Method for producing carrier particles for cultivating biological cells and use thereof for cultivating biological cells

Abstract

The invention relates to a method for producing carrier particles for cultivating biological cells, particularly dried hydrogel particles, and to applications and bioreactors comprising such carrier particles. The invention can be used in the cultivation of biological cells, in particular in the cultivation (expansion) of multipotent and pluripotent stem cells or differentiated cells, for example for tissue engineering. The invention further relates to a freeze dryer designed to carry out the method. The method for producing carrier particles for cultivating biological cells comprises the steps of: providing (S10) an aqueous suspension of hydrogel beads, freeze-drying (S30) the hydrogel beads such that dried hydrogel particles are formed, storing (S40) the freeze-dried hydrogel particles, and subsequently rehydrating (S50) the hydrogel particles by adding an aqueous cultivation medium for cultivating biological cells. The method is characterised in that the freeze-drying (S30) of the hydrogel beads and the storing (S40) and rehydrating (S50) of the hydrogel particles take place in the same container, which is a bioreactor (10) for cultivating biological cells. The hydrogel particles can advantageously be produced efficiently in high quality and in a sufficiently large amount as carrier particles for the cultivation of biological cells. A further advantage is that the method permits the production of a ready-to-use bioreactor with carrier particles as a finished kit.

Description

[0001] DESCRIPTION

[0002] METHOD FOR THE PREPARATION OF CARRIER PARTICLES FOR THE CULTURE OF BIOLOGICAL CELLS AND THEIR APPLICATION FOR THE CULTURE OF BIOLOGICAL CELLS

[0003] Technical field

[0004] The invention relates to methods for producing carrier particles, in particular dried hydrogel particles, for the cultivation of biological cells, and to applications and bioreactors comprising such carrier particles. Applications of the invention include the cultivation of biological cells, particularly the cultivation (expansion) of multipotent and pluripotent stem cells or of differentiated cells, e.g., for tissue engineering. The invention further relates to a dehydrator designed for carrying out the method.

[0005] Technical background

[0006] The use of pluripotent stem cells for artificial tissue replacement and regenerative medicine requires the standardized and reproducible multiplication of these cells. In sufficient quantities, these cells can be differentiated into specialized cell systems such as cardiomyocytes, and these cells can then be used in the application step for drug screening, toxicity analyses, or disease modeling. Performing these processes in traditional two-dimensional culture dishes (Petri dishes or T-flasks) requires significant material and personnel resources due to the limited growth area and the large volume of culture medium, thus reducing the overall economic efficiency. Bioreactors and bioreactor systems provide a standardized platform for the expansion and differentiation of stem cells, enabling, for example, the high-quality production of human induced pluripotent stem cells (hiPSCs).One way to provide cells with even more growth surface area in bioprocesses is through the use of carrier particles made of hydrogels.

[0007] Methods for producing such carrier particles are generally known and are described by way of example in German patent application DE 102020 116 108 Al. As also described there by way of example, the use of polysaccharide polymers, in particular alginate hydrogels (also referred to simply as alginates), as carriers for cell cultures is generally known. Alginates can be used in layers on two-dimensional substrates or as cell carriers (spherical particles, beads, carrier beads, microcarriers) in suspensions. Depending on the specific cultivation task and the cells to be cultivated, alginates can be modified by additives. Additives include, for example, peptides (e.g., RGD peptide), proteins (e.g., collagen I), or complex mixtures (e.g., Matrigel™) that influence cell adhesion. Cell cultivation on microcarriers in suspensions, e.g.,in bioreactors, it has advantages for the formation of three-dimensional cell arrangements (e.g. 3D aggregates, spheroids, micro-carrier cell hybrids) under near-physiological conditions, and it allows for efficient process control, as a more favorable surface-to-volume ratio can be achieved.

[0008] Until now, dried carrier particles for the cultivation of biological cells have been produced in small vials. These vials are advantageous because they allow water to evaporate easily during freeze-drying. However, they are unsuitable when large quantities of carrier particles are required.

[0009] Another problem with conventional techniques arises when the carrier particles adhere to the inner wall of the vial. The dried particles can also clump together in a cake-like fashion. This can impair the efficiency of the process and lead to undesirable results, particularly during further handling and transfer of the dried particles into a bioreactor for the cultivation of biological cells.

[0010] The object of the invention is to provide an improved method for producing carrier particles for the cultivation of biological cells, in particular an improved method for producing freeze-dried and subsequently rehydrated hydrogel particles, which overcomes the disadvantages of conventional techniques. The method is intended, in particular, to enable improved efficiency in connection with the application of the produced carrier particles in the cultivation of biological cells.

[0011] These tasks are solved by methods and devices with the features of the independent claims. Advantageous further developments are specified in the dependent claims and the description.

[0012]

[0013] the

[0014] According to a first general aspect of the invention, the above problem is solved by a method for producing carrier particles for the cultivation of biological cells, comprising the following steps: An aqueous suspension of hydrogel beads, preferably spherical hydrogel beads, is provided. Preferably, the hydrogel beads are freshly produced by crosslinking a precursor polymer with an ionic precipitating agent. The production of the hydrogel beads can be carried out using methods known per se. The suspension liquid of the aqueous suspension of hydrogel beads can comprise an aqueous solution containing the precipitating agent, in which the production of the hydrogel beads takes place, and / or a washing and / or buffer solution with which the hydrogel beads are optionally washed after precipitation.

[0015] The hydrogel spheres are then freeze-dried to form dried hydrogel particles. This freeze-drying process can involve subjecting the hydrogel spheres to a reduced temperature (below room temperature, preferably below 0°C) and a reduced pressure (lower than atmospheric pressure). The reduced temperature is preferably below the freezing point and below, for example, 10°C below, the collapse temperature of the hydrogel spheres. The freeze-drying of the (spherical) hydrogel spheres is preferably carried out in suspension with the suspension liquid, with the suspension liquid first being removed and then the hydrogel spheres being dried. Alternatively, the hydrogel spheres can be removed from the suspension liquid of the aqueous suspension before freeze-drying.The suspension liquid is separated from the hydrogel beads before freeze-drying.

[0016] The freeze-dried hydrogel particles are then stored and subsequently rehydrated by adding an aqueous culture medium for the cultivation of biological cells. This rehydration can be repeated after the desired storage period, for example, when the hydrogel particles are needed for biological cell cultivation at a later time. The resulting hydrogel particles can also be described as cell carriers for the cultivation of biological cells.

[0017] According to the invention, the freeze-drying of the hydrogel spheres, as well as the storage and rehydration of the hydrogel particles, takes place in the same container, wherein the container is a bioreactor for the cultivation of biological cells. This results in several advantages:

[0018] Producing the carrier particles directly in the bioreactor allows for the production of sufficient quantities for cultivating biological cells. Simultaneously, it eliminates the previously required process step of transferring freeze-dried hydrogel particles from a first container (e.g., a vial) to a second container for rehydration and / or cell cultivation. Instead, rehydration takes place in the first container, which then serves as the bioreactor. This not only increases process efficiency by eliminating an additional step but also prevents undesirable damage to the carrier particles during transfer, such as when they partially adhere to the inner wall of the first container, resulting in a loss of cultivation surface area.Another advantage is that the process enables the production of a "ready-to-use" bioreactor with carrier particles for the cultivation of biological cells as a finished kit.

[0019] A bioreactor is a container (culture vessel) in which specific biological cells are (or can be) cultivated. For cell cultivation, the bioreactor is placed within a bioreactor system. The bioreactor system serves to control, monitor, and optimize the cell culture processes in one or more bioreactors, for example, by controlling and monitoring parameters such as temperature, pH, and oxygen supply within each bioreactor. The bioreactor can be a suitable glass container and / or a tube, such as a centrifuge tube for a bioreactor system.

[0020] The bioreactor can have a diameter of at least 3 cm. Alternatively, or in addition, the bioreactor can have a receiving chamber with a receiving volume of at least 50 ml, preferably at least 100 ml, or in the range of 50 to 500 ml. Advantageously, the carrier particles required for the cultivation of biological cells can be produced in correspondingly large quantities.

[0021] For the freeze-drying of the hydrogel beads, the bioreactor is located in a freeze dryer (lyophilizer). The freeze dryer has a holding device for at least one bioreactor. According to a preferred embodiment, the holding device is designed to hold the at least one bioreactor at an angle to a vertical direction during the freeze-drying of the hydrogel beads in the freeze dryer, preferably to increase the surface area of ​​the hydrogel beads and / or the aqueous suspension during freeze-drying. An angled orientation means that a vertical or longitudinal axis of the bioreactor forms a non-zero angle with both a vertical and a horizontal axis. The angled orientation increases the surface area for sublimation during freeze-drying, allowing water to escape more easily during the process.The speed and quality of freeze-drying are increased. Furthermore, faster freezing and desorption are enabled. The holding device can be designed as a shelf or receiving surface for the at least one bioreactor, or it can include such a shelf or receiving surface. The shelf or receiving surface can either itself be inclined relative to the vertical direction or support the bioreactor in such a way that it can be positioned in the inclined orientation during freeze-drying.

[0022] According to a possible further development, the freeze dryer, preferably the holding device, has a tilt adjustment device by means of which the tilt angle of the inclined orientation of the at least one bioreactor relative to a vertical direction can be adjusted and / or changed. This offers the advantage that the tilt angle can be adjusted and optimized, for example, depending on the fill level of a bioreactor. At a low fill level, a greater tilt angle relative to the vertical can be set than at a high fill level without the aqueous suspension being able to flow out. The tilt adjustment device can, for example, be designed to change the position and / or spatial orientation of a shelf or receiving surface for the at least one bioreactor in order to adjust the tilt angle. The tilt adjustment device can, for example, be designed so that the tilt angle is continuously adjustable or adjustable in steps.

[0023] Another aspect is that the holding device can be designed to accommodate multiple bioreactors in an inclined orientation. This can further increase process efficiency. Optionally, the tilt adjustment device can be configured so that the inclined orientation can be set differently for each bioreactor.

[0024] In a further preferred embodiment, the holding device has at least one receiving surface or shelf for a bioreactor, which is made of a thermally conductive material and / or is temperature-controlled (heatable and / or coolable). This improves the temperature control of the bioreactor. The receiving surface or shelf can, for example, be thermally coupled to a temperature control circuit.

[0025] In a further preferred embodiment, the holding device has at least one receiving surface for a bioreactor, the surface shape of which is shaped to correspond to and / or be inverted from a section of the bioreactor's shell, so that the bioreactor rests flat against the receiving surface during freeze-drying. In other words, the receiving surface for a bioreactor can have a precisely fitting inverted shape of a section of the bioreactor's wall to increase the contact area. Advantageously, the freezing rate and / or the temperature rise, e.g., for desorption in the second drying stage, can be controlled more efficiently and thus improved.

[0026] The bioreactor has at least one outlet opening through which water vapor can escape during freeze-drying. This outlet opening for water sublimation can be, for example, the open end of the bioreactor vessel or integrated into a lid (end-drainage lid) that can be placed on the open end of the bioreactor vessel.

[0027] According to a preferred embodiment, at least one outlet opening is closed during the freeze-drying of the hydrogel beads with a barrier permeable to water vapor, preferably a membrane. This offers the advantage of reducing the risk of contamination of the carrier particles during freeze-drying.

[0028] A particularly preferred embodiment is one in which the barrier is a sterile barrier, preferably a sterile barrier designed as a membrane. Such a sterile barrier is understood to be a barrier, e.g., a membrane, that is permeable to water vapor and is made of a material and / or coated with a material that has microbial retention properties. This allows the risk of contamination of the carrier particles during freeze-drying to be reduced or eliminated even more reliably.

[0029] The membrane size is preferably adapted to the size of the bioreactor's outlet opening(s). Alternatively, or in addition, the membrane can be integrated into a lid, such as a screw-on lid, for the bioreactor. For example, the aforementioned at least one outlet opening of the bioreactor can be integrated into the lid and covered with the membrane. This allows for simple and quick equipping of the bioreactor with contamination protection for the freeze-drying step. All that is required is to attach the lid with the membrane.

[0030] In another preferred embodiment, a lid is provided for the bioreactor, designed to seal the bioreactor gas-tight in a closed position. During freeze-drying, this lid is either placed in an open position or stored at a distance from the bioreactor, allowing water vapor to escape through at least one outlet opening. After freeze-drying, the lid is returned to the closed position, in which the bioreactor is gas-tightly sealed by the lid. This allows the bioreactor to be easily brought into a storage-ready state after freeze-drying, protecting the dried hydrogel particles from moisture and other contamination. The at least one outlet opening can be integrated into the lid, allowing the lid to be in place even during the freeze-drying process.The lid can be positioned on the bioreactor in either an open or closed position with respect to the at least one outlet opening. In the open position, water vapor can escape through the at least one outlet opening, while in the closed position this is not possible and / or the at least one outlet opening is sealed. This further simplifies handling and improves process efficiency.

[0031] For example, the lid can be designed as a plug, with at least one outlet opening integrated into an insertion section of the plug. In the open position, the plug is positioned on the bioreactor such that the insertion section with the at least one outlet opening protrudes from the bioreactor. In the closed position, the insertion section, or at least the portion of the insertion section containing the at least one outlet opening, is covered by the wall of the bioreactor. This embodiment allows for particularly efficient handling of the bioreactor during and after the freeze-drying phase.

[0032] In another embodiment concerning the lid, which gas-tightens the bioreactor in a closed position, the freeze dryer features a locking device that moves the lid from the open to the closed position after the freeze-drying process is complete. This further simplifies handling and improves process efficiency. For example, the locking device can include a locking plunger that is movable towards the lid by means of a guide mechanism, such as a transmission. The locking device can be designed so that the locking plunger extends outwards from the drying chamber and can be moved into the open or closed position by an operator using manual force. Alternatively, the locking plunger can be driven by a motor, e.g., a motor.It can be driven by an electric motor to selectively move it into the open or closed position.

[0033] The closing device can, for example, be designed to move the lid from the open position to the closed position within a drying chamber or within a pre-chamber (with airlock) of the freeze dryer that can be filled with a protective gas, e.g., nitrogen. For example, it can be provided that the lid is moved into the closed position by an operator wearing protective gloves within a pre-chamber of the freeze dryer that can be filled with a protective gas. Such pre-chambers are also known as glove boxes and provide a workspace in which an operator can work wearing protective gloves without disturbing the controlled atmosphere.

[0034] In a further embodiment of the process, at least one adhesion-reducing substance is added to the suspension. The adhesion-reducing substance is a substance for reducing air bubbles on the hydrogel particles and / or for reducing the adhesion of the hydrogel particles to the bioreactor wall. The substance can also be referred to as a surfactant. This improves the quality of the produced hydrogel particles. The substance preferably comprises poloxamer. Poloxamer has proven particularly effective in reducing air bubble and cake formation in experiments.

[0035] In a further embodiment of the process, at least one lyoprotectant or adhesion-reducing substance is added to the suspension. The lyoprotectant is a substance that minimizes or prevents damage to the hydrogel spheres, particularly the macromolecules from which the hydrogel spheres are composed, caused by ice formation during freeze-drying. Furthermore, a lyoprotectant is used that causes the hydrogel spheres, i.e., the dried hydrogel particles, to have a shape approximating a spherical particle form after freeze-drying. The lyoprotectant may comprise poloxamer, trehalose, dimethyl sulfoxide (DMSO), and / or sucrose.

[0036] According to a further preferred embodiment, the concentration of the lyoprotectant substance and / or the adhesion-reducing substance in the suspension liquid containing the originally produced hydrogel beads is selected in the range of 1 mg / ml to 500 mg / ml. This concentration range is preferred because below 1 mg / ml the effect is insufficient, and above 500 mg / ml the lyoprotectant substance and / or the adhesion-reducing substance can cause excessive contamination of the culture medium in the bioreactor.

[0037] According to a further preferred embodiment, the freeze-dried hydrogel particles are stored in a storage container, preferably a bag, in an airtight manner by vacuum sealing after freeze-drying the hydrogel spheres, preferably until the start of rehydration. This variant is particularly advantageous if, for example, the bioreactor is only covered with a lid during freeze-drying, which has the water vapor-permeable membrane described above, and the lid cannot be brought into an airtight (gas-tight) closed position after freeze-drying.

[0038] The hydrogel spheres comprise or are preferably alginate spheres (alginate hydrogels). However, the practical implementation of the invention is not limited to alginate spheres, but can also be realized with ECM (extracellular matrix), collagen, gellan, or pectin hydrogels. Alginate has proven particularly advantageous because it exhibits a shape-retention effect (memory effect) and, after freeze-drying and rehydration, re-forms spherical alginate spheres and / or an approximately spherical particle shape.

[0039] The term "approximately spherical particle shape" includes a shape of a dried hydrogel particle that represents a spheroid (in particular a sphere or ellipsoid) and has a smooth or structured surface topology with characteristic structural dimensions such as steps, protrusions or depressions, which is smaller than one cross-sectional dimension, preferably smaller than 1 / 10 of the cross-sectional dimension, of the dried hydrogel particle.

[0040] According to a second general aspect of the invention, a method for cultivating biological cells in a bioreactor is provided, comprising the steps of:

[0041] Production of carrier particles for the cultivation of biological cells according to a method as described herein, and

[0042] Cultivation of biological cells in the same bioreactor in which the carrier particles were produced, wherein the bioreactor is introduced into a bioreactor system for cultivation.

[0043] The process is characterized by the fact that the freeze-drying of the hydrogel spheres, the storage and rehydration of the hydrogel particles, and the subsequent cultivation of the biological cells using the hydrogel particles as carrier particles for the cells to be cultivated all take place in the same container, which is a bioreactor for cultivating biological cells. As already mentioned, a particular advantage is that this allows for the provision of a ready-to-use bioreactor with carrier particles (cell carriers) as a complete kit. The dried hydrogel particles do not need to be transferred from a separate storage container to the bioreactor. This reduces the risk of damage and contamination of the carrier particles and makes handling more efficient.

[0044] According to a third general aspect of the invention, the above problem is solved by using the carrier particles, which are produced by a method as described herein, as carrier particles (cell carriers) for the cultivation of biological cells. Preferably, the carrier particles have a characteristic cross-sectional dimension, such as a diameter, in the range of 50 pm to 2 mm.

[0045] According to a fourth general aspect of the invention, the above problem is solved by a bioreactor for the cultivation of biological cells in a bioreactor system. The bioreactor comprises hydrogel particles as carrier particles for the biological cells to be cultivated, which are produced by a method as described herein. To avoid repetition, the features of the bioreactor described in this document in connection with the methods shall also be disclosed and claimable for the bioreactor as an object per se, and vice versa.

[0046] According to another aspect, a bioreactor is disclosed and claimed that comprises freeze-dried hydrogel particles as carrier particles for the biological cells to be cultured, which are produced by a process as described herein, but without the rehydration step having yet been carried out. That is, a bioreactor is provided in which the hydrogel particles are produced by freeze-drying the hydrogel spheres as described above and are stored in the dried state. Such a modified process can be described accordingly as follows:

[0047] Method for the production of dried carrier particles for the cultivation of biological cells, comprising the steps:

[0048] Provision of an aqueous suspension of hydrogel beads, freeze-drying of the hydrogel beads to form dried hydrogel particles, and

[0049] Storage of the freeze-dried hydrogel particles,

[0050] where the freeze-drying of the hydrogel beads and the storage of the hydrogel particles take place in the same container, which is a bioreactor for cultivating biological cells.

[0051] According to a further general aspect of the invention, a freeze dryer is provided for freeze-drying hydrogel beads suspended in an aqueous suspension. The freeze dryer comprises a holding device for at least one bioreactor, as described above in connection with the method according to the first general aspect. The freeze dryer thus comprises a holding device configured to hold the at least one bioreactor at an angle to a vertical direction during the freeze-drying of the hydrogel beads in the freeze dryer. To avoid repetition, the features of the freeze dryer described in this document in connection with the methods are also disclosed and claimable for the freeze dryer as an object per se, and vice versa.

[0052] The previously described preferred embodiments and features of the invention can be combined with one another in any way.

[0053] Brief description of the characters

[0054] Further details and advantages of the invention are described below with reference to the accompanying drawings. These show:

[0055] Figure 1 shows a flowchart illustrating features of preferred embodiments of the inventive method for producing carrier particles;

[0056] Figure 2 shows a flowchart illustrating features of preferred embodiments of the inventive method for cultivating biological cells in a bioreactor;

[0057] Figures 3A and 3B show schematic representations of bioreactors with different lids according to embodiments of the inventive method for producing carrier particles;

[0058] Figure 4 shows a schematic representation of an inclined position of a bioreactor during freeze-drying according to an embodiment of the inventive method for producing carrier particles;

[0059] Figures 5A and 5B are schematic representations of a freeze dryer according to an embodiment of the invention;

[0060] Figure 6 shows a schematic representation of a holding device for a freeze dryer according to an embodiment of the invention;

[0061] Figures 7A and 7B show a schematic representation of a holding device for a freeze dryer with a tilt adjustment device according to an embodiment of the invention; and

[0062] Figure 8 shows photographic images of rehydrated carrier particles with and without added poloxamer according to different embodiments of the method.

[0063] The embodiments shown in the figures are at least partially identical, so similar or identical parts are provided with the same reference numerals, and reference is made to the descriptions of the other embodiments or figures to avoid repetition. Furthermore, for the sake of clarity, structurally identical parts are not all individually marked with reference numerals.

[0064] Detailed

[0065]

[0066] en

[0067] The invention is described below with exemplary reference to alginate-based cell carriers (carrier particles) as an example of hydrogel spheres. Alginate spheres, which according to the invention are provided in an aqueous suspension in a bioreactor and subjected to freeze-drying, can be produced, for example, from commercially available alginate, which typically exhibits low viscosity due to the relatively short chain lengths of the polymer macromolecules. Alternatively, alginate with a higher viscosity compared to commercially available alginate, due to longer molecular chains, can be used. The selection of a specific alginate to be used depends, for example, on the desired elasticity of the cell carriers during cell culture.The invention is not limited to the use of alginate, but can also be implemented with other hydrogels, such as ECM (extracellular matrix), collagen, gellan or pectin.

[0068] Figure 1 schematically shows the main steps of the production of carrier particles for the cultivation of biological cells according to the invention.

[0069] In step S10, hydrogel beads, e.g., alginate beads, are provided in an aqueous suspension in a container. The alginate beads were previously produced using a separate process and transferred to the container after production. A bioreactor is used as the container, i.e., a vessel (culture vessel) in which biological cells can be cultivated using hydrogel particles, e.g., alginate beads, as cell carriers. An exemplary bioreactor 10 is shown in Figure 3. The hydrogel beads, e.g., alginate beads, contained in the aqueous suspension 2 are designated by reference numeral 1. The bioreactor 10 preferably has a diameter d of at least 3 cm or a receiving chamber (11) and / or a receiving volume of at least 50 ml or in the range of 50 to 500 ml.

[0070] The aforementioned production of alginate spheres is carried out, for example, in a manner known per se, by generating sodium alginate droplets with a nozzle and crosslinking the alginate droplets in an aqueous suspension liquid with an ionic precipitating agent. A BaCl₂ solution is used, for example, as the precipitating agent. The size and size distribution of the alginate spheres can be adjusted by the nozzle dimensions and operating parameters. The crosslinked alginate droplets form dimensionally stable alginate spheres suspended in the suspension liquid.

[0071] Optionally, the alginate beads can be functionalized after precipitation by coating them with tyramine and / or a protein, such as Matrigel. Coating is achieved by precipitation from the suspension liquid or by direct covalent coupling. The Matrigel coating offers advantages for cell adhesion during subsequent cell culture.

[0072] A lyoprotectant or an adhesion-reducing substance can be added to the suspension liquid either during the introduction of the alginate droplets into the suspension liquid or, alternatively, after crosslinking and formation of the alginate spheres. Preferably, the substance comprises poloxamer (trade name Pluronic F 68, 1 mg / ml). The suspended alginate spheres are loaded with the lyoprotectant substance, for example, by storing the alginate spheres in an aqueous solution containing the lyoprotectant substance, for example, for at least one day.

[0073] Optionally, in step S20, the alginate beads loaded with the lyoprotectant substance are removed from the suspension. For example, the suspension liquid is poured off, leaving the alginate beads surrounded by residual liquid in the container. Alternatively, a sieve is used for removal.

[0074] If step S20 is not performed, the alginate beads are freeze-dried immediately after being loaded with the lyoprotectant substance in step S30. The freeze-drying process is described in more detail below with reference to Figures 3 to 7.

[0075] Freeze-drying takes place in a freeze dryer in which the bioreactor 10 containing the hydrogel beads is placed. A freeze dryer 30 is shown in an exemplary and highly schematic form in Figure 5A. For sterile process control, the freeze dryer 30 can be located in a cleanroom. Alternatively, a sterile barrier (as described below) can be used. A special feature of the freeze dryer 30 is that it has a holding device 32 for at least one bioreactor 10, which is designed to hold the at least one bioreactor 10 at an angle to the vertical during the freeze-drying of the hydrogel beads in the freeze dryer 30.

[0076] Figure 4 shows an example of such a holding device 32. It can be seen that the bioreactor 10 is mounted on the holding device 32 at an angle to the vertical V, i.e., the longitudinal axis A of the bioreactor forms an angle α of less than 90° with the horizontal H. Accordingly, the tilt increases the surface area 4 of the aqueous suspension 2, thereby accelerating sublimation during freeze-drying. The speed and quality of the freeze-drying are thus increased.

[0077] During freeze-drying, the bioreactor 10 has at least one outlet opening through which water vapor can escape. Figure 3 shows two different embodiments. In the embodiment shown in Figure 3A, a lid 14 is arranged on the open end of the vessel or the opening 12 of the bioreactor, into which several outlet openings 18 are integrated. During freeze-drying, the lid is moved to an open position (as shown in Figure 3A) so that water vapor can escape from the bioreactor 10 through the outlet openings 18. After freeze-drying, the lid 14 is moved to a closed position in which the bioreactor is gas-tightly sealed by the lid (not shown in Figure 3A). In the closed position, the freeze dryer 30 can be vented (after completion of freeze-drying), and the bioreactors 10 with the dried carrier particles can be removed.This makes it easy to bring the bioreactor 10 into a storable state after freeze-drying, in which the dried hydrogel particles are protected from moisture and other contamination.

[0078] For example, the lid 14 can be designed as a stopper, e.g., a rubber stopper, in which the outlet openings 18 are integrated into an insertion section 16 of the stopper. The stopper is subsequently also referred to as a lyophilization stopper. In the open position, the stopper is positioned on the bioreactor such that the insertion section 16 with the outlet openings 18 protrudes from the bioreactor. In the closed position of the stopper, the insertion section 16, or at least the part of the insertion section having at least one outlet opening 18, is covered by the vessel wall of the bioreactor. This closure 14 has the advantage that the bioreactor, in the closed position of the lid 14, is in a storage-ready state until the later use of the bioreactor and the dried carrier particles stored therein for the desired cultivation of biological cells in the bioreactor system.For this purpose, the lid 14 could then be replaced by a standard cultivation lid (for use in the bioreactor system). This embodiment enables particularly efficient handling of the bioreactor 10 during and after the freeze-drying phase.

[0079] Figure 3B shows an embodiment in which the open end of the bioreactor 10 is sealed with a barrier permeable to water vapor. The barrier can, for example, comprise a screw cap 20 that encloses a water-permeable membrane 22. The membrane 22 can be quickly attached to the bioreactor 10 using the screw cap 20 and later removed again. The screw cap 20 (hereinafter also referred to as the drainage cap) and the membrane 22 are thus adapted to the size of the bioreactor to enable quick handling.

[0080] Membrane 22 is preferably designed as a sterile membrane, meaning that it is permeable to water vapor and made of a material and / or coated with a material that exhibits microbial retention properties. This further reduces the risk of contamination of the carrier particles during freeze-drying. Such membrane materials are offered, by way of example, by Tecklen GmbH, DE-85667 Oberpframmern, under the name LyoprotectO-Membrane. The special feature of the present membrane 22, however, is that its size is already adapted to the opening of the bioreactor 10 and it is integrated into the screw cap 20 to enable quick and safe handling. A lid, which can be arranged on the bioreactor in a different manner, can also be used instead of the screw cap.

[0081] The use of such a sterile membrane 22 offers the advantage that a special cleanroom environment is not required. Alternatively, instead of integrating a sterile membrane into the lid, it can be arranged around a holder for the bioreactor.

[0082] The drainage lid 20 shown in Figure 3B allows the water to sublimate and ensures sterility; however, this lid 20 does not allow the bioreactor 10 to be hermetically sealed after freeze-drying. In this case, the bioreactor 10 can be hermetically sealed or packaged, for example, by vacuum sealing in a storage bag (not shown) until use, in order to prevent rehydration of the carrier particles by atmospheric moisture.

[0083] The following phases can be used for freeze-drying: First, the alginate beads are cooled from room temperature to a final temperature of, for example, -45 °C (approx. 0.4 °C / min) in a freezing phase over 150 minutes. During the freezing phase, a linear time-temperature function is implemented, for example.

[0084] This is followed by a stabilization phase in which the alginate beads are stored at the final temperature for a stabilization interval of 120 minutes. The stabilization phase has the advantage that the sample containing the frozen alginate beads is completely frozen before the drying phases are carried out.

[0085] A subsequent first drying phase functions as a pre-drying phase, in which the suspension liquid is removed by sublimation. This first drying phase is carried out, for example, in a freeze dryer as illustrated in Figures 5A and 5B, equipped with a cooling unit and a condenser. During this first drying phase, the final temperature, for example -45 °C, is maintained, while the pressure is reduced from atmospheric pressure to an initial pressure of 50 pbar over a period of 10 minutes, following a linear time-pressure function. The frozen sample is then held at the final temperature and initial pressure for a stabilization period of, for example, 80 hours during the first drying phase.

[0086] In a subsequent second drying phase, the dried alginate particles are subjected to a second negative pressure, which is lower than the first negative pressure and, for example, 100 pbar. The reduction to the second negative pressure is achieved using a linear time-pressure function over a duration of, for example, 300 minutes. During the second drying phase, the temperature of the dried alginate particles is equal to the final temperature or an elevated temperature, such as room temperature (20 °C). After the reduction to the second negative pressure, the dried sample is maintained at this pressure for a stabilization period of, for example, 20 hours during the second drying phase.

[0087] A subsequent aeration phase is provided, in which the dried alginate particles are brought to atmospheric pressure according to a linear time-pressure function with a pressure rise interval of at least 1 minute. The aeration phase can be carried out with air or an inert gas. Using a relatively long pressure rise interval advantageously prevents damage to the dried alginate particles. When aeration is performed with air, the bioreactor is sealed beforehand in the drying chamber 44 of the freeze dryer 30, while when an inert gas is used, sealing takes place after aeration, e.g., within the drying chamber 44 or within the pre-chamber of the freeze dryer 30, which can be filled with the protective gas.

[0088] Dry nitrogen or argon is preferably used as the inert gas. The dried alginate particles are particularly preferably stored in the inert gas or under vacuum. Practical tests have shown that the dried alginate particles produced according to the invention can be stored, for example, at 4 °C for several months without loss of functionality.

[0089] To close the bioreactor after the drying process, the freeze dryer 30 can have a closure device 38. This is illustrated in Figures 5A and 5B. The closure device 38 allows the lid (plug) 14, as previously described in connection with Figure 3A, to be moved from the open position to the closed position. For example, the closure device 38 can have a closure plunger 40, which is movable towards the lid 14 by means of a guide mechanism, e.g., a transmission. The guide mechanism is shown here by way of example in the transmission unit 50. The transmission unit 50 is held on the holding device 32 by a bracket 52. The closure device 38 can be designed such that the closure plunger 40 extends out onto the drying chamber 44.The extended section serves as an actuating element 42, which can be selectively moved into the open or closed position by an operator using muscle power. Alternatively, the locking plunger 42 can be driven by a drive motor, e.g., an electric motor, to selectively move it into the open or closed position.

[0090] The freeze-drying process described as an example in step S30 can be modified with regard to the set temperatures and pressures and the form of the time-pressure and time-temperature functions, depending on the specific application conditions. For example, preparatory tests can be used to determine which time and pressure parameters yield optimal drying results for a specific hydrogel sample, particularly an alginate sample.

[0091] As a result of freeze-drying step S30, the dried alginate particles are present as finished cell carriers in bioreactor 10. Storage then follows (step S40 in Fig. 1) at, for example, 4°C or at room temperature until use. The dried alginate particles are stored in bioreactor 10 during this time.

[0092] For biotechnological use, the lid 14, e.g., the lyophilization stopper, is removed from the bioreactor 10 under sterile conditions. If the bioreactors 10 were previously stored vacuum-sealed in a bag, they are removed from the bag. The carrier particles are then rehydrated by adding culture medium (step S50 in Fig. 1) and optionally washed several times to remove the lyoprotective agent and / or the adhesion-reducing substance. If the substance has no effect on the cells, the washing step can be omitted.

[0093] Figure 2 shows a method for cultivating biological cells in a bioreactor according to an exemplary embodiment. The carrier particles are produced according to steps S10 to S50 described above. Optionally, additional culture medium is added to the rehydrated carrier particles in the bioreactor 10, if this has not already been done in step S50. The cells to be cultivated are added, and the bioreactor 10 containing the carrier particles and cells is sealed with the standard lid of the bioreactor 10 (or alternatively with the lid used during freeze-drying) and placed in the bioreactor system. In step S70, cell cultivation then takes place in the bioreactor 10, controlled by the bioreactor system. The bioreactor system can be designed in a manner known per se.

[0094] Figure 6 shows another advantageous embodiment of the holding device 32. The holding device 32 enables the holding of several bioreactors 10 (here, six bioreactors are shown by way of example) at an angle to the vertical during freeze-drying. Accordingly, the closure device 38 is designed such that the respective lid 14 of each of the six bioreactors 10 can be moved from the open position to the closed position after completion of freeze-drying. For this purpose, several, here six, closure plungers 40 of the closure device are provided.

[0095] Figures 7A and 7B schematically illustrate another embodiment of the holding device 32 for the at least one bioreactor. In this embodiment, the holding device 32 has a tilt adjustment device 34 by means of which the tilt angle α of the inclined orientation can be adjusted and / or changed. The front support section 48, on which the lid-side end region of the bioreactor 10 rests, is shown here as an example only and is horizontally adjustable. This support section 48 thus serves as an adjustment element. The rear support section 46 serves as a fixed holding element. Figure 7B shows an adjustment position in which the bioreactor 10 is oriented at a steeper angle than in the adjustment position shown in Figure 7A. The desired inclination of the bioreactor 10 during freeze-drying can therefore be flexibly adjusted by means of the tilt adjustment device.

[0096] Figure 8 shows photographic images of rehydrated carrier particles with and without added poloxamer according to different embodiments of the process. The upper images A and B of Figure 8 show alginate carrier particles produced by the process without added poloxamer as an adhesion-reducing agent. Here, there is an increased risk of air bubble formation, as indicated by the black dots. The lower images C and D of Figure 8 show alginate carrier particles produced by the process with added poloxamer. In comparison, a significantly reduced air bubble formation is evident.

[0097] The features of the invention disclosed in the foregoing description, the drawings, and the claims can be significant for the realization of the invention in its various embodiments, both individually and in combination or sub-combination. The invention is not limited to the preferred embodiments described above. Rather, a multitude of variants and modifications are possible, which also make use of the inventive concept and therefore fall within the scope of protection. In particular, the invention also claims protection for the subject matter and the features of the dependent claims independently of the referenced claims. Specifically, the individual features of independent claim 1 are each disclosed independently of one another. List of reference numerals

[0098] 1 Hydrogel beads, e.g. alginate beads 2 Aqueous suspension of hydrogel beads 4 Surface

[0099] 10 Bioreactor

[0100] 11 Recording Room

[0101] 12 Opening the bioreactor

[0102] 14 lids, e.g. stoppers

[0103] 16 Introductory section

[0104] 18 Outlet opening

[0105] 20 screw caps

[0106] 22 Barrier, e.g. membrane

[0107] 30 Freeze dryers

[0108] 32 Holding device

[0109] 34 Tilt adjustment device

[0110] 38 Locking device

[0111] 40 sealing stamps

[0112] 42 Actuating element

[0113] 44 drying chamber

[0114] 46 retaining element

[0115] 48 Adjustment element

[0116] 50 transmission units

[0117] 52 bracket

[0118] a tilt angle

[0119] d diameter bioreactor

[0120] A Longitudinal axis, orientation of bioreactor

[0121] V Vertical

[0122] H Horizontal

Claims

Claims 1. Method for the production of carrier particles for the cultivation of biological cells, comprising the steps: Provision (S10) of an aqueous suspension (2) of hydrogel beads (1), freeze-drying (S30) of the hydrogel beads (1) so that dried hydrogel particles are formed, Storage (S40) of the freeze-dried hydrogel particles, and subsequent rehydration (S50) of the hydrogel particles by adding an aqueous cultivation medium for the cultivation of biological cells; characterized in that the freeze-drying (S30) of the hydrogel spheres (1) and the storage (S40) and rehydration (S50) of the hydrogel particles take place in the same container, which is a bioreactor (10) for the cultivation of biological cells.

2. Method according to claim 1, wherein the biorector (10) a) has a diameter (d) of at least 3 cm; and / or b) has a receiving chamber (11) with a receiving volume of at least 50 ml, more preferably at least 100 ml or in a range of 50 to 500 ml.

3. Method according to one of the preceding claims, wherein the freeze dryer (30) for carrying out the freeze-drying has a holding device (32) for at least one bioreactor (10) which is configured to hold the at least one bioreactor (10) in an inclined orientation relative to a vertical direction during the freeze-drying (S30) of the hydrogel beads (1) in the freeze dryer (30).

4. Method according to claim 3, wherein the freeze dryer (30), preferably the holding device (32), has a tilt adjustment device (34) by means of which a tilt angle (a) of the inclined orientation can be adjusted and / or changed.

5. Method according to one of claims 3 or 4, wherein several bioreactors (10) can be held and / or are held in the inclined orientation by means of the holding device (32).

6. Method according to any one of claims 3 to 5, wherein the holding device (32) has at least one receiving surface for a bioreactor, a) which is made of a thermally conductive material and / or is temperature-controlled and / or b) whose surface shape is designed to correspond to and / or be inverted to a shell surface section of the bioreactor (10), so that the bioreactor (10) lies flat on the receiving surface with the shell surface section during freeze-drying.

7. Method according to one of the preceding claims, wherein during the freeze-drying of the hydrogel beads (1) the biorector (10) has at least one outlet opening which is closed with a barrier (22) permeable to water vapor, preferably a membrane (22).

8. Method according to claim 7, wherein the barrier (22) is a sterile barrier.

9. Method according to one of the preceding claims, wherein a lid (14) for the bioreactor (10) a) during freeze-drying (S30) is placed in an open position or stored at a distance from the bioreactor (10) so that water vapor can escape from the bioreactor (10) via at least one outlet opening (12, 18), and b) is brought into a closed position after freeze-drying, in which the bioreactor (10) is sealed gas-tight by the lid (14).

10. Method according to claim 9, wherein the at least one outlet opening (18) is integrated into the lid (14).

11. Method according to claim 10, wherein the lid (14) is designed as a plug and the at least one outlet opening (18) is integrated into an insertion section (16) of the plug, wherein the plug is positioned on the bioreactor (10) in the open position such that the insertion section (16) with the at least one outlet opening (18) protrudes from the bioreactor (10).

12. Method according to one of claims 9 to 11, wherein the freeze dryer (30) has a closing device (38) by means of which, after completion of the freeze-drying, the lid (14) is moved from the open position to the closed position.

13. Method according to claim 12, wherein the closure device (38) has a closure plunger (40) which is movable towards the lid (14) by means of a guide mechanism.

14. Method according to claim 12 or 13, wherein the closure device (38) is configured to move the lid (14) from the open position to the closed position within a drying chamber (44) or within a pre-chamber of the freeze dryer (30) that can be filled with protective gas.

15. Method according to one of claims 9 to 11, wherein the lid (14) is brought into the closed position by an operator using protective gloves within a pre-chamber of the freeze dryer (30) which can be filled with protective gas.

16. Method according to any of the preceding claims, wherein at least one lyoprotectant substance or an adhesion-reducing substance is added to the suspension (2), the substance preferably comprising poloxamer.

17. Method according to one of the preceding claims, in which, for storing the freeze-dried hydrogel particles, the bioreactor (10) is stored airtight in a storage container, preferably a bag, by vacuum sealing after the freeze-drying (S30) of the hydrogel spheres (1), preferably until the start of rehydration.

18. Method according to any of the preceding claims, wherein the hydrogel beads (1) comprise alginate beads.

19. Method for cultivating biological cells in a bioreactor, comprising the steps: Production of carrier particles for the cultivation of biological cells according to a method of the preceding claims, and Cultivation (S70) of biological cells in the same bioreactor (10) in which the carrier particles were produced, wherein the bioreactor (10) is introduced into a bioreactor system for cultivation.

20. Use of carrier particles produced by a method according to any one of claims 1 to 18 as carrier particles for the cultivation of biological cells.

21. Bioreactor (10) for the cultivation of biological cells in a bioreactor system, comprising Hydrogel particles (3) as carrier particles for the biological cells to be cultivated, which are produced by a method according to one of claims 1 to 18.

22. Freeze dryer (30) for freeze-drying hydrogel beads (1) provided in an aqueous suspension (2), comprising: a holding device (32) for at least one bioreactor (10), preferably a holding device (32) according to claims 3 to 6, which is designed to hold the at least one bioreactor (10) in an oblique orientation relative to a vertical direction during the freeze-drying (S30) of the hydrogel beads in the freeze dryer (30).

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