Methods and devices for targeted binding of matrix of cell culture

Abstract

The present invention relates to a method of binding a matrix (20) of a cell culture, comprising the steps of: a. Providing (100) a cell culture comprising at least one cell (10) wherein the cell culture comprises a matrix (20); and b. Contacting the cell culture with at least one first component (31) of a biological, chemical and / or physical capture system (30) and incubating (300), wherein the first component (31) of the capture system (30) is a component bound to the substrate (20, 21); and c. Contacting a tool (40) with the cell culture and incubating (500) wherein the tool (40) comprises at least one capture molecule (41), and wherein the capture molecule (41) binds or is bound to a second component (32) of a capture system (30, 31, 32); and d. Obtaining (600) the substrate (20, 21) bound by means of the capture system (30, 31, 32).

Description

Technical Field

[0001] The present invention relates to a method for binding a substrate for cell cultures, particularly a cell-loaded substrate, and an apparatus for binding a substrate for cell cultures, particularly a cell-loaded substrate. Existing technology

[0002] 3D cell models include, for example, spheroids and organoids, which are aggregates of cells composed of cell lines (spheroids) or stem cells (organoids). Spheroids are typically spherical and compact, while 3D cell cultures form structures and functions similar to the original tissue. Unlike 2D cell cultures (where cells are cultured on a flat surface or in a suspension), here cells are cultured in a synthetic and / or biological matrix, typically in the form of a three-dimensional artificial extracellular matrix. For expansion of 3D cell models and other experiments, they must be harvested from the matrix again after several days of culture.

[0003] The standard amplification procedure known in the art consists of the following sequence, adjusted according to the matrix used: First, seeding begins by placing cells and / or miniature 3D cell models in, for example, a hydrogel precursor solution, and then the precursor / cell mixture is transferred to the desired culture vessel, for example, in droplet form to a cell culture plate. Optionally, the 3D matrix is ​​gelled and / or polymerized under specific conditions known to those skilled in the art, and then covered with a culture medium. Amplification is optionally set up, wherein the cell-loaded hydrogel is incubated under cell culture conditions for several days to several weeks, wherein the culture medium is optionally changed occasionally to remove metabolic waste and provide fresh nutrients. The next step is harvesting, i.e., removing the cell culture medium, for example by mechanical and / or enzymatic degradation of the 3D matrix to release the 3D cell models contained therein, and performing phase separation by centrifugation, thereby producing at least two separate phases: a liquid phase containing matrix components and / or matrix residues, and a cell precipitate. The precipitate is resuspended in a washing buffer. Optionally, the above steps are repeated. The final optional step is to perform division, i.e., to pulverize the 3D cell models, for example by enzymatic and / or mechanical means, and reseed for further amplification.

[0004] Reliable binding of cell culture substrates, especially cell-bearing substrates, is a highly challenging task, particularly when preventing unintentional entrainment of substrate residues while simultaneously preventing cell loss to maximize cell yield. Existing methods generally fall short in achieving this adequately. When performing phase separation of substrate / cell / culture medium mixtures by centrifugation for cell harvesting and / or purification, complete separation of the intermediate substrate phase and the underlying cell phase is often not achieved; substrate residues remain in solution, and / or the substrate is collected along with the cells. Manually collecting the upper culture medium phase and intermediate substrate phase using a pipette is also difficult and challenging, both visually and operationally, to clearly and distinctly identify and “touch” the different phases. Furthermore, significant inter-operator variability exists, and there is a risk of substrate residues remaining in solution and / or cell loss. Additionally, subsequent washing with appropriate buffer solutions, including the addition of buffer and thorough mixing of the remaining mixture, also presents problems, as there is again a risk of substrate residues remaining and / or cell loss. In addition, the process is time-consuming and reagent-intensive, and the frequent centrifugation and the resulting shear forces cause cell load problems, which may lead to decreased cell viability and / or cell lysis.

[0005] Invention disclosure The methods and apparatus of the present invention provide reliable and on-demand solutions for combining cell culture matrices, by which they selectively and specifically retain components to be removed from cell cultures, regardless of their density, while simultaneously protecting the cells. Furthermore, this enables their use in automated processes, particularly in automated microprocesses (e.g., (micro)fluidic) systems), without the clumping phenomenon known in the prior art and the resulting partial or complete blockage of the apparatus suitable for implementing the method.

[0006] In the context of the above explanation, a method for incorporating a cell culture matrix is ​​proposed according to the present invention, comprising the following steps: a. Providing a cell culture containing at least one, preferably multiple, cells of the same or different species, wherein the cell culture comprises a matrix; and b. Contact the cell culture with at least one 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 the tool with and / or incubating it with the cell culture, wherein the tool comprises at least one capture molecule, and wherein the capture molecule binds to or is bound to a second component of the capture system; and d. Obtain the matrix bound by the capture system.

[0007] In the first step a. of the method for combining a matrix, particularly a cell-carrying matrix, a cell culture containing at least one cell and a matrix is ​​provided in a suitable container and / or dish. Preferably, more than one cell is provided, for example, different cells or cell clusters. Furthermore, it is conceivable that the cells exist as single cells, encapsulated cells, or cell clusters containing multiple cells. Preferably, the at least one cell is connected to the matrix, forms a mixture therewith, and / or is embedded and / or encapsulated in the matrix. The embedding and / or encapsulation of the at least one cell in the matrix preferably occurs immediately.

[0008] In step b., the cell culture is contacted and incubated with at least one first component of a biological, chemical, and / or physical capture system. The first component binds to the matrix (e.g., matrix molecules, matrix fragments, or a portion thereof) in a covalent or non-covalent manner and / or directly or indirectly (e.g., by means of at least one, preferably two or more, identical or different designed linkers). Preferably, the first component of the capture system forms a specific bond. In the preparation of the 3D matrix, the first component is embedded in the matrix network in a covalent or non-covalent manner. Here, it is found that the first component is bound to the matrix (e.g., matrix molecules, matrix fragments, or a portion thereof), preferably the technical structure of the matrix. Preferably, the at least one cell is embedded and / or encapsulated in the matrix. For this purpose, biological, chemical, and / or physical capture systems, particularly two-component capture systems, can be used, for example, as they have known advantages, such as high affinity and specificity. The “contact” can be performed, for example, by dripping, injecting, spreading, introducing, instilling, immersing, and / or other methods known to those skilled in the art for contacting the capture system with the cell culture. The term "incubation" in the method of this invention refers to a method step in which the cell culture is contacted with the capture system or a first component of the capture system for a certain period of time, preferably under suitable conditions, so that the capture system (especially the first component of the capture system) binds to the matrix (e.g., matrix molecules, matrix fragments, or a portion thereof) as described elsewhere. Preferably, incubation is performed for several days to several weeks, more preferably with occasional changes of the cell culture medium. In addition to incubation, the method step also includes, for example, monitoring it. Monitoring can be performed, for example, based on a predetermined time period, such as by observing the cell culture. Alternatively, colored or otherwise visually visible indicators can be provided, which are achieved by means of chemical and / or biochemical indicators, such as staining, color change of staining, or disappearance of staining, upon achieving the binding as described elsewhere. The incubation and monitoring steps can be performed alternately herein.

[0009] In step c., at least one, preferably two or more, tools of the same or different design are introduced into or brought into contact with and incubated with the cell culture. The “introduction” can be achieved, for example, by immersion, submersion, pouring, transferring, or other methods of introducing the tool into the cell culture. The contact and incubation are described in detail elsewhere. The tool can be, for example, but not limited to, rods, pestles, membranes, containers, chambers, vessels, surfaces (e.g., surfaces of (micro)fluidic) systems) and / or mixtures thereof. Important to the invention, the tool comprises at least one trapping molecule, preferably an immobilized trapping molecule, wherein the trapping molecule is covalently or non-covalently bound or attached directly or indirectly (e.g., by means of at least one, preferably two or more, identical or different design linkers), preferably specifically bound or attached to a second component of the trapping system. Preferably, the trapping molecule is a linker. It is conceivable that the second component is attached to any tool of any shape and / or size. Furthermore, it is conceivable that the second component has at least one, preferably two, three, four, five, six, seven, eight, nine, or ten identical or different binding sites for the first component.

[0010] In the final step d., the matrix bound by the capture system is obtained, particularly the cell-bearing matrix and / or matrix fragments. That is, the matrix preferably contains cells bound thereto. This step is affected, for example, by the contact and incubation in steps b. and c. More preferably, the acquisition in step d. may include purification and / or separation. This removes the matrix, particularly the cell-bearing matrix, matrix fragments, and / or polymer molecules, from the cell culture, and immobilizes the matrix, for example, the formed three-dimensional matrix, in the desired location by the contained ligands. Thus, for example, cell-bearing microbeads or layers can be spatially immobilized after their formation. Therefore, by eliminating one or more purification steps, particularly washing and / or centrifugation steps, this method not only saves time, cost, and resources, but also has selectivity and specificity to bind the at least one cell in a cell-friendly and reliable manner, i.e., trapping and thus spatially immobilizing it in a specific location, while simultaneously ensuring the removal of unwanted components of the cell culture without cell loss to maximize cell yield. Therefore, the method of binding the matrix (i.e., spatially immobilizing the matrix) of the present invention overcomes the above-mentioned disadvantages.

[0011] The method of the present invention may include one or more further steps, which may be performed before and / or after the explicitly mentioned steps. Furthermore, all or a single step may be repeated arbitrarily frequently. These steps may also be performed simultaneously. The method is also suitable for partial or full automation.

[0012] Advantageous extensions of the apparatus of the present invention are set forth in the dependent claims.

[0013] In an extended scheme, it can be envisioned that step a1 is performed after step a.: a1. To culture at least one cell from the cell culture.

[0014] The term "culture" in the method of this invention refers to a method step in which a cell culture is "incubated" for a certain period of time, preferably under suitable conditions, to achieve the proliferation of the at least one cell. The culture is preferably carried out under cell culture conditions for several hours, days, or weeks, more preferably in which the cell culture medium is sometimes changed. It is conceivable that the cell culture is transferred to a suitable culture vessel and / or covered with at least one cell culture medium. More preferably, the at least one cell is cultured in a substrate. In addition to culture, the method step also includes, for example, monitoring of it, which will be described in detail elsewhere.

[0015] In another extended scheme, it can be envisioned that step a2 is performed after step a. and / or a1. a2. To gel the matrix.

[0016] In this invention, "gelation" refers to the partial or complete solidification of the matrix and / or the adjustment of its strength. Gelation can occur, for example, through enzymatic and / or chemical means. Gelation is, for example, temperature-dependent. In this way, the strength and / or shape can be adjusted as needed.

[0017] Furthermore, it can be envisioned that step b1 is performed before and / or after step b.: b1. The matrix is ​​pulverized into matrix fragments and the at least one cell is released, wherein in step d., matrix and / or matrix fragments bound to the capture system are obtained.

[0018] In this invention, "pulverization" refers to altering, preferably reducing, and / or adjusting the size and / or range of the matrix to release the at least one cell and / or to break down the matrix into matrix fragments. The pulverization is carried out, for example, by enzymatic and / or chemical method steps. This allows the matrix to be pulverized into any desired shape, such as microspheres, smaller fragments down to individual molecules, and then fixed in a specific location in a desired container using a capture system after formation, for example, for subsequent cell culture, such as encapsulated cells. It is found that in this step, the at least one cell contained within the matrix is ​​released from the matrix and remains in the cell culture for further method steps (e.g., purification, separation, and / or reculturing), and / or the matrix is ​​broken down into matrix fragments.

[0019] Furthermore, it is conceivable that the capture system is selected from: key / lock principles, receptor / ligand systems (e.g., biotin / (streptavidin), where biotin and / or streptavidin peptides serve as ligands and streptavidin and / or avidin serve as receptors), enzyme / substrate reactions, antigen / antibody reactions, nucleic acids and nucleic acid-binding proteins, glycoproteins as ligands and lectins as receptors, polyhistidine as ligands and nickel and / or cobalt ions as receptors (preferably immobilized by binding to a solid-coupled chelating agent), magnets and magnetic particles and / or magnetic components (particularly magnetic beads and / or magnetic matrix components, such as nanoparticles and / or microparticles), and mixtures and / or combinations of the above receptor / ligand pairs (e.g., antibodies immobilized on magnetic beads). With capture systems such as key / lock systems or receptor / ligand systems, molecules (ligands) can be targeted and captured and bound by their "belonging" specific molecules (receptors). Specific sequences (antigens) naturally present in the matrix molecules are preferably avoided, as there is a risk that the corresponding sequences may also be located on / potentially located on cells cultured in the matrix. This is especially important for natural matrix molecules (e.g., proteins, particularly collagen and / or gelatin). It is preferable to avoid artificially introducing specific sequences (antigens) onto / into matrix molecules, for example, via linkers, because there is a risk that the corresponding sequences may also be located on / potentially located on cells cultured in the matrix (e.g., protein A, protein G, protein L). This is particularly important for synthetic matrix molecules, such as PEG.

[0020] In another extended embodiment, it is conceivable that the at least one cell belongs to a cell model, a cell line (e.g., a spheroid and / or organoid), a cell aggregate, a 3D cell model, a miniature 3D cell model, and / or a mixture thereof. The term "3D cell model" refers to cells cultured in a microstructured three-dimensional cell culture under in vitro conditions. In a 3D cell model, the three-dimensional structure is achieved through a matrix.

[0021] Furthermore, it is conceivable that the matrix is ​​a hydrogel (which is made, for example, from skeletal proteins such as collagen, gelatin methacrylate and / or commercially available Matrigel), a hydrogel precursor and / or a mixture thereof.

[0022] The invention also includes a device for binding a matrix to a cell culture, as described in detail elsewhere. This device is preferably suited for carrying out methods of the invention. The device is suitable for accommodating a cell culture containing at least one cell and a matrix, a biological, chemical, and / or physical capture system, wherein the capture system comprises a first component (which binds to the matrix (e.g., a matrix molecule or a portion thereof) covalently or nonvalently and / or directly or indirectly (e.g., through at least one, preferably two or more linkers of the same or different designs) – preferably specifically bound) and a second component, and, as described elsewhere, a tool containing at least one capture molecule (preferably an immobilized capture molecule). The device is characterized in that the second component binds to the capture molecule covalently or nonvalently and / or directly or indirectly (e.g., through at least one, preferably two or more linkers of the same or different designs), preferably specifically bound.

[0023] In another extended embodiment, the device can be envisioned as a (micro)fluidic system, a laboratory-on-a-chip, a cell-on-a-chip, and / or a bioreactor. The term "(micro)fluidic system" refers to a system that mounts the full functionality or a portion thereof of a macroscopic laboratory onto a substrate, such as a chip made of glass and / or plastic. The substrate can have various sizes. The microfluidic system may preferably mimic the channels and functions of a laboratory-on-a-chip or a cell-on-a-chip.

[0024] Other advantages, features and details of the invention will become apparent from the following description of preferred embodiments of the invention and with the aid of the accompanying drawings. Brief description of the attached diagram Figure 1 shows a schematic diagram of the method for combining matrix fragments according to the present invention. Figures 1a to 1c );and Figure 2 shows a schematic diagram of the cell-loaded matrix combined according to the method of the present invention. Figure 2a and Figure 2b ).

[0026] Invention Implementation Scheme In the figures, identical parts and components with the same function are equipped with the same reference numerals.

[0027] Figures 1a to 1c The diagram shows a schematic of combining matrix fragment 21 with a suitable tool 40 according to the first method of the present invention. Figure 1a As can be seen, a plurality of cells 10 are provided in a matrix 20 of 100 cell cultures, and these cells 10 are cultured in the matrix 20 for 200. Furthermore, these cells 10 cultured in the matrix 20 are contacted with and incubated for 300 by a first component 31 of a capture system 30. The first component 31 of the capture system 30 is designated as ligand 31. Figure 1bIn this process, matrix 20 is pulverized into matrix fragments 21, for example, by enzymatic and / or mechanical means. Thus, as... Figures 1a to 1c As shown, cells 10 contained in matrix 20 are released, and ligand 31 specifically binds to matrix fragment 21. Figure 1c In this process, a tool 40 is introduced into the cell culture, brought into contact with it, and incubated for 500°C. The tool 40 is formed in the form of a surface immobilized with a capture molecule 41, which specifically binds to the second component 32 of the capture system 30, 32. In this case, the capture molecule 41 is a linker 41 through which the second component 32 of the capture system 30 binds to the tool 40. The first component 31 of the capture system 30 may be bound to the second component 32, which in turn binds to the matrix fragment 21. The second component 32 is referred to herein as a receptor 32, which is specific for ligands 31 containing the bound matrix fragment 21. The receptor 32 has four binding sites for each ligand 31. Thus, 600 matrix fragments 21 are obtained, in which cells 10 released from the matrix 20, i.e., the matrix fragments 21, remain in the cell culture.

[0028] Figure 2a and 2b The diagram shows a schematic of combining a cell-loaded matrix 20 with a suitable tool 40 according to the method of the present invention. Figure 2a It can be seen from this that, with Figure 1a Similarly, a plurality of cells 10 in a substrate 20 of 100 cell cultures are provided and cultured therein for 200. Subsequently, these cells 10 are contacted with and incubated for 300 by a first component 31 of a capture system 30. The first component 31 is designated as ligand 31. Figure 2b In, with Figure 1c Similarly, tool 40 is introduced into the cell culture, contacted with it, and incubated for 500°. Tool 40 is formed in the form of a surface immobilized with a capture molecule 41, which specifically binds to the second component 32 of the capture systems 30 and 32. In this case, the capture molecule 41 is a linker 41 through which the second component 32 of the capture system 30 binds to tool 40. The first component 31 of the capture system 30 may be bound to the second component 32, which in turn binds to the matrix 21. The second component 32 is also referred to herein as receptor 32, which is specific for ligands 31 containing the bound matrix 20. Receptor 32 has four binding sites for each ligand 31. Thus, 600°, spatial immobilization and binding of the cell-bearing matrix 20 is achieved.

Claims

1. A method for combining cell culture substrates (20, 21), comprising the following steps: a. Provide (100) a cell culture containing at least one cell (10), wherein the cell culture comprises a matrix (20); and b. Contacting and incubating (300) the cell culture with at least one first component (31) of a biological, chemical, and / or physical capture system (30), wherein the first component (31) of the capture system (30) is a component that binds to the matrix (20, 21); and c. Contact the tool (40) with the cell culture and incubate (500), wherein the tool (40) contains at least one capture molecule (41), and wherein the capture molecule (41) is bound to or is bound to a second component (32) of the capture system (30); and d. Obtain (600) the matrix (20, 21) bound by the capture system (30, 31, 32).

2. The method according to claim 1, Step a1 is performed after step a.: a1. Cultivate (200) the at least one cell (10) in the cell culture.

3. The method according to claim 1 or 2, Step a2 is performed after step a. and / or a1. a2. Gel the matrix (20).

4. The method according to any one of the preceding claims, Step b1 is performed before and / or after step b.: b1. The matrix (20) is crushed (400) into matrix fragments (21), and at least one cell (10) is released.

5. The method according to any one of the preceding claims, wherein the capture system is selected from key / lock principles, receptor / ligand systems, enzyme / substrate reactions, antigen / antibody reactions, nucleic acids and nucleic acid-binding proteins, glycoproteins / lectins, polyhistidines and nickel and / or cobalt ions, magnets and magnetic particles and / or magnetic components, particularly magnetic matrix components, and / or mixtures and / or combinations thereof.

6. The method according to any one of the preceding claims, wherein the at least one cell (10) belongs to a cell model, cell line, cell aggregate, 3D cell model, micro 3D cell model and / or mixture thereof.

7. The method according to any one of the preceding claims, wherein the matrix (20, 21) is a hydrogel, a hydrogel precursor and / or a mixture thereof.

8. An apparatus for binding a matrix (20, 21) of a cell culture, wherein the cell culture comprises at least one cell (10), a matrix (20, 21), and a biological, chemical, and / or physical capture system (30, 31, 32), wherein the capture system (30, 31, 32) comprises a first component (31) and a second component (32) that bind to the matrix (20, 21), and the apparatus has a tool (40) comprising capture molecules (40, 41), characterized in that, The second component (32) binds to the capturing molecules (40, 41).

9. The apparatus according to claim 8, characterized in that, The device is a (micro)fluidic system, a laboratory-on-a-chip, a cell-on-a-chip, and / or a bioreactor.

Images