (31) of the capture system is a component that binds to the matrix; and c. Introducing and/or contacting and/or incubating an agent with the cell culture, wherein the agent comprises at least one capture molecule and wherein the capture molecule binds or is bound to a second component of the capture system; and d. Obtaining the matrix bound by the capture system.
  • a cell culture is provided in a suitable container and/or vessel, which comprises at least one cell and a matrix.
  • a suitable container and/or vessel which comprises at least one cell and a matrix.
  • more than one cell is provided, for example different cells or a cell network.
  • the cell it is conceivable for the cell to be present individually, encapsulated, or in a cell network with several cells.
  • the at least one cell enters into a bond with the matrix, forms a mixture with it, and/or is incorporated and/or embedded in the matrix. The incorporation and/or embedding of the at least one cell in the matrix preferably takes place immediately.
  • the cell culture is brought into contact with at least a first component of a biological, chemical, and/or physical capture system and incubated.
  • the first component binds covalently or non-covalently and/or directly or indirectly, for example by means of at least one, preferably two or more, identically or differently configured linkers, to the matrix, such as a matrix molecule, a matrix fragment, or a part thereof.
  • the first component of the capture system preferably forms a specific bond.
  • the first component is embedded in the matrix network, covalently or non-covalently. It has been recognized that the first component binds to the matrix, such as a matrix molecule, a matrix fragment, or a part thereof, preferably the technical structures thereof.
  • the at least one cell is embedded and/or embedded in the matrix.
  • biological, chemical, and/or physical capture systems in particular 2-component capture systems, with their known advantages, such as high affinity and specificity, are used.
  • the “bringing into contact” can be carried out, for example, by dripping, injecting, laying on, introducing, dropping, dipping and/or any other method known to the person skilled in the art for bringing into contact bringing the capture system into contact with the cell culture.
  • incubation in the context of the method according to the invention refers to a method step in which the cell culture, together with the capture system or with a first component of the capture system, is in contact over a certain period of time, preferably under suitable conditions, in such a way as to enable the binding of the capture system, in particular the first component of the capture system, with the matrix, such as a matrix molecule, a matrix fragment, or a part thereof, as described elsewhere.
  • Incubation is preferably carried out for several days to weeks, more preferably with intermittent replacement of a cell culture medium.
  • This method step includes, in addition to incubation, for example, also monitoring the latter. Monitoring can be carried out, for example, based on a fixed period of time, for example by observing the cell culture.
  • a colored or otherwise optically visible indication can be provided, which occurs when the binding described elsewhere is achieved by means of a chemical and/or biochemical indicator, e.g. by means of a color, a color change of the color, or loss of color.
  • a chemical and/or biochemical indicator e.g. by means of a color, a color change of the color, or loss of color.
  • the steps of incubation and monitoring can be carried out alternately.
  • step c. at least one, preferably two or more, identically or differently configured agents are introduced into the cell culture or brought into contact with it and incubated.
  • the "introduction” can be carried out, for example, by immersion, submersion, pouring, pouring in, and/or another method for introducing the agent into the cell culture. The bringing into contact and incubation is described in detail elsewhere.
  • the agent can be, for example, but by no means exclusively, a rod, a pestle, a membrane, a vessel, a chamber, a container, a receptacle, a surface, such as the surface of a (micro)fluidic system, and/or a mixture thereof.
  • the agent comprises at least one capture molecule, preferably an immobilized capture molecule, wherein the capture molecule binds or is bound, preferably specifically, covalently or non-covalently and/or directly or indirectly, for example by means of at least one, preferably two or more, identical or differently configured linkers to a second component of the capture system.
  • the capture molecule is a linker. It is conceivable to link the second component to any agent of any shape and/or Dimension. Furthermore, it is conceivable that the second component has at least one, preferably two, three, four, five, six, seven, eight, nine or ten identically or differently configured binding sites for the first component.
  • the matrix bound by the capture system is obtained, in particular cell-laden matrix and/or matrix fragments.
  • the matrix preferably contains the cells bound to it.
  • This step is influenced, for example, by the contacting and incubation from steps b. and c. More preferably, the obtaining in step d. can include purification and/or separation. In this way, it is possible to remove the matrix, in particular the cell-laden matrix, matrix fragments and/or polymer molecules from the cell culture, as well as to hold the matrix, such as a formed three-dimensional matrix, in the desired position via the ligands it contains. This allows, for example, cell-laden beads or layers to be spatially fixed after their production.

Landscapes

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

Abstract

L'invention concerne un procédé pour lier une matrice (20) d'une culture cellulaire, comprenant les étapes consistant à : a. fournir (100) une culture cellulaire comprenant au moins une cellule (10), cette culture cellulaire comprenant une matrice (20) ; et b. mettre en contact et incuber (300) la culture cellulaire avec au moins un premier composant (31) d'un système de capture biologique, chimique et/ou physique (30), ledit premier composant (31) dudit système de capture (30) étant un composant se liant à ladite matrice (20) ; et c. mettre en contact et incuber (500) un agent avec la culture cellulaire, cet agent (40) comprenant au moins une molécule de capture (41), cette molécule de capture (41) se liant ou étant liée à un deuxième composant (32) du système de capture (30, 31, 32) ; et d. à obtenir (600) la matrice (20) liée au moyen du système de capture (30, 31, 32).
PCT/EP2024/077578 2023-10-02 2024-10-01 Procédé et appareil pour la liaison ciblée d'une matrice de culture cellulaire Pending WO2025073684A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102023209675.7A DE102023209675A1 (de) 2023-10-02 2023-10-02 Verfahren und Vorrichtung für das gezielte Binden einer Matrix einer Zellkultur
DE102023209675.7 2023-10-02

Publications (1)

Publication Number Publication Date
WO2025073684A1 true WO2025073684A1 (fr) 2025-04-10

Family

ID=92973464

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/077578 Pending WO2025073684A1 (fr) 2023-10-02 2024-10-01 Procédé et appareil pour la liaison ciblée d'une matrice de culture cellulaire

Country Status (2)

Country Link
DE (1) DE102023209675A1 (fr)
WO (1) WO2025073684A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69221484T2 (de) * 1991-04-25 1998-02-19 Univ Brown Res Found Implantierbare, biokompatible immunisolator-trägersubstanz zum abgeben ausgesuchter, therapeutischer produkte
US20050054101A1 (en) * 2003-07-17 2005-03-10 Global Cell Solutions Llc Automated cell culture system and process
DE102010012252A1 (de) * 2010-03-22 2011-09-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Thermoresponsives Substrat mit Mikrogelen, Verfahren zu dessen Herstellung und Kultivierungsverfahren für biologische Zellen
US20180312924A1 (en) * 2017-04-26 2018-11-01 Michael Joseph Pugia Protein and gene analysis from same sample
US20220145355A1 (en) * 2019-03-13 2022-05-12 Evorion Biotechnologies Gmbh Methods and kits for determining cell secreted biomolecules
US20220325240A1 (en) * 2021-01-12 2022-10-13 Berkeley Lights, Inc. Systems, apparatuses, and methods for cellular therapeutics manufacture
EP4141096A1 (fr) * 2021-08-25 2023-03-01 Sartorius Stedim Fmt Sas Procédés de production de cellules modifiées
WO2023076629A1 (fr) * 2021-10-29 2023-05-04 Slingshot Biosciences, Inc. Particules d'hydrogel en tant que cellules nourricières et en tant que cellules présentatrices d'antigènes synthétiques

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69221484T2 (de) * 1991-04-25 1998-02-19 Univ Brown Res Found Implantierbare, biokompatible immunisolator-trägersubstanz zum abgeben ausgesuchter, therapeutischer produkte
US20050054101A1 (en) * 2003-07-17 2005-03-10 Global Cell Solutions Llc Automated cell culture system and process
DE102010012252A1 (de) * 2010-03-22 2011-09-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Thermoresponsives Substrat mit Mikrogelen, Verfahren zu dessen Herstellung und Kultivierungsverfahren für biologische Zellen
US20180312924A1 (en) * 2017-04-26 2018-11-01 Michael Joseph Pugia Protein and gene analysis from same sample
US20220145355A1 (en) * 2019-03-13 2022-05-12 Evorion Biotechnologies Gmbh Methods and kits for determining cell secreted biomolecules
US20220325240A1 (en) * 2021-01-12 2022-10-13 Berkeley Lights, Inc. Systems, apparatuses, and methods for cellular therapeutics manufacture
EP4141096A1 (fr) * 2021-08-25 2023-03-01 Sartorius Stedim Fmt Sas Procédés de production de cellules modifiées
WO2023076629A1 (fr) * 2021-10-29 2023-05-04 Slingshot Biosciences, Inc. Particules d'hydrogel en tant que cellules nourricières et en tant que cellules présentatrices d'antigènes synthétiques

Also Published As

Publication number Publication date
DE102023209675A1 (de) 2025-04-03

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