EP4100509A1

EP4100509A1 - Three-dimensional cell culture

Info

Publication number: EP4100509A1
Application number: EP21703904.9A
Authority: EP
Inventors: Manfred TASCHNER; Volker LORBER
Original assignee: Lifetaq Analytics GmbH
Current assignee: Lifetaq Analytics GmbH
Priority date: 2020-02-05
Filing date: 2021-02-05
Publication date: 2022-12-14
Also published as: CN115052966A; US20230078423A1; WO2021156443A1

Abstract

The invention relates to a cell culture device for a three-dimensional cell culture comprising one or more cell culture units, characterised in that a cell culture unit comprises: i. at least one matrix holder having a central opening for receiving a matrix; ii. a carrier that is fixedly or reversibly connected to the at least one matrix holder; and iii. a strip of wells, consisting of a vessel with at least one recess, that can encompass at least one or more matrix holders up to the upper end of the central opening, wherein the matrix holder is oriented vertically.

Description

THREE-DIMENSIONAL CELL CULTURE

TECHNICAL AREA

The present invention relates to the technical field of cell culture, in particular the three-dimensional cell culture of animal and human cells.

BACKGROUND OF THE INVENTION

Animal and human cells are traditionally cultivated in the second dimension in biomedical and pharmaceutical research. This involves transferring cells to a plastic or glass surface where they can attach and multiply. However, this environment does not correspond to the natural, physiological conditions of the cells and therefore very often leads to a loss of function, to dedifferentiation of the cells and, in the worst case, to cell death. (DE102007020302A1). Cells in the human body behave fundamentally different in their environment than those in culture. The reasons are both complex intercellular and extracellular processes, which are influenced, for example, by neighboring cells, the tissue or also by soluble factors such as growth factors or hormones. 2D cell cultures allow cells to attach and grow outside of an organism, but they do not adequately reflect the natural growth conditions of cells in their natural environment.

In order to reduce these rifts between two-dimensional cell culture and the in vivo environment, numerous three-dimensional cell culture systems have been developed as alternatives over the past decade. 3D cultures bring numerous advantages, as the cultivated cells enable a more physiologically authentic model of human tissue. These include, for example, systems with solid supports, such as porous support structures that imitate the extracellular matrix, and support-independent systems, such as spheroid cultures. There are currently around 150 different 3D culture systems worldwide, 76% of which use a scaffold-based framework for cultivation (BCC Research: “3D Cell Cultures: Technologies and Global Markets”, (BIO140A), Publ. Date: Jan. 2015 ). Examples are hanging-drop multiwell plates that are used to produce spheroids (US 2013/236924 A1), systems for producing three-dimensional matrices that include a magnetic core (US 2017/137766 A1) or a cell culture system that has a three-dimensional biocompatible scaffold structure and Includes nanoparticles. These structures are supposed to simulate the extracellular matrix as the natural environment of the cells (DE102007020302A1).

DE10003521A1 describes a device for producing three-dimensional matrix bodies, which consist of a carrier substance and cell cultures stored therein, and their use for cultivating cells in multiwell plates.

US 2019/276784 A1 discloses various complex cell culture devices which are intended to imitate the joint of a mammal. The cell culture device comprises a plurality of hollow scaffolds with many openings for receiving a matrix in the cavity of the scaffold.

US 2007/237683 A1 discloses a device which is used for better optical imaging of two-dimensional cell cultures. The device comprises a multiwell plate with cylindrical bottomless inlets that hold the corrugated strips. The corrugated strips also comprise cylindrical wells that fit into the inlets of the multiwell plate, but have a transparent bottom on which the cells are cultivated.

CA 2579680 A1 discloses a bioreactor with a perfusion unit and a multiwell plate, which comprises a plurality of bioreactor units, in each of which different experiments can be carried out.

WO 2004/027016 A1 discloses a perfusion incubator with a peristaltic pump, which can be used in particular for growing embryos.

However, the handling of scaffold-based systems and already existing three-dimensional cell culture systems is often difficult and automation of the cell culture is very difficult or even impossible. There is therefore a need for three-dimensional cell culture systems that are robust and easy to handle and can preferably also be managed in an automated system.

BRIEF DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide improved devices for the in vitro cultivation of animal and human cells. In particular, it is an object of the present invention to provide cell culture devices which enable simplified handling and the handling of which can optionally also be automated.

This object is achieved by the subject matter of the invention. The use of scaffold-based matrices in automated cell culture processes turns out to be difficult due to the complexity with regard to different materials and techniques. For this reason, a new cell culture system was developed in which both hydrogels and porous support structures can be used. In particular, a cell culture device with a vertical orientation was developed with which cells can be cultivated in a three-dimensional (3D) environment. The aim here is to reduce the manual effort as much as possible and to open up possibilities for 3D cultures made of hydrogels or porous support structures for medium to high-throughput applications.

The present invention comprises a cell culture device for a three-dimensional cell culture with one or more cell culture units, characterized in that a cell culture unit comprises the following: i. at least one matrix holder (12) with a central opening (13) for receiving a matrix; ii. a carrier (10) which is fixedly or reversibly connected to the at least one matrix holder (12); and iii. a corrugated strip (11) consisting of a vessel with at least one depression which can comprise at least one matrix holder (12) up to the upper end of the central opening (13) or more, the matrix holder (12) being oriented vertically.

The compact structure of the cell culture unit allows easy handling of not just one but also several matrix holders.

The central opening (13) of the matrix holder (12) can have a through opening (13a and 13c) or a recess with a wall (13b).

The inner walls of the opening (13) can have an angle of 1-30 °, so that the two ends of the opening (13) can have different diameters. In particular, they can have an angle of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28 or 29 °.

The present invention also includes an inoculating device (30, 33).

In one embodiment of the matrix holder (12), the opening (13) of the matrix holder has a recess (13c) for sealing and reversible attachment to a colonization device (30, 33). According to a particular embodiment, the colonization device (30, 33) comprises sealing elements (31). The colonization device can have elevations (32).

In a further embodiment, the opening (13a) has an upper depression (13d) in order to avoid the inclusion of air bubbles in the matrix during the horizontal colonization.

According to a preferred embodiment of the cell culture device, the central opening (13) comprises a matrix which is a solid porous support or a hydrogel, or a combination thereof. For example, the matrix can be a solid, porous carrier to which a hydrogel is applied and / or which is filled with a hydrogel.

A cell culture unit can comprise several matrix holders (12). In particular, the cell culture unit can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 matrix holders (12). The matrix holders (12), which are encompassed by a cell culture unit, can contain the same or different matrices.

In particular, the matrix holders (12) of a cell culture unit each contain a matrix of hydrogel or a matrix that is a solid, porous carrier. This allows the simultaneous culture of cells in the same three-dimensional environment, whereby the medium in the wells of the corrugated strip (11) can be identical or different. On the one hand, the composition of the medium can be different, for example to investigate the influence of the medium on cell growth. On the other hand, if the medium contains a test active ingredient, different active ingredients or different concentrations of an active ingredient can be tested.

In particular, a cell culture unit comprises matrix holders (12) whose matrix is a hydrogel and matrix holders whose matrix is a solid, porous carrier. If a cell culture unit comprises various matrix holders (12), this enables, for example, rapid screening of different three-dimensional environments.

In particular, the matrix consists of natural, semi-synthetic or synthetic material or a combination thereof. The matrix can optionally be modified or coated with protein or peptide sequences. For example, the matrix can have antibodies or antibody fragments or RGD sequences. In particular, any material compatible with the culture of animal or human cells can be used as the material for the matrix described herein. In particular, the natural material is selected from collagen, fibrin, glycosamine glycans and hyaluronic acid, and the synthetic or semi-synthetic material is selected from polymeric components such as polymethyl methacrylate, polylactic acid, polyglycol, polystyrene and ceramic components such as hydroxyapatite or tricalcium phosphate.

According to a further embodiment, the carrier (10) has an element (15) for receiving the matrix holder (12) and the matrix holder (12) has notches (14a, 14b) at its upper end, via which it can be reversibly connected to the element ( 15) of the carrier (10) can be connected.

According to a further particular embodiment of the carrier (10) of the cell culture device, the carrier comprises an opening at at least one end, preferably at both ends, for fastening a magnetizable nut (45), for example by means of a screw. In particular, the carrier has several additional openings for matrix holders (46) which can be connected to the carrier, for example by means of fastening pins (47).

According to a particular embodiment, the device additionally comprises a transport unit (20) which can transport one or more cell culture units (21).

In particular, the transport unit (20) facilitates the use of the cell culture device in question in an automated environment. One or more cell culture units can be moved and operated by means of the transport unit. In particular, this makes it possible to automate certain processes, such as automated media change or automated settlement of the units. In particular, the automation can take place by means of a robot.

According to a certain embodiment, the transport unit comprises: i. a support rail (22) for fastening one or more cell culture units (21) to the transport unit (20), ii. a receiving unit (20a) for a support rail (22), iii. a lid (23), and iv. two spacer elements (24), which are optionally directly connected to one another, for the orderly reception of at least two or more carriers (10) of a matrix holder, v. optionally at least one identification element (26) such as an RFID chip or a barcode, and vi. optionally a centering unit (27).

In particular, the corrugated strip (11) comprises a position (25) for attachment to the support rail (20).

The spacer elements serve to accommodate and orderly arrange the cell culture units that are attached in the spacer above the carrier (10) of the matrix holders. In particular, the cell culture units are arranged in parallel in the spacer (24). The spacer elements can be connected to one another in a fixed or reversible manner, or they can also not be connected to one another directly, but only via the carrier of the matrix holders.

According to a particular embodiment, the transport unit (20) comprises: i) a plate (48) which forms the underside of the transport unit, ii) openings in the plate (48) for centering the transport unit on the plate (49), iii) openings in the plate (48) for screw connections (50), iv) a further plate (51) which is attached approximately at mid-height of the transport unit, v) magnetizable metal rods (52), vi) a centering bracket (54), vii) openings in the plate (51) for the centering bracket (54), viii) and a centering object (55).

In particular, the magnetizable metal rods (52) are used for reversible connection with a cell culture robot. According to a special example, the magnetizable metal rods (52) serve as a docking point for reversible connection with a robotized unit for transporting the transport unit, for example for reversible connection with magnets of the Oli-MAT (available from LifeTaq-Analytics GmbH, Tulln an der Donau). The Z-axis of the Oli-MAT has two permanent magnets with which the transfer unit can be carried from position to position. A short pulse can briefly demagnetize the magnets, which leads to the transfer unit being switched off.

The present invention also encompasses a method of culturing cells using the cell culture device disclosed herein.

In particular, the method comprises the following steps: i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Colonizing the matrix in the opening (13) of the matrix holder (12) with the cell suspension, iii. Transferring the matrix holder into a corrugated strip (11), in particular using the carrier of the matrix holder (10), and iv. Incubate the cell culture device containing the cells under conditions that allow cell growth.

According to a particular embodiment of the method disclosed herein, the colonization of the matrix with the cell suspension takes place in a horizontal arrangement of the matrix holder (12).

In particular, the matrix holder (12) of the cell culture device for use in this method has an opening (13) with a depression (13c), the matrix holder being reversibly connected to a colonization device (30) via a complementary sealing element (31), wherein the matrix used is a hydrogel and the cell cultivation method comprises in particular the following steps: i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with one or more ingredients of a hydrogel to produce a cell suspension-hydrogel mixture, iii. Populating the opening (13) of the matrix holder (12), which is horizontally aligned in the inoculating device (30), with the cell suspension-hydrogel mixture, iv. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% CO 2, until the matrix has polymerized, v. Separating the matrix holder (12) from the inoculating device (30), vi. Transferring the matrix holder (12) into a corrugated strip (11) filled with medium, preferably using the carrier of the matrix holder (10), vii. Incubate the cell culture device containing the cell suspension-matrix mixture under conditions that allow cell growth.

Polymerization of the hydrogel can in particular last approximately 5 minutes and up to 24 hours. The duration of the complete polymerization typically depends on the composition of the hydrogel, in particular its components and their components Concentration dependent, as well as the environment, for example the humidity and the air temperature. The person skilled in the art is able accordingly to determine the time required for the gel to harden.

After the cell suspension-hydrogel mixture has fully polymerized, a further layer of a cell suspension-hydrogel mixture can be introduced into the matrix. The further cell suspension-hydrogel mixture can contain cells other than the first mixture, such as, for example, modified variants of the cells of the first mixture. The further cell suspension-hydrogel mixture can contain the same cells as the first mixture, but for example a different hydrogel composition.

According to a particular embodiment, the colonization device (30) has an inner elevation (32) which ensures that a depression is created in the opening (13) when the cell suspension-hydrogel mixture is fully polymerized, which after removal of the colonization device (30) the further cell suspension-hydrogel mixture can be filled. This enables, for example, the co-culture of different cell types or the culture of one cell type in a different three-dimensional environment in a matrix holder (12).

According to a further embodiment of the method disclosed herein, the colonization of the matrix with the cell suspension taking place in a horizontal arrangement of the matrix holder (12), the opening (13) of the matrix holder (12) has a recess with a wall (13b), and the matrix is a solid porous support. In particular, the method comprises the following steps: i. Transferring a solid porous support into the recess (13b), ii. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, iii. Transfer the cell suspension into the opening (13b) of the matrix holder

(12), iv. Incubating the inoculation device for at least 30 minutes to 24 hours under conditions which allow the cells to grow on the solid porous support, v. Transferring the matrix holder into a corrugated strip (11) previously filled with medium, in particular using the carrier of the matrix holder (10), and vi. Incubating the cell culture device containing the attached cells on the porous support under conditions that allow cell growth.

According to a further embodiment of the method disclosed herein, the matrix is populated with the cell suspension in a vertical arrangement of the matrix holder (12).

In particular, the matrix holder (12) of the cell culture device for use in this method has an upper opening (13d), the matrix holder (12) is reversibly connected to a colonization device (33) and the matrix is a hydrogel. In particular, the method has the following steps: i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with a hydrogel to produce a cell suspension-hydrogel mixture, iii. Filling the wells of the colonization device (33) with the cell suspension-hydrogel mixture, iv. Populating the opening (13) of the matrix holder by immersing the matrix holder (12) in the inoculating device (33), v. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% CO 2, until the matrix has polymerized, vi. Transferring the matrix holder (12) from the inoculation device (33) into a corrugated strip (11) filled with nutrient medium, and vii. Incubate the cell culture device containing the cell suspension-matrix mixture under conditions that allow cell growth.

In order to keep the volume as low as possible, the volume of the wells of the inoculation device (33) can be reduced in contrast to the corrugated strip (11). The colonization device (33) can additionally have filling elements (40). The wells of the inoculation device (33) can be filled with medium via the filling elements (40).

In particular, the method has the following steps when a filling device (33) is used which has filling elements (40): i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with a hydrogel to produce a cell suspension-hydrogel mixture, iii. Introducing the matrix holder into the inoculating device (33), iv. Filling the wells of the colonization device (33) with the cell suspension-hydrogel mixture via the filling elements (40), whereby the opening (13) of the matrix holder is colonized with the cell suspension-hydrogel mixture, v. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% 5% CO 2, until the matrix polymerizes completely vi. Transferring the matrix holder (12) from the inoculation device (33) into a corrugated strip (11) filled with nutrient medium, and vii. Incubate the cell culture device containing the cell suspension-matrix mixture under conditions that allow cell growth.

According to a further particular embodiment, the matrix holder is aligned horizontally for colonization. In this embodiment, the opening (13) of the matrix holder (12) has a recess with a wall (13b) and the matrix is a solid, porous carrier. The method for colonizing the matrix holder in this embodiment comprises the steps: i. Transferring a solid porous support into the recess (13b), ii. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, iii. Transfer of the cell suspension into the opening (13b) of the matrix holder (12), iv. Incubating the inoculation device, in particular for at least 30 minutes to 24 hours, under conditions which allow the cells to grow on the solid, porous support.

The matrix holder can then be transferred into a corrugated strip (11) filled with cell culture medium, in particular using the carrier of the matrix holder (10), and the cells can be incubated in the cell culture device, which contains the cells attached to the porous carrier, under conditions which enable cell growth.

According to a particular embodiment of the method according to the invention, the transfer of the matrix holders into a corrugated strip, the transfer of an entire cell culture device and / or the transfer of a corrugated strip can be automated by a robot-supported gripper arm.

According to a particular embodiment of the cell culture device, the corrugated strip is a perfusion device (34), the hose connections (41) to the At the end of a pump. A continuous flow of a cell-compatible liquid, such as, for example, cell culture medium, can be passed through the perfusion device via these hose connections by means of a pump.

According to a further embodiment, the cell culture device further comprises a box (44) and an insert (42) for receiving one or more

Cell culture units (21) in the box (44).

According to a further embodiment, the cell culture device further comprises a box (44) and an insert (43) for receiving one or more

Colonization devices (33) in the box (44).

CHARACTERS

FIG. 1 shows components of an embodiment of the cell culture unit (21), in particular matrix holders (12) with various openings (13a, 13b, 13c, 13d), a corrugated strip (11) and a carrier (10).

FIG. 2 shows components of an embodiment of the transport unit (20), in particular a receiving unit (20a), several cell culture units (21), a support rail (22), a cover (23), spacer elements (24), a marking element (26) and a centering unit (27).

Figure 3 shows colonization devices. FIG. 3a) shows a colonization device (30) with sealing elements (31) which can be reversibly connected to the matrix holder (12) via the recess of the opening (13c). FIG. 3b) shows a colonization device (30) which has elevations (32).

FIG. 4 shows components of a cell culture unit and the colonization device

(33) with filling elements (40).

FIG. 5 shows components of a cell culture unit and a perfusion device

(34) with hose connections (41).

FIG. 6 shows components of a cell culture unit (10, 11, 12) and a box (44) and an insert (42) for receiving one or more cell culture units in the box.

FIG. 7 shows components of a cell culture unit (10, 12) with a colonization device (30), as well as a box (44) for receiving one or more colonization devices.

FIG. 8 shows a further embodiment of a carrier (10) of the cell culture device with an opening for fastening a magnetizable nut by means of a screw (45), several openings in the carrier for matrix holders (46), fastening pins of a matrix holder (47) and the matrix holder (12).

FIG. 9 shows a further embodiment of the transport unit (20) comprising several cell culture devices (21). The figure shows a plate (48) which forms the underside of the transport unit, openings in the plate (48) for centering the transport unit on the plate (49), openings in the plate (48) for screw connections (50), another plate ( 51) which is attached approximately at the middle of the transport unit, magnetizable metal rods (52), a centering bracket (54) and openings in the plate (51) for the centering bracket (54) and a centering object (55).

FIG. 10 shows a further embodiment of a colonization device (30) comprising sealing elements (31). The figure shows a carrier (10) with a matrix holder (12) which is introduced into the inoculation device.

DETAILED DESCRIPTION OF THE INVENTION

The use of different matrices in automated cell culture processes turns out to be difficult due to the complexity with regard to different materials and techniques. For this reason, a system was developed in which both hydrogels and solid porous supports can be used. The aim when using automated processes is to keep the manual effort as low as possible.

According to the invention, the cell culture device in question opens up the possibility of using three-dimensional cell cultures with a matrix of hydrogel and / or porous support structures in automated medium to high-throughput applications. For example, toxicology studies can thus be carried out in an automated environment in a three-dimensional cell culture.

The invention relates to cell culture devices for the in vitro cultivation of biological cells and tissue and to cultivation methods using these cell culture devices. In particular, the cell culture devices in question are robust and easy to handle and enable cells to be cultivated in vitro in a three-dimensional environment. Another particular advantage of the cell culture devices in question is that they are suitable for use in an automation-supported environment, for example in combination with a robot. Due to the compact structure of the cell culture device according to the invention, in particular the cell culture units, the cultivation of different cell types can take place simultaneously in different environments, with variations in the cultivation of the cells, in particular through human interaction with the cell culture device, being reduced. In particular, several replicates of a cell culture system can be cultivated at the same time.

The cell culture device in question comprises at least one matrix holder, a carrier for fixing the matrix holder and a vessel with at least one generally trough-shaped depression (well), which is called a corrugated strip. The cell culture device according to the invention is particularly characterized in that the at least one matrix holder is oriented vertically.

The matrix holder is particularly characterized in that it consists of an outer basic shape and comprises an inner central opening. This opening can have various modifications and shapes, for example it can be a through opening or a recess with a rear wall which is therefore only open on one side. This enables both hydrogels and solid porous supports to be used. The opening of the matrix holder can be continuous or discontinuous; the matrix holder can, for example, have a rectangular or round opening and be shaped, for example, as a cylinder, cuboid, pyramid, or cube.

The interior walls of the opening of the matrix holder can be straight or at an angle of about 1 to 25 degrees. In particular, the inner walls of the opening of the matrix holder can form an angle of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24 or 25 degrees. Optionally, the outer wall of the opening can also have such an angle.

As a result, the two ends of the opening can have different diameters. For example, if the opening is a recess with a wall, the rear wall can thus have a smaller diameter than the front opening. This has the effect that hydrogels adhere less to the rear wall and can be removed more easily from the opening of the matrix holder. This can prevent damage to the hydrogel and the cells when they are removed from the matrix holder.

In a particular embodiment, the matrix holder also has modifications around the inner central opening. These modifications can for example, further depressions around the opening, creating a wall around the opening. This wall is in particular a raised curve with an inner trough-shaped opening, which optionally has a wall, for example a rear wall, or is continuous. Modifications of this type are particularly suitable for reversibly connecting the matrix holder to a correspondingly compatible colonizing device, for example via a plug-in system.

In a further special embodiment, the matrix holder has a depression or also an indentation on the upper edge of the opening, whereby air bubbles can escape from the well. In this way, for example, the inclusion of air bubbles in the matrix, in particular during colonization, can be avoided.

It is known that mechanical stimulation stimulates the formation of extracellular matrix proteins. According to a particular embodiment, the matrix holder is formed from soft, elastic silicone of different Shore hardness, whereby mechanical forces, such as pulling, stretching or pressing, can act on the matrix holder and thus on the cells in the matrix without a negative effect.

The cell culture device in question has at least one carrier which is fixedly or reversibly connected to at least one matrix holder. The carrier is preferably suitable for more than one matrix holder, in particular at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 25, 30, 35, 40, 45, 50 or more matrix holders fixed or reversible. The matrix holders are preferably arranged in the longitudinal direction, in particular in a line or in parallel lines.

According to a particular embodiment in which the matrix holders are reversibly connected to the carrier, the carrier comprises constructions or elements for receiving one matrix holder in each case. These constructions or elements are preferably arranged in the longitudinal direction, in particular in a line or in parallel lines. In particular, the matrix holders have elements, such as notches, for example, which are compatible with the constructions or elements of the carrier and can thus be reversibly connected to the carrier. For example, the elements of the carrier and the notches of a matrix holder can be a Represent a plug-in system by inserting or pushing the notches of the matrix holder into the elements of the carrier.

According to a further particular embodiment, the carrier has elements for fixed or reversible connection and attachment to a transport unit. In particular, the carrier and the transport unit have compatible elements via which the carrier and the transport unit can be connected to one another in a fixed or reversible manner. For example, they can be connected by pushing or plugging one of the elements into the matching element.

According to a further particular embodiment of the carrier of the cell culture device, the carrier comprises an opening at at least one end, preferably both ends, for fastening a magnetizable nut, for example by means of a screw. In particular, the carrier additionally has several openings for matrix holders, which can be connected to the carrier, for example, by means of fastening pins.

The nut is preferably made of iron or another magnetizable metal. The nut can be magnetized by permanent magnets, for example in a separate structure such as a robot. The separate construction can for example be a cell culture robot, for example Oli-Mat (available from LifeTaq-Analytics GmbH, Tulln an der Donau). The carrier can be reversibly connected to the separate construction via the magnetizable nut and thus enable the cell culture unit (for example in fresh nutrient medium) to be moved over the carrier. To park at the destination, the carriers can be separated from the magnet by physical force or by a short impulse.

The cell culture device in question has at least one corrugated strip, which consists of a vessel with at least one depression. This depression can completely encompass at least one matrix holder, at least up to the upper end of the central opening of the matrix holder. The corrugated strip preferably has depressions, the number of which corresponds to the number of matrix holders on a carrier. Alternatively, the corrugated strip can also have more depressions than matrix holders are contained on a carrier, and / or at least one of the depressions of the corrugated strip can accommodate more than one matrix holder. Optionally, the corrugated strip has only one depression which is large enough to accommodate all of the matrix holders of a carrier or a plurality of carriers. If a recess in the corrugated strip comprises several matrix holders, the matrix holders are in contact with the same medium, whereby, for example, variations in the composition of the medium between different wells can be avoided.

According to a particular embodiment, the corrugated strip has an element for fixed or reversible attachment to the transport unit. Optionally, the corrugated strip also has a sanding or wiping construction. An abrasive construction is particularly helpful when using the cell culture device in an automated environment, for example with a robot, since in this way the dripping of cell culture medium when transporting individual units can be avoided.

According to a particular embodiment, at least one of the depressions has an insert, as a result of which the volume of the depression is reduced. For example, the required volume of cell culture medium or hydrogel can be reduced as a result, as a result of which the costs of the cell culture can be reduced considerably. By reducing the volume, the amount of reagents that are tested or used on the cells in the cell culture system can also be reduced.

The components of the subject cell culture device can consist of any material that can be used in a cell culture system. In particular, the components of the cell culture device consist of biocompatible and sterilizable material. These include, for example, materials such as silicone, polycarbonate, polystyrene, polyethylene, polysulfone (PSU), polyphenylsulfone (PPSU) and other materials commonly used in cell culture systems.

The matrix described herein can be a solid porous support or a hydrogel, or a combination thereof. For example, the matrix can be a solid, porous carrier to which a hydrogel is applied and / or which is filled with a hydrogel. For example, the porous matrix can also be dehydrated. In particular, the porous matrix can be a poly (L-lactide) (PLLA) that is processed into microfibers that are very consistent in terms of fiber diameter and pore size. Alternatively, it can be polystyrene scaffolds that are suitable for 3D cell culture. In particular, the matrix consists of natural, semi-synthetic or synthetic material, or a combination thereof. The matrix can optionally be modified or coated with proteins or peptides. For example, the matrix can have antibodies, antibody fragments or RGD sequences. RGD sequences are amino acid sequences containing arginine / glycine / asparagine. This Sequences occur in proteins of the EC matrix, for example

Fibronectin / vitronectin. Cells can bind to these via integrins.

The porous matrix can be transferred into the matrix holder, but alternatively it can also be integrated into the matrix holder by means of lyophilization.

In particular, any material compatible with the culture of animal cells can be used as the material for the matrix described herein. In particular, the natural material is selected from collagen, fibrin, glycosamine glycans and hyaluronic acid, and the synthetic or semi-synthetic material is selected from polymeric components such as polymethyl methacrylate, polylactic acid, polyglycol, polystyrene and ceramic components such as hydroxyapatite or tricalcium phosphate.

According to a particular embodiment, the matrix is prepared in the matrix holder with proteins or protein components. Here, the hydrogel or the solid porous support is formed in the matrix holder. The matrix can then be functionalized using traditional cross-linking techniques such as NHS-EDC or click chemistry or other variants with functional proteins such as antibodies, growth factors or extracellular protein as well as with functional peptides, whereby the different liquids are stored in different corrugated strips and the chemical reactions via the manual change from one corrugated strip to the next takes place. After that, cells can be colonized or an animal organism can be operated on.

Both adherent cells and suspension cells can be cultivated in the cell culture system in question.

The cells to be cultivated are preferably eukaryotic cells, in particular animal cells. The cells are preferably mammalian cells, particularly preferably human but also non-human, such as rodent cells, for example from mouse, hamster, opossum, potorous, or rat, insect, bovine cell lines, dog such as MDCK cell lines, pigs, etc.

The cells can be stem cells, for example omnipotent, pluripotent, multipotent or unipotent, or differentiated tissue cells or blood cells or lymphocytes. The cells can be from immortalized cell lines, for example tumor cell lines. It is possible for the cells to be cultivated in such a way that differentiation takes place. Thus, cells can also change the degree of differentiation. This should preferably go through the culture conditions are determined (e.g. growth or differentiation factors).

Adherent cells include any animal, in particular human, cells that are typically used in biomedical or pharmaceutical research. These include, for example, stem cells from various sources such as mesenchyme, fat, liver, brain or other sources or differentiated cells such as fibroblasts, chondrocytes, osteoblasts, epithelial cells of different origins, endothelial cells, neuronal cells, hepatocytes as well as induced pluripotent stem cells of embryonic origin and cell lines.

Suspension cells can include, for example, blood precursor cells or differentiated blood cells. Suspension cells can either be embedded in a hydrogel matrix or cultivated as a suspension in the context of co-culture studies in the corrugated strip, with an adherent cell culture being embedded in a matrix in the matrix holder in the second case. For example, interaction studies between blood cells as a suspension and mesenchymal stem cells in a hydrogel can be carried out in the matrix holder.

A three-dimensional cell culture system describes the cultivation of cells in a microstructured three-dimensional cell culture under in vitro conditions. The culture or its cells should adopt a spatial orientation. This takes place mainly in the form of hydrogels made from structural proteins such as fibrin, collagen, gelatin (poly) methacrylate or matrigel, as well as from solid scaffolds such as polystyrene, polylactic acid or other chemical substances. In a three-dimensional environment, many cell lines form spheroids, the diameter of which increases over time after the cells are embedded. Even cells that do not form spheroid often show a morphology that differs greatly from 2D cultures.

A cell-compatible liquid, in particular cell culture medium, is preferably used in the cell culture device according to the invention and in the method according to the invention. The above-described steps ii) to v) are preferably carried out in this liquid. This fluid is used as part of the complex cellular environment. It can be a medium suitable for the growth of the cells or for the cell supply. Defined cell culture media are based on the component groups amino acids, carbohydrates, inorganic salts and vitamins. Frequently contained salts are, for example, calcium chloride, potassium chloride, Magnesium sulfate, sodium chloride and monosodium phosphate. Frequently contained vitamins are for example folic acid, nicotinamide, riboflavin and B12. In addition, the cell culture medium can contain FCS (Fetal Calf Serum) or FBS (Fetal Bovine Serum). Preferred cell culture media are MEM, α-MEM, DMEM, RPMI and variations or modifications thereof.

If the cell culture device according to the invention also comprises a cell-compatible liquid, such as cell culture medium, and living cells, it is also referred to as a cell culture system.

In particular, the cell culture system comprises the container and the culture medium, but not the specific atmospheric composition. Thus, in step ii) of the method in question, the cells can be removed from the incubator in which atmospheric conditions prevail which are intended to promote the best possible growth. The preferred atmospheric conditions for the best possible cell growth for animal cells are 35-38 ° C., particularly preferably approximately 37 ° C., and 0-10% CO2, particularly preferably 3-6% CO2.

A transport unit described here for transporting the cell culture units preferably comprises a support rail (22) for fastening one or more cell culture units, a receiving unit (20a) for a support rail (22), a cover (23), and two spacer elements (24), which are optionally connected directly to one another, for the orderly reception of at least two or more carriers (10) of a matrix holder. Optionally, the transport unit can include one or more identification elements (26) such as an RFID, barcode or QR code, and / or a centering unit (27), especially for automated processes. The transport unit is preferably made of a solid material, for example a plastic, which is preferably temperature-resistant and / or acid-resistant. The individual elements of the transport unit can consist of the same material or of different materials.

Optionally, the transport unit described herein can also have a plate (48) which forms the underside of the transport unit, openings in the plate (48) for centering the transport unit on the plate (49), openings in the plate (48) for screw connections (50), another plate (51) which is attached approximately mid-height of the transport unit, magnetizable metal rods (52), a centering bracket (54), openings in the plate (51) for the centering bracket (54), and / or a centering object (55) include. The magnetizable metal rods (52) enable a reversible connection to an external construction, for example a robot, which is used for the automated manipulation of the cell cultures. For example, the magnetizable metal rods (52) serve as a docking point for reversible connection with a robotized unit for transporting the transport unit, for example for reversible connection with magnets of the Oli-MAT (available from LifeTaq-Analytics GmbH, Tulln an der Donau). The Z-axis of the Oli-MAT has two permanent magnets with which the transfer unit can be carried from position to position. The magnets can be briefly demagnetized with a short pulse, which leads to the transport unit being switched off.

In addition, the system also includes a device for colonization in a horizontal and in a vertical orientation.

In the case of colonization in a vertical arrangement, the cells are mixed, for example, with a hydrogel and transferred into the device for vertical colonization, a corrugated strip, by means of a pipette. The matrix holder with the device for the matrix holder is then dipped into the hydrogel until the latter has polymerized. The matrix holder is then carefully transferred into a corrugated strip filled with medium, only a defined amount being transferred along with the corresponding cells in the inner opening of the fully polymerized hydrogel. Depending on the concentration of the hydrogel components, the polymerization can take a few minutes to hours. Accordingly, the culture may have to be transferred to an incubator. In order to avoid air bubbles in the opening during the polymerization, the matrix holder can in this case have a small opening in the vertical surface above the opening.

In another variant of vertical colonization, the matrix holder is first transferred into the device for vertical colonization and then the hydrogel with cells is added. After the polymerisation is complete, it is again transferred to a corrugated strip filled with medium.

Alternatively, in the case of hydrogels, the colonization can take place in a horizontal arrangement with a horizontal colonization device. In this case, the matrix holder with the device for fastening the matrix holder to the inoculation device is fastened beforehand. The cells are then mixed with the hydrogel and this mixture is transferred into the opening of the matrix holder. After the polymerization has finished, the device is removed manually from the matrix holder and transferred to a corrugated strip filled with medium. With this arrangement, the volume can be reduced by at least a factor of 5 compared to the vertical arrangement.

In the case of horizontal colonization, the colonization device can be provided with a plateau. After colonization as described above, the matrix holder is detached from the horizontal colonization device, whereby a depression is created on the rear side of the hydrogel. In a special embodiment, colonization with a further cell type, for example of epithelial origin such as endothelial cells of a blood vessel or epithelial cells of the intestine, is possible on this depression and can form a barrier-like structure.

In the case of porous solid supports, the colonization takes place preferably in a horizontal arrangement. In this case the central opening has a recess with a wall. The porous carrier on which cells can be colonized directly rests on this wall. In this arrangement, the construct can be transferred to an incubator for several hours up to a day. After colonization has taken place, the matrix holder can be transferred to a well strip filled with medium. Depending on the carrier, the carrier can be dehydrated in several steps before colonization. This can be done in advance by transferring the device with the matrix holder from one corrugated strip filled with liquid to another corrugated strip filled with liquid. After colonization, a subsequent change of media can be carried out by transferring the device with the matrix holder and the three-dimensional cell culture from the old corrugated strip with old medium to a new corrugated strip previously filled with fresh medium. In a special embodiment, the transfer of the corrugated strips with the matrix holders can also take place in an automated manner with the aid of the transport unit or a robot-supported gripper arm.

For a long-term cultivation of cells in the matrix as a hydrogel or porous degradable carrier, the culture can be decellularized according to a particular embodiment in order to obtain an extracellular matrix cell-free. For example, mesenchymal stem cells can be differentiated into osteoplasts, chondrocytes or adipocytes over several weeks and finally the matrix obtained can be made cell-free using a standard decellularization protocol.

In another possible variant, a preculture in 2D is completely dispensed with and the passage of the cells in 2D is avoided. In this case it will be Cells in low concentration (<5000-10000 / well) on the matrix holder, colonized either as a hydrogel or as a solid porous carrier and cultured to a high concentration. The matrix is then dissolved or the cells are detached from the carrier by means of an enzymatic reaction and recovered for the next colonization.

In another embodiment, the matrix holder can be filled with cells and a hydrogel matrix and transferred into a device for perfusion, wherein a continuous or periodic flow can be initiated.

The cell culture device according to the invention can be stored or transported in a box, optionally with a special insert for receiving the devices. In particular, the box can have further elements for temperature control or cooling or elements for temperature control and / or temperature recording or sensors for CO2 or O2 measurement. The box can also be an isothermal box.

The matrix holder, for example made of silicone, can alternatively be transferred into a bioreactor system with perfusion. For example, cells can be grown in low concentration, if the number of cells becomes too large for a purely static culture, the matrix holder can be transferred to a larger vessel or bioreactor system with perfusion. This allows initiation of a continuous or periodic flow.

Various methods of using the cell culture device of the invention are described herein. For this, the matrix posture can be aligned horizontally or vertically. In particular, the elements used for cultivation are sterilized, in particular autoclaved. The cells used can be prepared for cultivation, in particular by washing and contact with proteases that break down those proteins that maintain the cell structure, for example trypsin. As a result, the cells can be detached from a carrier, isolated and suspended in the further process. The cells can also be centrifuged to increase the cell density. The number of cells for embedding or colonizing the matrix can be determined by a person skilled in the art; the number of cells can, for example, be around 1000-100000 cells per matrix. The matrix holder can be transferred to a corrugated strip (11), in particular using the carrier of the matrix holder (10). The conditions for the colonization of the matrix in the opening (13) of the matrix holder (12) with the cell suspension can be for the respective Cell types are individually adapted, for example the temperature can be about 30 ° C to 38 ° C, for example about 37 ° C for mammalian cell cultures.

According to a further embodiment, the cell culture device can comprise one or more electrodes. In particular, the electrodes are arranged in such a way that the cells to be cultivated can be electrically stimulated. The electrodes are preferably located in the corrugated strip, in particular in one or more of the depressions in the corrugated strip.

The invention further comprises the following embodiments:

1 . Cell culture device for a three-dimensional cell culture with one or more cell culture units, characterized in that a cell culture unit (21) comprises: i. at least one matrix holder (12) with a central opening (13) for receiving a matrix; ii. a carrier (10) which is fixedly or reversibly connected to the at least one matrix holder (12); and iii. a corrugated strip (11) consisting of a vessel with at least one depression which can comprise at least one matrix holder (12) up to the upper end of the central opening (13) or more, the matrix holder (12) being oriented vertically.

2. The cell culture device according to item 1, wherein the central opening (13) of the matrix holder (12) is a through opening (13a and 13c) or a recess with a wall (13b).

3. The cell culture device according to item 1 or 2, wherein the central opening (13) comprises a matrix which is a solid porous support or a hydrogel, or a combination thereof.

4. The cell culture device according to any one of items 1 to 3, wherein the matrix consists of natural, semi-synthetic or synthetic material, or a combination thereof.

5. The cell culture device according to item 4, wherein the natural material is selected from collagen, fibrin, glycosamine glycans and hyaluronic acid, and the synthetic or semi-synthetic material is selected from polymeric components such as polymethyl methacrylate, polylactic acid, polyglycol, polystyrene and ceramic components such as hydroxyapatite or tricalcium phosphate. 6. The cell culture device according to one of points 1 to 5, wherein the carrier (10) has an element (15) for receiving the matrix holder (12) and the matrix holder has notches (14a, 14b) at its upper end, via which it is reversible can be connected to the element (15) of the carrier (10).

7. The cell culture device according to one of items 1 to 6, wherein the device comprises at least 5, in particular at least 10, matrix holders (12).

8. The cell culture device according to one of items 1 to 7, wherein the device additionally comprises a transport unit (20) which can transport one or more cell culture units (21).

9. The cell culture device according to item 8, wherein the transport unit (20) comprises i. a support rail (22) for fastening one or more cell culture units (21) to the transport unit (20), ii. a receiving unit (20a) for a support rail (22), iii. a lid (23), and iv. two spacer elements (24), which are optionally directly connected to one another, for the orderly reception of at least two or more carriers (10) of a matrix holder, v. optionally at least one identification element (26) such as an RFID chip or a barcode, and vi. optionally a centering unit (27).

10. The cell culture device according to item 8 or 9, wherein the corrugated strip (11) comprises a position (25) for attachment to the support rail (20).

11. The cell culture device according to item 8 or 9, wherein the cell culture units are arranged in parallel in the spacer (24).

12. A method for culturing cells using the cell culture device according to any one of items 1 to 11.

13. The method according to item 12, comprising the steps i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Colonization of the matrix in the opening (13) of the matrix holder (12) with the cell suspension, iii. Transferring the matrix holder into a corrugated strip (11), in particular using the carrier of the matrix holder (10), and iv. Incubate the cell culture device containing the cells under conditions that allow cell growth.

14. The method according to item 13, wherein the colonization of the matrix with the cell suspension takes place in a horizontal arrangement of the matrix holder (12).

15. The method according to item 14, wherein the opening (13) of the matrix holder (12) has a recess (13c) for attachment and is reversibly connected to a colonization device (30) via a complementary sealing element (31), and the matrix is a Hydrogel, comprising the steps i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with one or more ingredients of a hydrogel to produce a cell suspension-hydrogel mixture, iii. Populating the opening (13) of the matrix holder (12), which is horizontally aligned in the inoculating device (30), with the cell suspension-hydrogel mixture, iv. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% CO 2, until the matrix has polymerized, v. Separating the matrix holder (12) from the inoculating device (30) vi. Transfer the matrix holder (12) to one filled with medium

Corrugated strips (11), preferably using the carrier of the matrix holder (10), vii. Incubate the cell culture device containing the cell suspension matrix

Mixture contains, under conditions that allow cell growth.

16. The method according to item 13, wherein the colonization of the matrix with the cell suspension takes place in a vertical arrangement of the matrix holder (12).

17. The method according to item 16, wherein the opening (13) of the matrix holder (12) has an upper opening (13d) and the matrix holder (12) is reversibly connected to a colonization device (33), and the matrix is a hydrogel, comprising the Steps i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mix the cell suspension with a hydrogel to obtain a

To produce cell suspension-hydrogel mixture, iii. Filling the wells of the inoculation device (33) with the cell suspension-hydrogel mixture, iv. Populating the opening (13) of the matrix holder by immersing the matrix holder (12) in the inoculating device (33), v. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% 5% CO 2, until the matrix has polymerized, vi. Transferring the matrix holder (12) from the inoculation device (33) into a corrugated strip (11) filled with nutrient medium, and vii. Incubate the cell culture device containing the cell suspension matrix

Mixture contains, under conditions that allow cell growth.

18. The method according to item 16, wherein the opening (13) of the matrix holder (12) has an upper opening (13d) and the matrix holder (12) is reversibly connected to a colonization device (33) which comprises filling elements (40) and which The matrix is a hydrogel comprising the steps of i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mix the cell suspension with a hydrogel to obtain a

Prepare cell suspension-hydrogel mixture, iii. Introducing the matrix holder into the inoculating device (33), iv. Filling the wells of the colonization device (33) with the cell suspension-hydrogel mixture via the filling elements (40), whereby the opening (13) of the matrix holder is colonized with the cell suspension-hydrogel mixture, v. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% 5% CO2, until the matrix has polymerized out, vi. Transferring the matrix holder (12) from the inoculation device (33) into a corrugated strip (11) filled with nutrient medium, and vii. Incubate the cell culture device containing the cell suspension matrix

Mixture contains, under conditions that allow cell growth.

19. Cell culture device according to one of items 1 to 7, wherein the corrugated strip is a perfusion device (34), and the perfusion device has hose connections (41), optionally for connection to a pump. 20. Cell culture device according to one of items 1 to 7, additionally comprising a box (44) and an insert (42) for receiving one or more

Cell culture units (21) in the box (44).

21. Cell culture device according to one of items 1 to 7, additionally comprising a box (44) and an insert (43) for receiving one or more

Settlement devices (30).

EXAMPLES

The present invention is further described by the following examples, without necessarily being limited to these specific embodiments of the invention.

Example 1:

Colonization of a hydrogel matrix with cells using the horizontal colonization device (30) and subsequent cultivation of the cells in a cell culture unit

The carrier with the matrix holders (13c) was connected to the inoculation device (30) and autoclaved together. A 2D culture from mesenchymal stem cells was then washed twice with DPBS without Ca / Mg and trypsinized. The detached cells were then taken up in medium with serum and transferred to 50 ml Falcon. Next, these were centrifuged in an ultracentrifuge at 0.3RPM for 5 minutes and the supernatant was discarded. In the next step, new medium without serum was added and, in addition, a cell count was carried out. The cells were then centrifuged again at 0.3 RPM for 5 minutes and the supernatant was discarded. A 5 ml cell-fibrinogen-thrombin solution was prepared for 50 matrix holders. Different volumes are used depending on the initial concentration of fibrinogen and thrombin. In the present example, the cell pellet was taken up in 1.75 ml medium without serum, so that a cell number between 1000-100000 cells per matrix was incorporated. This was followed by the addition of 750 ml of reconstituted fibrinogen, so that a concentration of 5 mg / ml of fibrinogen was achieved. Next, 2 ml of a thrombin stock solution (8000U / ml) was transferred to 250 ml of 40mM CaCh (~ 64U / ml). 40 ml of this solution were transferred to 2.5 ml of 40 mM CaCh (~ 1 U / ml). 45 ml of the cell fibrinogen solution were pipetted into a matrix holder (12) using a pipette. Then 45 ml of thrombin solution (target concentration Fibrinogen ~ 2.5mg / ml thrombin 0.5U / ml) added to each cell fibrinogen plug without air bubbles and transferred to an incubator at 37 ° C with 5% CO2 for complete polymerization. After the polymerization, the inoculation device (30) was manually detached from the matrix holder (12) and the carrier with the fully polymerized matrix-cell solution was transferred to a new corrugated strip (11) with ~ 1 -1.5 ml of fresh medium each and transferred to an incubator (37 ° C / 5% CO 2) transferred.

Example 2:

Colonization of a hydrogel matrix with cells using the vertical colonization device (33) and subsequent cultivation of the cells in a cell culture unit

The inoculation device (33) and a carrier with the matrix holders (13a) were autoclaved together. A 2D culture from mesenchymal stem cells was washed twice with DPBS without Ca / Mg and trypsinized. The detached cells were then taken up in medium with serum and transferred to 50 ml Falcon. This was followed by centrifugation using an ultracentrifuge at 0.3RPM for 5 minutes and the supernatant then discarded. In the next step, new medium without serum was added and a cell count was carried out. The cells were then centrifuged again at 0.3 RPM for 5 minutes and the supernatant was again discarded. 5 ml of cell-fibrinogen-thrombin solution were prepared for 5 matrices. Different volumes are used depending on the initial concentration of fibrinogen and thrombin. In this case, the cell pellet was taken up in 1.75 ml medium without serum, so that a cell number between 50000-250000 cells per matrix could be incorporated. This was followed by the addition of 750 ml of reconstituted fibrinogen, so that a concentration of 5 mg / ml of fibrinogen was achieved. Then 2 ml of a thrombin stock solution (8000U / ml) were transferred to 250 ml of 40 mM CaCh (~ 64U / ml). 40 ml of this solution were transferred to 2.5 ml of 40 mM CaCh (~ 1 U / ml). Next, the fibrinogen cell solution was mixed with the thrombin solution and 0.5 ml-1 ml was transferred into the colonization device (33). As soon as the solution was in the inoculation device, the carrier was quickly immersed in the matrix holder and the inoculation device was stored in an incubator at 37 ° C. and 5% CO 2 until polymerization was complete. After the polymerization, the carrier was carefully pulled up out of the inoculation device and transferred to a new corrugated strip with fresh medium and transferred to an incubator (37 ° C./5% CO ₂ ). Example 3:

Colonization of a solid porous matrix and subsequent cultivation of the cells in a cell culture unit

A carrier with the matrix holders without opening (13b) and several corrugated strips (11) were autoclaved together. In this case, a porous dehydrated matrix (for example “Alvetex” Reprocell or “Mimetix” TheElectroSpinningCompany) was transferred into the matrix holder using tweezers. Alternatively, the matrix could already be integrated in the matrix holder by means of lyphilization steps. In the next step, according to the manufacturer's instructions, the matrix was rehydrated using ethanol and washed. For efficient handling, the liquids used were transferred into several parallel corrugated strips so that the carrier could quickly be dipped from one corrugated strip into the other.

A 2D culture from mesenchymal stem cells was washed twice with DPBS without Ca / Mg and trypsinized. The detached cells were then taken up in medium with serum and transferred to 50 ml Falcon. This was followed by another centrifugation by means of an ultrafuge, centrifuged at 0.3RPM for 5 minutes and subsequent removal of the supernatant. In the next step, new medium with serum was added and a cell count was carried out. The cells were then centrifuged again at 0.3 RPM for 5 minutes and the supernatant was discarded. A 5 ml cell solution was prepared for 50 matrix holders, the number of cells being between 1000 and 20,000 cells per matrix. The carrier with the matrix holders was placed horizontally and 90 ml of cell suspension was transferred into the opening on the matrix. The construct was then cultivated in an incubator for 12-24 hours at 37 ° C. and 5% CO 2 until the cells had attached to the matrix. After the attachment, the carrier was transferred to a new corrugated strip with fresh medium and its subsequent cultivation in an incubator (37 ° C / 5% CO 2).

Claims

PATENT CLAIMS

1 cell culture device for a three-dimensional cell culture with one or more cell culture units, characterized in that a cell culture unit (21) comprises: i. at least one matrix holder (12), with a central opening or recess (13) for receiving a matrix; ii. a carrier (10) which is fixedly or reversibly connected to the at least one matrix holder (12); and iii. a corrugated strip (11) consisting of a vessel with at least one depression which can comprise at least one matrix holder (12) up to the upper end of the central opening (13) or more, the matrix holder (12) being oriented vertically.

2. The cell culture device according to claim 1, wherein the central opening (13) of the matrix holder (12) is a through opening (13a and 13c) or a recess with a wall (13b), in particular wherein the central opening (13) comprises a matrix which is a solid porous support or a hydrogel, or a combination thereof.

3. The cell culture device according to one of claims 1 or 2, wherein the matrix consists of natural, semi-synthetic or synthetic material, or a combination thereof, in particular the natural material is selected from collagen, fibrin, glycosamine glycans and hyaluronic acid, and the synthetic or semi-synthetic material selected from polymeric components such as polymethyl methacrylate, polylactic acid, polyglycol, polystyrene and ceramic components such as hydroxyapatite or tricalcium phosphate.

4. The cell culture device according to one of claims 1 to 3, wherein the carrier (10) has an element (15) for connection to the matrix holder (12) and the matrix holder at its upper end has notches (14a, 14b) through which it can be reversibly connected to the element (15) of the carrier (10).

5. The cell culture device according to one of claims 1 to 4, wherein the device comprises at least 5, in particular at least 10 matrix holders (12).

6. The cell culture device according to one of claims 1 to 5, wherein the device additionally comprises a transport unit (20) which can transport one or more cell culture units (21).

7. The cell culture device of claim 6, wherein the transport unit (20) comprises i. a support rail (22) for fastening one or more cell culture units (21) to the transport unit (20), ii. a receiving unit (20a) for a support rail (22), iii. a lid (23), and iv. two spacer elements (24), which are optionally directly connected to one another, for the orderly reception of at least two or more carriers (10) of a matrix holder, v. optionally at least one identification element (26) such as an RFID chip or a barcode, and vi. optionally a centering unit (27).

8. The cell culture device according to claim 6 or 7, wherein the corrugated strip (11) comprises a position (25) for attachment to the support rail (20), and / or the cell culture units are arranged in parallel in the spacer (24).

9. The cell culture device according to any one of claims 6 to 8, wherein the transport unit (20) comprises: i. a plate (48) which forms the underside of the transport unit, ii. Openings in the plate (48) for centering the transport unit on the plate (49), iii. Openings in the plate (48) for screw connections (50), iv. a further plate (51) which is attached approximately at mid-height of the transport unit, v. magnetizable metal rods (52), vi. a centering bracket (54), vii. Openings in the plate (51) for the centering bracket (54), viii. and a centering object (55).

10. A method for cell cultivation using the cell culture device according to any one of claims 1 to 9 comprising the steps: i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Introducing the cell suspension into the cell culture device according to one of Claims 1 to 9, iii. Incubate the cell culture device containing the cell suspension under conditions that allow cell growth.

11. The method of claim 10, comprising the steps of i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Colonizing the matrix in the opening (13) of the matrix holder (12) with the cell suspension by introducing the cell suspension into the cell culture device according to one of claims 1 to 9, iii. Transferring the matrix holder into a corrugated strip (11), in particular using the carrier of the matrix holder (10), and iv. Incubate the cell culture device containing the cells under conditions that allow cell growth.

12. The method according to claim 11, wherein the opening (13) of the matrix holder (12) has a recess (13c) for attachment and is reversibly connected to a colonization device (30) via a complementary sealing element (31), and the matrix is a Hydrogel, comprising the steps i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with one or more ingredients of a hydrogel to produce a cell suspension-hydrogel mixture, iii. Populating the opening (13) of the matrix holder (12), which is horizontally aligned in the inoculating device (30), with the cell suspension-hydrogel mixture, iv. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% CO 2, until the matrix has polymerized, v. Separating the matrix holder (12) from the inoculating device (30) vi. Transferring the matrix holder (12) into a corrugated strip (11) filled with medium, preferably using the carrier of the matrix holder (10), vii. Incubate the cell culture device containing the cell suspension-matrix mixture under conditions that allow cell growth.

13. The method according to claim 12, wherein the opening (13) of the matrix holder (12) has an upper opening (13d) and the matrix holder (12) is reversibly connected to a colonization device (33), and the matrix is a hydrogel comprising the Steps i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with a hydrogel to produce a cell suspension-hydrogel mixture, iii. Filling the wells of the inoculation device (33) with the cell suspension-hydrogel mixture, iv. Populating the opening (13) of the matrix holder by immersing the matrix holder (12) in the inoculating device (33), v. Incubating the cell batch in a suitable cultivation environment, preferably at 37 ° C. and 5% CO 2, until the matrix has polymerized, vi. Transferring the matrix holder (12) from the inoculation device (33) into a corrugated strip (11) filled with nutrient medium, and vii. Incubate the cell culture device containing the cell suspension-matrix mixture under conditions that allow cell growth.

14. The method according to claim 12, wherein the opening (13) of the matrix holder (12) has an upper opening (13d) and the matrix holder (12) is reversibly connected to a colonization device (33) which comprises filling elements (40) and which The matrix is a hydrogel comprising the steps of i. Suspending the cells to be cultivated in an appropriate amount of a cell culture medium to form a cell suspension, ii. Mixing the cell suspension with a hydrogel to produce a cell suspension-hydrogel mixture, iii. Introducing the matrix holder into the inoculating device (33), iv. Filling the wells of the colonization device (33) with the cell suspension-hydrogel mixture via the filling elements (40), whereby the opening (13) of the matrix holder is colonized with the cell suspension-hydrogel mixture, v. Incubation of the cell batch in a suitable cultivation environment, preferably at 37 ° C and 5% CO2, until the matrix polymerizes, vi. Transferring the matrix holder (12) from the inoculation device (33) into a corrugated strip (11) filled with nutrient medium, and vii. Incubate the cell culture device containing the cell suspension-matrix mixture under conditions that allow cell growth.

15. Cell culture device according to one of claims 1 to 9, wherein the corrugated strip is a perfusion device (34), and the perfusion device has hose connections (41), optionally for connection to a pump.

16. Cell culture device according to one of claims 1 to 9, additionally comprising a box (44) and an insert (42) for receiving one or more cell culture units (21) or colonization devices (30).