Method and device for the targeted binding of a matrix of a cell culture

Abstract

The invention relates to a method for the binding of a matrix (20) of a cell culture, comprising the following steps: a. providing (100) a cell culture having at least one cell (10), the cell culture comprising a matrix (20); and b. bringing in contact and incubating (300) the cell culture with at least one first component (31) of a biological, chemical and / or physical catcher system (30), the first component (31) of the catcher system (30) being a component which binds to the matrix (20); and c. bringing in contact and incubating (500) a means (40) with the cell culture, the means (40) having at least one catcher molecule (41), and the catcher molecule (41) binding or being bound to a second component (32) of the catcher system (30, 31, 32); and d. obtaining (600) the matrix (20) bound by means of the catcher system (30, 31, 32).

Description

[0001] Description

[0002] Method and device for the targeted binding of a cell culture matrix

[0003] Technical area

[0004] The invention relates to a method for binding a matrix, in particular a cell-laden matrix, to a cell culture and a device for binding a matrix, in particular a cell-laden matrix, to a cell culture.

[0005] State of the art

[0006] Examples of 3D cell models include spheroids and organoids, i.e., cell aggregates composed of cells from a single cell line (spheroids) or stem cells (organoids). While spheroids are typically spherical and compact, 3D cell cultures develop structures and functions similar to the original tissue. In contrast to 2D cell cultures, in which the cells are cultivated on a flat surface or in a suspension solution, the cells are cultivated in a three-dimensional artificial extracellular matrix in the form of synthetic and / or biological matrices, often hydrogels. For the expansion of the 3D cell models and other experiments, they must be harvested from the matrix after several days of cultivation.A standard procedure for expansion known from the prior art is the following sequence, with adaptations depending on the matrix used: First, seeding takes place, beginning with the inclusion of cells and / or mini 3D cell models, e.g. in a hydrogel precursor solution, followed by transferring the precursor / cell mixture into the desired cultivation vessel, e.g. in the form of drops in a cell culture plate. If necessary, the 3D matrix is ​​gelled and / or polymerized under specific conditions known to those skilled in the art, before it is covered with culture medium. Optionally, expansion is provided, in which the cell-laden hydrogels are incubated under cell culture conditions for several days to weeks, if necessary with temporary exchange of the culture medium to remove metabolic waste products and to provide fresh nutrients. Next comes harvesting, i.e.h the removal of the cell culture medium, the degradation of the 3D matrix, e.g., mechanically and / or enzymatically, to release the 3D cell models contained therein, and phase separation by centrifugation, which yields at least two separate phases, i.e., a liquid phase containing matrix components and / or matrix residues and a cell pellet. This is resuspended in wash buffer. These steps are repeated if necessary. The final and optional step is splitting, i.e., the comminution of the 3D cell models, e.g., enzymatically and / or mechanically, and reseeding for further expansion.

[0007] Reliably binding 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. When phases are separated by centrifugation of the matrix / cell / medium mixture for cell harvesting and / or purification, a clean separation of the middle matrix phase and the lower cell phase often does not occur; 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 using a pipette is also difficult, as well as visually and manually challenging to clearly identify and "hit" the different phases.In addition, there is high variability between individual experimenters and the risk of matrix residues in the solution and / or cell loss. Furthermore, the subsequent washing with a suitable buffer solution, including the addition of buffer and mixing of the remaining mixture, is problematic, as there is again a risk of matrix residues remaining and / or cell loss. Furthermore, this procedure is time-consuming and reagent-intensive. Furthermore, there is the problem of stress on the cells due to frequent centrifugation and the resulting shear forces, which can lead to decreased cell viability and / or cell lysis.

[0008] Disclosure of the invention

[0009] 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.

[0010] Against this background of the above explanations, 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 means of the capture system.

[0011] In the first step a. of the method for binding a matrix, in particular a cell-laden matrix, a cell culture is provided in a suitable container and / or vessel, which comprises at least one cell and a matrix. Preferably, more than one cell, for example different cells or a cell network, is provided. Furthermore, it is conceivable for the cell to be present individually, encapsulated, or in a cell network with several cells. Preferably, 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.

[0012] In step b., 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 designed linkers, to the matrix, such as a matrix molecule, a matrix fragment or a part thereof. Preferably, the first component of the capture system forms a specific bond. During the production of 3D matrices, the first component is incorporated into 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. Preferably, the at least one cell is incorporated and / or embedded in the matrix.For this purpose, biological, chemical, and / or physical capture systems, in particular two-component capture systems, with their known advantages such as high affinity and specificity, are used. The "bringing into contact" can be achieved, for example, by dripping, injecting, applying, introducing, dropping, immersing, and / or any other method known to the person skilled in the art for bringing the capture system into contact with the cell culture. The term "incubating" 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 for 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 the cell culture medium. This method step includes, for example, incubation and monitoring of the medium. Monitoring can be carried out, for example, based on a fixed period of time, for example, by observing the cell culture. Alternatively, a colored or otherwise optically visible indication can be provided, which occurs when the binding described elsewhere is achieved using a chemical and / or biochemical indicator, e.g., by means of a color, a color change of the color, or loss of color. The incubation and monitoring steps can occur alternately.

[0013] In 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, and / or another method for introducing the agent into the cell culture. The bringing into contact and incubation are 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.It is essential to the invention that the agent has 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 preferably a linker. It is conceivable to attach 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.

[0014] In the final step d., the matrix bound by means of the capture system is obtained, in particular cell-laden matrix and / or matrix fragments. This means that the matrix preferably has 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, and to hold the matrix, such as a shaped three-dimensional matrix, in the desired position using the ligands it contains. For example, cell-laden beads or layers can be spatially fixed after their production.Thus, by eliminating one or more purification steps, particularly washing and / or centrifugation steps, this method is not only time-, cost-, and resource-saving, but also selective and specific, allowing the at least one cell to be bound, i.e., retained and thus spatially fixed, to a defined location in a cell-protecting and reliable manner. At the same time, it is ensured that undesirable components of the cell culture are removed without cell loss to maximize cell yield. Thus, the described disadvantages are eliminated by means of the method according to the invention and the binding of the matrix, i.e., its spatial fixation.

[0015] The method according to the invention can comprise one or more additional steps that can be performed before and / or after the explicitly mentioned steps. Furthermore, all or individual steps can be repeated as often as desired, and the steps can also be performed simultaneously. Furthermore, the method is suitable for partial or complete automation.

[0016] Advantageous developments of the device according to the invention are listed in the subclaims. In one development, it is conceivable that step a1 follows after step a: a1. Cultivation of the at least one cell in the cell culture.

[0017] The term “cultivation” in connection with the method according to the invention refers to a method step in which the cell culture is “incubated” over a certain period of time, preferably under suitable conditions, in order to achieve proliferation of the at least one cell. Cultivation preferably takes place for several hours, days to weeks under cell culture conditions, more preferably with intermittent replacement of a cell culture medium. It is conceivable to transfer the cell culture into a suitable cultivation vessel and / or to overlay the cell culture with at least one cell culture medium. More preferably, the cultivation of the at least one cell takes place in the matrix. In addition to cultivation, this method step also comprises, for example, the monitoring of this, as described in detail elsewhere.

[0018] In a further development, it is conceivable that after step a. and / or a1., a step a2. follows: a2. Gelling of the matrix.

[0019] In the context of the invention, "gelling" refers to the partial or complete solidification and / or adjustment of the strength of the matrix. Gelling occurs, for example, enzymatically and / or chemically. Gelling is, for example, temperature-dependent. In this way, it is possible to adjust the strength and / or shape as required.

[0020] Furthermore, it is conceivable that a step b1. is carried out before and / or after step b.: b1. Crushing the matrix into matrix fragments and releasing the at least one cell, wherein in step d. the matrix and / or the matrix fragments are obtained bound to the capture system.

[0021] In the context of the invention, "comminution" refers to the change, preferably the reduction, and / or adjustment of the size and / or extent of the matrix in order to release the at least one cell and / or to break the matrix into matrix fragments. The comminution is carried out, for example, by means of enzymatic and / or chemical process steps. In this way, it is possible to comminute the matrix into any desired shape, such as small beads, smaller fragments, or even individual molecules. After shaping, these can be fixed in a desired vessel using a capture system at a defined location, e.g., for subsequent cultivation of the cells, for example, encapsulated cells.It has been recognized that in this step, at least one cell contained in the matrix is ​​released from the matrix and remains in the cell culture for further process steps, such as purification, separation and / or re-cultivation, and / or the matrix is ​​broken down into matrix fragments.

[0022] Furthermore, it is conceivable that the capture system is selected from the list comprising a key / lock principle, a receptor / ligand system, such as biotin / (strept)avidin with biotin and / or streptavidin peptide as ligand and streptavidin and / or avidin as receptor, an enzyme / substrate reaction, an antigen / antibody reaction, a nucleic acid and a nucleic acid-binding protein, a glycoprotein as ligand and lectin as receptor, polyhistidine as ligand and nickel and / or cobalt ions as receptor, preferably immobilized by binding to a solid-coupled chelator, a magnet and a magnetic particle and / or a magnetic component, in particular a magnetic bead and / or a magnetic matrix component, such as a nano- and / or microparticle, and a mixture and / or combination of the aforementioned receptor / ligand pairs, such as antibodies immobilized on magnetic beads.Using capture systems, such as the key / lock or receptor / ligand system, specific molecules (ligands) can be captured and bound by a "corresponding" specific molecule (receptor). Specific sequences (antigens) that are naturally present in the matrix molecules should preferably be avoided, as there is a risk that the corresponding sequences are / could be found (also) on the cells cultured in the matrix. This is particularly relevant for natural matrix molecules, such as proteins, especially collagen and / or gelatin. Specific sequences (antigens) that have been artificially introduced onto / into the matrix molecules, for example via a linker, should preferably be avoided, as there is a risk that the corresponding sequences are / could be found (also) on the cells cultured in the matrix (such as protein A, protein G, protein L).This is particularly relevant for synthetic matrix molecules such as PEG etc.

[0023] In a further development, it is conceivable that the at least one cell belongs to a cell model, a cell line, such as spheroids and / or organoids, a cell aggregate, a 3D cell model, a mini 3D cell model, and / or a mixture thereof. The term "3D cell model" refers to the cultivation of cells in a microstructured three-dimensional cell culture under in vitro conditions. In the 3D cell model, the three-dimensionality is achieved using the matrix.

[0024] Furthermore, it is conceivable that the matrix is ​​a hydrogel, for example made of scaffold proteins such as collagen, gelatin methacrylate and / or commercial Matrigel, a hydrogel precursor and / or a mixture thereof.

[0025] The present invention also encompasses a device for binding a matrix of a cell culture, as described in detail elsewhere. The device is preferably suitable for carrying out the method according to the invention. The device is suitable for receiving a cell culture comprising at least one cell and a matrix, a biological, chemical and / or physical capture system, wherein the capture system comprises a first component that 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 or a part thereof, preferably specifically binding, and a second component, and an agent described elsewhere comprising at least one capture molecule, preferably an immobilized capture molecule.The device is characterized in that the second component binds, preferably specifically, to the capture molecule 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. In a further development, it is conceivable that the device is a (micro)fluidic system, a lab-on-chip, a cell-on-chip, and / or a bioreactor. The term “(micro)fluidic system” refers to a system that accommodates all or part of the functionality of a macroscopic laboratory on a substrate, such as a chip made of glass and / or plastic. The substrate can have different sizes. The microfluidic system can preferably replicate the channels and functions of a lab-on-chip or cell-on-chip.

[0026] Further advantages, features and details of the invention will become apparent from the following description of preferred embodiments of the invention and from the drawings.

[0027] Short description of the drawings

[0028] Fig. 1 shows a schematic representation (Fig. 1a to Fig. 1c) of the binding of matrix fragments according to the method of the invention; and

[0029] Fig. 2 shows a schematic representation (Fig. 2a and Fig. 2b) of the binding of the cell-loaded matrix according to the method of the invention.

[0030] Embodiments of the invention

[0031] Identical components and elements with the same function are provided with the same reference symbols in the figures.

[0032] Fig. 1a to Fig. 1c show a schematic representation of the binding of the matrix fragments 21 to a suitable agent 40 according to a first method according to the invention. In Fig. 1a it can be seen that several cells 10 are provided 100 in a matrix 20 in a cell culture and that the cells 10 are cultivated 200 in the matrix 20. Furthermore, these cells 10 cultivated in the matrix 20 were brought into contact with a first component 31 of a capture system 30 and incubated 300. The first component 31 of the capture system 30 is shown as ligand 31. In Fig. 1b the matrix 20 is comminuted 400 into matrix fragments 21, for example enzymatically and / or mechanically. In this way, as can be seen in Fig. 1a to Fig. 1c, the cells 10 contained in the matrix 20 are released and the ligand 31 binds specifically to the matrix fragments 21. In Fig.1c, an agent 40 is introduced into the cell culture, brought into contact, and incubated 500, wherein the agent 40 is designed in the manner of a surface with capture molecules 41 immobilized thereon, which specifically binds a second component 32 of the capture system 30, 32. In the present case, the capture molecule 41 is a linker 41, via which the second component 32 of the capture system 30 is bound to the agent 40. The first component 31 of the capture system 30, which in turn is bound to the matrix fragments 21, can bind to the second component 32. The second component 32 is represented here as a receptor 32, which is specific for the ligand 31 with the matrix fragments 21 bound thereto. The receptor 32 has four binding sites, each for a ligand 31. In this way it is possible to obtain the matrix fragments 21 600, whereby the cells 10 released from the matrix 20, ie the matrix fragments 21, remain in the cell culture.

[0033] 2a and 2b show a schematic representation of the binding of the cell-laden matrix 20 to a suitable agent 40 according to a method according to the invention. In Fig. 2a it can be seen that, as in Fig. 1a, several cells 10 are provided 100 in a matrix 20 in a cell culture and cultivated 200 therein. Furthermore, these cells 10 were brought into contact with a first component 31 of a capture system 30 and incubated 300. The first component 31 is shown as ligand 31. In Fig. 2b, as in Fig. 1c, an agent 40 is introduced into the cell culture, brought into contact and incubated 500, wherein the agent 40 is designed in the manner of a surface with capture molecules 41 immobilized thereon, which specifically binds a second component 32 of the capture system 30, 32. In the present case, the capture molecule 41 is a linker 41, via which the second component 32 of the capture system 30 is bound to the agent 40.The first component 31 of the capture system 30, which in turn is bound to the matrix 20, can bind to the second component 32. The second component 32 is also depicted here as a receptor 32, which is specific for the ligand 31 with the matrix 20 bound to it. The receptor 32 has four binding sites, each for a ligand 31. In this way, it is possible to obtain the cell-laden matrix 20, i.e., to spatially fix and bind it.

Claims

Claims 1. A method for binding a matrix (20, 21) of a cell culture, comprising the following steps: a. providing (100) a cell culture with 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 a 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. contacting and incubating (500) an agent (40) with the cell culture, wherein the agent (40) has at least one capture molecule (41) and wherein the capture molecule (41) binds or is bound to a second component (32) of the capture system (30); and d. Obtaining (600) the matrix (20, 21) bound by means of the capture system (30, 31, 32).

2. The method according to claim 1, wherein step a1. is carried out after step a.: a1. Cultivating (200) the at least one cell (10) in the cell culture.

3. The method according to claim 1 or 2, wherein after step a. and / or a1., a step a2. is carried out: a2. Gelling of the matrix (20).

4. Method according to one of the preceding claims, wherein before and / or after step b. a step b1. is carried out: b1. Crushing (400) the matrix (20) into matrix fragments (21) and releasing the at least one cell (10).

5. The method according to any one of the preceding claims, wherein the capture system is selected from the list comprising a key / lock principle, a receptor / ligand system, an enzyme / substrate reaction, an antigen / antibody reaction, a nucleic acid and a nucleic acid-binding protein, a glycoprotein lectin, polyhistidine and nickel and / or cobalt ions, a magnet and a magnetic particle and / or a magnetic component, in particular a magnetic matrix component, and / or a mixture and / or combination thereof.

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

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

8. Device 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) has a first component (31) binding to the matrix (20, 21) and a second component (32), and with an agent (40) with a capture molecule (40, 41), characterized in that the second component (32) binds to the capture molecule (40, 41).

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

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