Method for producing carrier particles for culturing living cells, carrier particles, and their use

Abstract

The present invention relates to a method for producing carrier particles for culturing biological cells, comprising the steps of providing an aqueous suspension of hydrogel beads and freeze-drying the hydrogel beads to form dry hydrogel particles, wherein at least one lyoprotectant substance is added to the suspension, and the hydrogel beads in the suspension are loaded with the lyoprotectant substance, and the dry hydrogel particles, under the action of the lyoprotectant substance, acquire a shape that approximates the shape of spherical particles and that is maintained after rehydration. The present invention also relates to carrier particles for culturing biological cells, preferably comprising dry hydrogel particles having a protein coating, and to the use of the carrier particles.

Description

Technical Field

[0001] The present invention relates to a method for producing carrier particles for culturing living cells, particularly a method for producing dry hydrogel particles. The present invention further relates to carrier particles for culturing living cells, particularly dry hydrogel particles produced using the above method, and the use of the carrier particles. The present invention is applicable to the culture of living cells, particularly the culture (proliferation) of pluripotent stem cells and totipotent cells or differentiated cells, for example, culture for tissue engineering.

Background Art

[0002] This specification refers to the following prior art representing the technical background of the present invention. [1] Paper "Investigation of structural cellular processes in the context of tissue engineering: alginate-based scaffold structures", Michael Gepp, Saarland University, 2017 [2] European Patent Application Publication No. 2361968 [3] US Patent No. 6642363 [4] M. Gepp et al., "J. Appl. Phycol." (2017) 29: 2451 - 2461

[0003] The use of polysaccharide polymers, particularly alginate hydrogels (also abbreviated as "alginates"), as carriers for cell culture is well known (see [1] and the references cited therein). Alginates can be used as cell carriers (spherical particles, beads, carrier beads, microcarriers) in layers on two-dimensional substrates or in suspensions (see, for example, [1] and [2]). Depending on the specific culture task and the cells being cultured, alginates may be modified with additives. Additives include, for example, peptides or collagen that affect cell adhesion (see, for example, [3] or [4]). Culturing cells in suspension, for example on microcarriers in a bioreactor, has advantages for forming three-dimensional arrangements of cells (e.g., 3D aggregates, spheroids, hybrids of microcarriers and cells) under physiologically close conditions and enables efficient execution of the process by allowing the setting of a preferred surface-to-volume ratio.

[0004] Conventional cell culture using alginate as a microcarrier in a bioreactor includes, for example, the following steps: First, alginate beads are prepared by crosslinking, which precipitates liquid alginate into spherical particles (see, for example, [1]). Depending on the application, a suspension of alginate beads is formed with a particle size distribution having a diameter in the range of 200 μm to 500 μm. The width of the particle size distribution, which can be set based on droplet formation conditions, may affect subsequent cell culture. After washing the crosslinked alginate beads, they are modified (activated and / or functionalized), for example, by binding tyramine or by adjusting the elasticity of the beads with an alginate mixture selected before the alginate beads were prepared. The completed modified alginate beads are suspended in culture medium and placed in a bioreactor. Cells to be cultured are added to the suspension in the bioreactor, where they colonize the alginate beads and are subjected to a specified culture protocol. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] European Patent Application Publication No. 2361968 [Patent Document 2] U.S. Patent No. 6,642,363 [Non-patent literature]

[0006] [Non-Patent Document 1] Dissertation “Untersuchung von strukturbildenden zellulaeren Prozessen im Kontext des Tissue Engineerings:Alginat‐basierte Gerueststrukturen”Michael Gepp,Universitaet des Saarlandes,2017 [Non-Patent Document 2] M. Gepp et.al., “J. Appl. Phycol.” (2017) 29:2451-2461 [Overview of the project] [Problems that the invention aims to solve]

[0007] Conventional methods have the following drawbacks and limitations: Preparing modified alginate beads with specific properties requires specialized knowledge, making it difficult to routinely prepare alginate beads in the laboratory for cell culture or industrial applications. Alginate beads are typically prepared according to user specifications, for example, using a specific particle size distribution, diameter, and / or modification, delivered to the user in suspension, and initially stored at the user's location. However, a drawback is that transportation and storage of the suspension at the user's location can lead to undesirable changes in the alginate beads.

[0008] Alginate beads can clump together or disintegrate, which is detrimental to accurate and reproducible weighing by the user. The sterility of alginate beads can be lost during storage. Furthermore, the properties of finished alginate beads can only be inspected or altered to a very limited extent, or not at all, by the user. For example, the particle size distribution of alginate beads in a suspension can only be altered afterward, if possible, with considerable effort. The water content of alginate beads can distort the culture medium in the bioreactor. To prevent this, the number of alginate beads can be kept low, but this increases the consumption of medium and reduces the number of cultureable cells. Therefore, conventional methods for cell culture are characterized by low efficiency, high process costs, lack of scalability, low success rates, and low reproducibility.

[0009] It is also known that alginate beads can be dried ([1]). Dried alginate beads can be stored better than in suspension. Furthermore, dried alginate beads can be rehydrated in culture medium in a bioreactor. However, in practice, the limitations of conventional use of alginate beads have not yet been overcome. Even when using conventional dried and rehydrated alginate beads, dried alginate beads are difficult to handle, and the reliability of successful cell culture and the reproducibility of culture results are limited, even when using consistent culture protocols.

[0010] The object of the present invention is to provide an improved method for producing carrier particles for culturing living cells, in particular an improved method for producing dried hydrogel particles that enables overcoming the shortcomings of the prior art. The object of the present invention is also to provide improved carrier particles for culturing living cells that enable overcoming the shortcomings of conventional carrier particles. The present invention is particularly intended to provide carrier particles that have reproducible properties, improved storage properties, can be accurately measured, are easily modifiable, and / or are suitable for routine use in bioreactors of different structures and purposes. Carrier particles produced according to the present invention are particularly intended to enable cell culture with increased efficiency, success rate, and / or reproducibility.

[0011] These objectives are each achieved by a method for producing carrier particles for culturing living cells and by carrier particles having the features of an independent claim. Preferred embodiments and applications of the present invention are described in the dependent claims. [Means for solving the problem]

[0012] According to a first general aspect of the present invention, the above objective is achieved by a method for producing carrier particles for culturing living cells, the method comprising the following steps: an aqueous suspension of spherical hydrogel beads is prepared. Preferably, the hydrogel beads are newly produced by crosslinking a precursor polymer with an ionic precipitating agent. The hydrogel beads may be produced using methods known to themselves. The suspension of the aqueous suspension of hydrogel beads comprises an aqueous solution containing the precipitating agent from which the hydrogel beads are produced, and / or a washing solution and / or buffer used if the hydrogel beads are optionally washed after precipitation. The hydrogel beads are then lyophilized to form dried hydrogel particles. Lyophilization of the hydrogel beads involves applying a low temperature (below room temperature, preferably below 0°C) to the hydrogel beads and applying negative pressure (a pressure lower than atmospheric pressure). Lyophilization of the hydrogel beads is preferably carried out while suspended in a suspension, the suspension is removed first, and then the hydrogel beads are dried. Alternatively, the hydrogel beads can be removed from the aqueous suspension before lyophilization; that is, the suspension is separated from the hydrogel beads before lyophilization.

[0013] According to the present invention, at least one lioprotectant substance is added to the suspension, and the hydrogel beads in the suspension are filled with the lioprotectant substance before freeze-drying. The lioprotectant substance (or lioprotectant agent) is a substance that minimizes or prevents damage to the hydrogel beads, particularly to the polymer that makes up the hydrogel beads, by ice formation during freeze-drying. Alternatively, a lioprotectant substance is used that causes the hydrogel beads after freeze-drying, i.e., the dried hydrogel particles, to approximate a spherical particle shape under the action of the lioprotectant substance.

[0014] Hydrogel beads preferably include alginate beads (alginate hydrogels). However, the invention is not actually limited to alginate beads and can also be carried out with collagen hydrogels, gellan gum hydrogels, or pectin hydrogels. Alginates have proven particularly advantageous because they have a shape memory effect and reform into spherical alginate beads after freeze-drying and rehydration.

[0015] The term “nearly spherical particle shape” includes shapes of dry hydrogel particles that exhibit a spheroidal shape (particularly a sphere or ellipsoid) and have characteristic structural dimensions smaller than the cross-sectional dimensions of the dry hydrogel particles, preferably less than 1 / 10 of the cross-sectional dimensions, such as smooth or structured surface topology with steps, protrusions or depressions.

[0016] According to a second general aspect of the present invention, the objective is achieved by carrier particles for culturing living cells, the carrier particles comprising dried hydrogel particles having a substantially spherical shape and containing a lioprotectant substance. The carrier particles are preferably prepared using a method according to the first general aspect of the present invention or one of its embodiments. Preferably, the carrier particles have characteristic cross-sectional dimensions, for example, a diameter, in the range of 50 μm to 2 mm.

[0017] According to a third general aspect of the present invention, the above object is achieved by using carrier particles according to the second general aspect of the present invention or its embodiments as cell carriers for culturing living cells. According to preferred modifications, the use of carrier particles in suspension bioreactors, particularly culture vessels, microtiter plates, hanging drops, cell culture bags, (suspension) bioreactors and / or dishes, such as Petri dishes, may be provided. The use of carrier particles preferably includes the steps of preparing dried particles, rehydrating and washing, cell inoculation, cell proliferation and cell passage / cell harvesting.

[0018] In accordance with the present invention, the inventors have found that by filling hydrogel beads with a lyoprotectant substance in which the particle shape of the dried hydrogel particles is substantially spherical, the disadvantages of conventional suspended or dried alginate beads in terms of storage stability, metering property, and handling property are avoided. Gentle freeze-drying that retains the structure and function of the spherical hydrogel beads is achieved.

[0019] Since the storage stability is improved and adhesion and aggregation are prevented, the sterility in the dry state can be maintained better, and contamination of the prepared carrier beads can be prevented. These advantages also apply particularly to conventional approaches to the freeze-drying of alginate beads, for example, according to [1]. Therefore, the inventors have found that conventional alginate beads in the dry state have an irregularly deformed shape rather than a spherical shape, for example, a folded shape and / or a shape provided with cracks or protrusions, and the quality of the dried alginate beads is impaired, for example, due to density fluctuations of the bulk material and irregular mass formation. The dried hydrogel particles prepared in accordance with the present invention promote aseptic freeze-drying and rehydration.

[0020] The dried hydrogel particles form a homogeneous material that is completely fluid or a cake that can be cut (a freeze-dried cake (Lyo-Kuchen), also called a dried product matrix), so the metering property is improved. The accuracy and reproducibility in setting the concentration of the carrier beads in the bioreactor are improved. The dried hydrogel particles can be quantified, for example, by volume measurement, weight measurement, and / or optical measurement. Since the dried hydrogel particles are opaque, the rehydration in the bioreactor can be confirmed by optical measurement.

[0021] The dried hydrogel particles can be processed in a simple manner. The processing is preferably carried out in a sterile cabinet, but pipetting is not required for metering. The dried hydrogel particles can be pre-distributed in a simple manner. Improved quality control is possible in the process of preparing and using the dried hydrogel particles.

[0022] The dried hydrogel particles can be used as an injectable bulk material or in pellet form, and can be introduced directly into a bioreactor (a container in which cell culture is carried out and which enables the setting of biochemical and physical culture conditions). Advantageously, the dried hydrogel particles do not distort the total volume of the culture medium in the bioreactor.

[0023] It is preferred to use the rehydrated hydrogel beads by directly adding the dried hydrogel particles to the culture medium in the bioreactor. The cells to be cultured may already be suspended in the culture medium at the time when the dried hydrogel particles are supplied, or may be added into the bioreactor after the rehydration of the dried hydrogel particles. The degree of rehydration of the dried hydrogel particles is preferably determined using light transmission measurement or scattered light measurement on the culture medium. It is particularly preferred that the cells are supplied to the culture medium after the dried hydrogel particles are completely hydrated.

[0024] For example, the rehydration of the dried hydrogel particles in water can optionally be considered as a further sub-step of the method according to the invention. Advantageously, the hydrogel beads formed upon rehydration (rehydrated hydrogel beads) have a spherical particle shape even after rehydration. Preferably, the rehydrated hydrogel beads are characterized by the spherical shape before lyophilization. This promotes the culturing of cells on the hydrogel beads in the inoculation stage. Furthermore, the spherical rehydrated hydrogel beads are advantageously characterized by a preferable floating behavior in the suspension and a lower tendency to undesirable aggregation as compared to deformed hydrogel beads.

[0025] According to a preferred embodiment of the present invention, a protein layer is supported on a hydrogel. This modification is preferably performed after the preparation of the hydrogel beads and before lyophilization. The protein layer may include, for example, extracellular matrix proteins, thereby advantageously promoting cell adhesion to the rehydrated hydrogel beads. The protein layer may consist of a sub-monolayer, monolayer, or multilayer of protein molecules on the hydrogel beads. For example, after the hydrogel beads are prepared in a suspension, at least one protein to be bound is added to the suspension and incubated with the hydrogel beads. Advantageously, incubation allows for the covalent bonding of at least one protein or mediator molecule to the entire surface of the hydrogel beads, including any pores.

[0026] Protein layer loading is preferably performed before freeze-drying. In selected applications of the present invention, protein layer loading may be performed after freeze-drying, particularly after rehydration, for example, in a bioreactor. For example, at least one protein may be supplied from the culture medium in the bioreactor or from cells already present in the bioreactor.

[0027] Preferably, the hydrogel beads are supported with at least one mediator molecule (e.g., tyramine) and / or at least one protein comprising laminin (recombinant and tissue-specific), vitronectin (recombinant and compatible with pluripotent stem cells), collagen (tissue-specific and compatible with pluripotent stem cells), decellularized tissue-derived proteins (highly tissue-specific), and a complex protein mixture derived from the basal matrix (e.g., Geltrex, Matrigel, denovoMatrix).

[0028] According to a more preferred embodiment of the present invention, the lioprotectant residue is removed after freeze-drying. Separating the lioprotectant residue from the dried hydrogel particles provides advantages for subsequent rehydration and subsequent cell culture. Advantageously, the effects of the lioprotectant on cells are prevented or minimized. Removal of the lioprotectant residue includes complete removal of the lioprotectant from the dried hydrogel particles, or removal of the lioprotectant from the surface of the dried hydrogel particles. The surface of the dried hydrogel particles is particularly preferably residue-free with respect to the lioprotectant.

[0029] The lioprotectant material, such as the "freeze-dried cake" residue, is preferably removed by mechanical treatment of the dried material, including grinding and sieving. Advantageously, the lioprotectant material is separated from the dried hydrogel while the dried hydrogel particles are separated from each other.

[0030] Alternatively or additionally, according to further modifications of the present invention, after rehydration of the dried hydrogel particles, the residue of the dried lioprotectant material can be removed, particularly before using the particles in a washing solution composed of sodium chloride. Washing the rehydrated hydrogel beads advantageously further suppresses the effect of the lioprotectant material on subsequent cell culture.

[0031] A further advantage of the present invention lies in the availability of different lioprotectant materials for modifying the hydrogel beads according to the present invention. These lioprotectant materials may include, in particular, trehalose, dimethyl sulfoxide (DMSO), sucrose, and / or poloxamer. These lioprotectant agents can be used alone or in combination and have the advantage that their effects on living cells have been well studied. Therefore, undesirable effects on cell culture can be avoided or minimized. Trehalose and / or sucrose are particularly preferred as lioprotectant materials because, when used, they yield carrier particles characterized by particularly comprehensive shape and function retention, and particularly good cell adhesion in cell cultures, after rehydration.

[0032] According to a more preferred embodiment of the present invention, the concentration of the lioprotectant substance in the suspension containing the initially prepared hydrogel beads is selected in the range of 1 mg / mL to 500 mg / mL. This concentration range is preferable because concentrations below 1 mg / mL result in insufficient approximation of the spherical particle shape, while concentrations above 500 mg / mL may cause an excessive load on the culture medium in the bioreactor due to the lioprotectant substance.

[0033] A further advantageous embodiment of the present invention may provide functionalization of cell carriers prepared according to the present invention for culturing living cells. Preferably, the functionalization of the hydrogel beads is carried out before or during their preparation (before or during encapsulation of active ingredients, magnetic particles, etc.), for example, before or during dropping to prepare the hydrogel beads by precipitation, so as to encapsulate the substance in the hydrogel matrix, or after provision and before lyophilization (e.g., before surface functionalization with tyramine and / or proteins). Functionalization involves the addition of particles and / or substances that impart further properties to the hydrogel beads beyond carrier function. According to a preferred variant of the present invention, the hydrogel beads are filled with magnetic particles and / or bioactive substances (active ingredients). It is particularly preferable that the magnetic particles and / or active ingredients are supplied before lyophilization. The inventors have found that functionalized hydrogel beads in a dry state also form substantially spherical particles, and functionalized hydrogel beads in a rehydrated state also form spherical cell carriers.

[0034] Hydrogel beads, each containing one or more magnetic particles, advantageously offer the possibility of manipulating cell carriers within a bioreactor by a magnetic field. The magnetic particles can consist of permanent magnet materials known to exist.

[0035] Preferably, the bioactive substance used to modify the hydrogel beads is a differentiation factor, i.e., a bioactive substance that induces cell differentiation at a specific point in time and / or influences the direction of cell differentiation.

[0036] According to a more preferred embodiment of the present invention, if the initially prepared dispersion of hydrogel beads contains an adhesion-reducing substance, adhesion between dry hydrogel particles is suppressed. This advantageously promotes the injectability of the dry hydrogel particles. The adhesion-reducing substance particularly preferably contains polyethylene glycol, and its effects on living cells have been advantageously well studied.

[0037] For the freeze-drying of hydrogel beads, known freeze-drying methods can be selected. Particularly for freeze-drying alginate beads, a protocol having the following steps is preferably applied. First, there is a freezing step in which the hydrogel beads, brought into suspension, are frozen according to a predetermined time-temperature function having a freezing interval of at least 120 minutes and an end temperature greater than -80°C and less than -20°C. Preferably, the freezing rate is set in the range of 0.1 to 1°C / min (slow freezing), from 50°C / min (rapid freezing) to 1 to 1.5°C / min (medium freezing). Setting the time-temperature function advantageously allows for gentle freezing of the hydrogel beads. Subsequently, a stabilization step is provided in which the hydrogel beads are stored at the end temperature for a stabilization duration interval of at least 90 minutes. Then, a first negative pressure selected in the range of 30 μbar to 60 μbar is applied to the frozen hydrogel beads at the end temperature in a first drying step for forming dry hydrogel particles. Next, in the second drying stage, a second negative pressure lower than the first negative pressure is applied to the dried hydrogel particles at a temperature above the final temperature. Finally, an aeration stage follows in which the dried hydrogel particles are transferred to atmospheric pressure according to a time-pressure function with a pressure increase interval of at least 0.5 to 1 minute (1 bar / min). The aeration stage is carried out in a container using an inert gas or air.

[0038] Further details and advantages of the present invention will be described below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0039] [Figure 1] This flowchart shows the features of a preferred embodiment of the carrier particle production method according to the present invention. [Figure 2] This flowchart shows the characteristics of a preferred embodiment of the use of carrier particles prepared according to the present invention. [Figure 3] This is a photograph of rehydrated carrier particles compared to conventional carrier particles. [Figure 4] This is a schematic process diagram illustrating an example of the use of carrier particles prepared according to the present invention. [Modes for carrying out the invention]

[0040] The present invention is described below with illustrative reference to alginate-based cell carriers (carrier particles). Alginate beads filled with a lioprotectant material and subjected to lyophilization according to the present invention can be made, for example, from commercially available alginates that typically have low viscosity due to the relatively short chain length of the polymer polymer. Alternatively, alginates with higher viscosity than commercially available alginates may be used due to longer molecular chains. The selection of the specific alginate used is preferably based on the desired elasticity of the cell carrier during the period of cell culture. The present invention is not limited to the use of alginates and may further be carried out using other hydrogels, such as collagen, gelan, or pectin.

[0041] Embodiments of the present invention are described below with particular reference to examples for filling alginates with lioprotectant substances, examples for modifying alginate beads, dried alginate particles and / or rehydrated alginate beads, and protocols for lyophilization. Details of the use of rehydrated alginate beads in the culture of living cells can be carried out as is known from conventional cell cultures.

[0042] Figure 1 schematically shows the main steps for producing dried alginate particles according to the present invention. In step P1, alginate beads are produced in an aqueous suspension in a container. The alginate beads are produced by generating sodium alginate droplets using a nozzle, for example, and crosslinking the alginate droplets in the aqueous suspension with an ionic precipitant, as described in [1], for example. As the precipitant, a BaCl2 solution is used, for example. The particle size and particle size distribution of the alginate beads can be set by the dimensions and operating parameters of the nozzle. The crosslinked alginate beads form dimensionally stable alginate beads suspended in the suspension.

[0043] Alginate beads are preferably functionalized by coating them with tyramine and / or protein, such as Matrigel, after precipitation. Coating is carried out by precipitation from the suspension or by direct covalent bonding. Matrigel coating is advantageous for cell adhesion in subsequent cell culture.

[0044] The lioprotectant substance is added to the suspension while the alginate droplets are being added to the suspension, or alternatively, after the crosslinking and formation of the alginate beads. In the inventors' tests, trehalose (100 mg / mL), poloxamer (trade name Pluronic F-68, 1 mg / mL), and / or sucrose (100 mg / mL) in an aqueous solution of NaCl (0.9%) were used as the lioprotectant substance. The suspended alginate beads are filled with the lioprotectant substance, for example, by storing the alginate beads in an aqueous solution containing the lioprotectant substance for at least one day.

[0045] In step P2, the alginate beads filled with the lioprotectant are optionally removed from the suspension. For example, the suspension is decanted, leaving the alginate beads surrounded by the residual liquid in the container. Alternatively, a screen is used for removal.

[0046] If step P2 is not provided, in step P3, the lyophilization of the alginate beads is performed immediately after the alginate beads are filled with the lioprotectant material. For this purpose, the following steps are provided.

[0047] First, the alginate beads are cooled to a final temperature of, for example, -45°C (approximately 0.4°C / min) over a freezing phase of 150 minutes. During the freezing phase, a linear time-temperature function is applied, for example.

[0048] Next, a stabilization step follows, in which the alginate beads are stored at the final temperature for a stabilization duration interval of 120 minutes. The stabilization step has the advantage that the sample is completely frozen by the frozen alginate beads before the drying step is performed.

[0049] The subsequent first drying stage functions as a pre-drying stage in which the suspension is removed by sublimation. The first drying stage is carried out, for example, in a freeze dryer equipped with a cooling unit and a condenser. During the first drying stage, a termination temperature, e.g., -45°C, is maintained, while the pressure decreases from atmospheric pressure to a first negative pressure of 50 μbar over a period of 10 minutes according to a linear time-pressure function. Subsequently, in the first drying stage, the frozen sample is held at the termination temperature and the first negative pressure for a stabilization period, e.g., 80 hours.

[0050] In the subsequent second drying stage, a second negative pressure, lower than the first negative pressure, for example, 100 μbar, is applied to the dried alginate particles. The reduction to the second negative pressure occurs over a period of, for example, 300 minutes using a linear time-pressure function. During the second drying stage, the temperature of the dried alginate particles is equal to the final temperature or the elevated temperature, for example, room temperature (20°C). After being reduced to the second negative pressure, the dried sample is maintained at the second negative pressure during the second drying stage for a stabilization period of, for example, 20 hours.

[0051] Next, an aeration step is provided in which the dry alginate particles are transferred to atmospheric pressure according to a linear time-pressure function having a pressure rise interval of at least 1 minute. The aeration step may be provided using air or an inert gas. Applying a relatively long pressure rise interval advantageously prevents damage to the dry alginate particles. When aeration is performed with air, the storage container is sealed in the chamber beforehand, but when an inert gas is used, the sealing is performed after aeration.

[0052] It is preferable to use dry nitrogen or argon as the inert gas. It is particularly preferable to store the dried alginate particles in an inert gas or under vacuum. In actual tests, dried alginate particles prepared according to the present invention exhibited storage properties at, for example, 4°C without losing functionality for several months.

[0053] The freeze-drying method for P3 described as an example may be modified with respect to the set temperature and pressure, as well as the form of the time-pressure function and time-temperature function, based on specific application conditions. For example, preliminary tests may be employed to determine which time and pressure parameters yield the optimal drying results for a particular hydrogel sample, especially an alginate sample.

[0054] As a result of freeze-drying P3, the dried alginate particles exist as completed cell carriers. According to the present invention, the addition of a lioprotectant substance gives the dried alginate particles a spherical shape (see, for example, the photograph in Figure 4), which is advantageously retained even after rehydration. The dried particles can be joined together as a cake or form an injectable bulk material composed of individual particles. When an adhesion-reducing substance, such as polyethylene glycol, is added to the suspension of alginate beads in addition to the lioprotectant substance, the formation of an injectable bulk material is promoted. To minimize adhesion between dried alginate particles, polyethylene glycol, trade name PEG-600, at a concentration of, for example, 50 mg / mL is used.

[0055] Depending on the concentration of the lioprotectant substance used, the aforementioned substance may form a residue on the surface of the dried alginate particles following freeze-drying P3. Therefore, as shown in Figure 1, an optional step P4 may be provided to remove the lioprotectant substance, particularly from the surface of the dried alginate particles, for example, by sieving or grinding.

[0056] Figure 2 schematically illustrates the application of dried alginate particles as cell carriers in the culture of living cells. First, in step K1, an aqueous culture medium is prepared in a bioreactor. The composition of the culture medium is selected based on the specific application of cell culture using methods known in themselves. Alginate particles prepared according to the present invention are added to the culture medium. The dried alginate particles are weighed, for example, by weighing. Tests using dried alginate particles prepared according to the present invention showed that particles not filled with polyethylene glycol initially lay on the surface of the culture medium and only sank into the culture medium after centrifugation, while particles modified with polyethylene glycol were not observed to float on the culture medium.

[0057] In step K2, the alginate particles are rehydrated in an aqueous culture medium. For example, as shown in Figure 3, the dried alginate particles are converted into alginate beads by absorbing water from the culture medium. Finally, in step K3, cell culture, cell incubation, and / or cell preservation of living cells are carried out in a culture medium containing alginate beads.

[0058] Figure 3 shows optical microscope images of lyophilized and rehydrated alginate beads filled with trehalose (A) or sucrose (B) according to a preferred modification of the present invention, compared with lyophilized and rehydrated alginate beads without a lioprotectant (C) and newly prepared alginate beads in suspension (D) (approximately 400 μm in diameter). The rehydrated particles (A, B) prepared according to the present invention advantageously exhibit the same spherical shape as the suspended untreated alginate beads (D). In contrast, the untreated lyophilized and rehydrated particles (C) (e.g., according to [1]) have an uneven, irregular surface, unlike the untreated alginate beads.

[0059] Figure 4 schematically illustrates a further use of dried alginate particles as a cell carrier in the proliferation of hiPS cells (human induced pluripotent stem cells). In the first proliferation stage, dried alginate particles 1 are added to a first culture vessel 2, for example, a suspension bioreactor having a volume of several milliliters (e.g., 10 mL) to several liters (e.g., 3 L). Alginate beads 3 (shown as an example) are formed by rehydration from the alginate particles 1. HiPSC cells are then added from vessel 4 to culture vessel 2. Cell culture is then carried out under specified culture conditions, and the cells first attach and adhere to the microcarriers, and then proliferate at a specific rate (e.g., 7 times) over several days (e.g., 7 days). In the final step of the process, alginate beads to which cells 5 have adhered are obtained, i.e., after they are obtained (e.g., by enzymatic treatment), the used microcarriers 6 and the proliferated hiPS cells 7 are present. These cells are then stored in a cell bank 10 and may be further investigated and / or processed 9 (e.g., differentiated into cardiomyocytes) and / or transferred for direct use 8 (e.g., bioprinting).

[0060] The features of the present invention disclosed in the above description and drawings, as well as in the claims, may be significant both individually and in combination or in partial combination, for carrying out the present invention in various configurations.

Claims

1. A method for producing carrier particles for culturing living cells, The steps include: preparing an aqueous suspension of hydrogel beads selected from the group consisting of alginate, collagen, gelan, and pectin; The steps include freeze-drying the hydrogel beads so that dry hydrogel particles are formed, Includes, At least one lioprotectant substance is added to the aqueous suspension, the lioprotectant substance fills the hydrogel beads in the aqueous suspension, and under the action of the lioprotectant substance, the dried hydrogel particles obtain a shape approximating a spherical particle shape. The lioprotectant substance comprises at least one of trehalose, dimethyl sulfoxide, sucrose, and poloxamer. The freeze-drying step of the hydrogel beads is, A freezing step in which the hydrogel beads are frozen according to a time-temperature function having a freezing interval of at least 120 minutes and an end temperature of less than -40°C, A stabilization step in which the hydrogel beads are stored at the termination temperature for a stabilization duration interval of at least 90 minutes, A first drying step for forming the dried hydrogel particles is to apply a first negative pressure, selected in the range of 30 μbar to 60 μbar, to the hydrogel beads frozen at the termination temperature, A second drying step in which a second negative pressure lower than the first negative pressure is applied to the dried hydrogel particles at a temperature equal to or greater than the completion temperature, The dry hydrogel particles undergo an aeration step in which they transition to atmospheric pressure according to a time-pressure function having a pressure increase interval of at least 0.5 minutes. including, Characterized by, method.

2. A protein layer is supported on the hydrogel beads. The method according to claim 1.

3. After the freeze-drying step, the residue of the lioprotectant substance is removed. The method according to claim 1 or 2.

4. The aforementioned residue is removed by mechanical treatment of the dried hydrogel particles, including crushing and sieving. The method according to claim 3.

5. The concentration of the lioprotectant substance in the aqueous suspension is selected to be in the range of 1 mg / mL to 500 mg / mL. The method according to any one of claims 1 to 4.

6. The dried hydrogel particles are subjected to rehydration. The dried residue of the lioprotectant substance is removed in a washing solution composed of sodium chloride before the carrier particles are used. The method according to any one of claims 1 to 5.

7. The hydrogel beads contain at least one of magnetic particles and a physiologically active substance. The magnetic particles and / or the physiologically active substance are dried together with the hydrogel beads during the freeze-drying step. The method according to any one of claims 1 to 6.

8. The hydrogel beads contain a differential factor. The method according to claim 7.

9. At least one adhesion-reducing substance that promotes the injectability of the dried hydrogel particles is added to the aqueous suspension. The method according to any one of claims 1 to 8.

10. The adhesion-reducing substance includes polyethylene glycol. The method according to claim 9.

11. Carrier particles for culturing living cells, It contains dried hydrogel particles having a protein coating, The dried hydrogel particles are Dry hydrogel particles selected from the group consisting of alginate, collagen, gellan, and pectin, and It contains a lioprotectant substance and has a shape that approximates a spherical shape. The lioprotectant substance comprises at least one of trehalose, dimethyl sulfoxide, sucrose, and poloxamer. The carrier particles are obtained by the following process: The steps include: preparing an aqueous suspension of hydrogel beads selected from the group consisting of alginate, collagen, gelan, and pectin; The steps include supporting a protein layer on the hydrogel beads, The steps include freeze-drying the hydrogel beads so that dry hydrogel particles having the protein coating are formed, Includes, At least one lioprotectant substance is added to the aqueous suspension, the lioprotectant substance fills the hydrogel beads in the aqueous suspension, and under the action of the lioprotectant substance, the dried hydrogel particles obtain a shape approximating a spherical particle shape. The freeze-drying step of the hydrogel beads is, A freezing step in which the hydrogel beads are frozen according to a time-temperature function having a freezing interval of at least 120 minutes and an end temperature of less than -40°C, A stabilization step in which the hydrogel beads are stored at the termination temperature for a stabilization duration interval of at least 90 minutes, A first drying step for forming the dried hydrogel particles is to apply a first negative pressure, selected in the range of 30 μbar to 60 μbar, to the hydrogel beads frozen at the termination temperature, A second drying step in which a second negative pressure lower than the first negative pressure is applied to the dried hydrogel particles at a temperature equal to or greater than the completion temperature, The dry hydrogel particles undergo an aeration step in which they transition to atmospheric pressure according to a time-pressure function having a pressure increase interval of at least 0.5 minutes. including, Carrier particles.

12. The protein coating comprises Matrigel, collagen, laminin and / or vitronectin. The carrier particle according to claim 11.

13. The surface of the dried hydrogel particles is residue-free with respect to the lioprotectant substance. The carrier particle according to claim 11 or 12.

14. It has characteristic cross-sectional dimensions in the range of 50 μm to 2 mm. A carrier particle according to any one of claims 11 to 13.

15. A material containing at least one of magnetic particles and a physiologically active substance, A carrier particle according to any one of claims 11 to 14.

16. Use of carrier particles prepared by the method described in any one of claims 1 to 10, as carrier particles for culturing pluripotent stem cells and pluripotent cells.

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