Method and cryopreservation device for cryopreserving a plurality of cell aggregates of biological cells

Abstract

Method for cryopreserving a plurality of cell aggregates (1, 2, 3) of biological cells, comprising the steps: - fractionation of the cell aggregates (1, 2, 3) depending on at least one property of the cell aggregates (1, 2, 3) into at least two fractions (4), - Collection of the fractions (4) in different containers (21), and - cryopreservation of the cell aggregates (1, 2, 3) of at least two fractions (4), specific pretreatment and / or freezing procedures being applied for each fraction.

Description

[0001] The invention relates to a method and a cryopreservation device for cryopreserving a plurality of cell associations of biological cells (also referred to as cell aggregates), e.g., cell tissue or organoids. Applications of the invention include, for example, biomedicine and / or biotechnology.

[0002] In biotechnological / pharmacological research and in biomedicine, such as transplantation medicine, there is interest in the applications of cell aggregates composed of a variety of biological cells. In particular, cell aggregates offer the ability to replicate specific properties or functions of organs of a biological organism without forming a complete organ. Cell aggregates include, for example, native biological tissues (cell-matrix aggregates grown in vivo), spheroids (spherical cell clusters), or organoids (artificially cultured in vitro cell-matrix aggregates). The formation of cell aggregates can require cultivation periods of days, weeks, or even months, with individual cell aggregates developing at different rates. Therefore, a cultivation process typically produces inhomogeneous samples with cell aggregates at different stages of maturity or development, particularly in terms of size.

[0003] Cryopreservation of biological materials, such as cells, cell components, and / or cell groups, is a well-known process for preserving the vitality of biological materials. Freezing is performed according to predetermined freezing protocols, typically with the addition of a cryoprotectant (CPA), which prevents or suppresses the formation of ice crystals during freezing. Process parameters (conditions of pretreatment and subsequent freezing) are selected primarily depending on the properties of the biological materials (see, for example, the review article by MA Taylor et al., "New Approaches to Cryopreservation of Cells, Tissues, and Organs," in "Transfus. Med. Hemother." 46: 2019, pp. 197-215).

[0004] For the effective cryopreservation of suspensions of single cells with a high viability yield, cell-type-specific process parameters, such as excipient composition, exposure time, and cooling rate, must be optimized. This can be a significant effort and require considerable experience. When cryopreserving cell clusters, the preservation success depends even more sensitively on the choice of process parameters, so the effort required to select optimized freezing parameters also increases.

[0005] While the cryopreservation of individual cells in suspension, particularly through slow freezing, is widely used, inhomogeneous samples from extended, three-dimensional cell aggregates, such as tissue, spheroids, or organoids, of various sizes can currently only be frozen with limited yield using slow freezing. For example, the required exposure time of excipients that leads to a desired concentration within the cell aggregate increases quadratically with its diameter. Since the above-mentioned inhomogeneity, particularly polydispersity, typically occurs in conventional (scalable) production processes for cell aggregates, losses would be expected even with a preservation protocol optimally selected with regard to the cell type and a specific size due to exposure times and conditions that are unsuitable for different sizes.In conventional cryopreservation of a polydisperse sample of cell aggregates, compromises in the choice of freezing parameters must always be accepted.

[0006] It is also known to preserve smaller cell clusters by vitrification (vitrification through ultrafast cooling). However, vitrification has strict technical limitations for providing very high concentrations of excipients and for ultrafast reaching temperatures below the glass transition point at any location within the three-dimensional tissue cluster. This primarily concerns the sample volume, which is limited by the limited thermal conductivity of aqueous media (λ = 0.56 W / Km, α = 0.14 mm). 2 / s). However, the excipient concentrations and exposure times are also limited due to their cytotoxicity. Furthermore, the likelihood of damage from thermal stress cracking increases with increasing sample size. Vitrification is therefore unsuitable for the routine preservation of samples containing numerous cell clusters of various sizes.

[0007] In practice, the aforementioned limitations in the choice of freezing parameters occur not only in the cryopreservation of samples containing cell aggregates of different sizes. The inhomogeneity of a sample can also result from the occurrence of other different properties, such as different shapes or elasticities, of the cell aggregates provided together.

[0008] The object of the invention is to provide an improved method and an improved cryopreservation device for cryopreserving a variety of cell aggregates from biological cells, which avoid the disadvantages of conventional techniques. The cryopreservation of cell aggregates is to be improved, in particular with regard to the optimization of process parameters, yield, effectiveness, and / or applicability to different cell types.

[0009] This object is achieved by a method and a cryopreservation device for cryopreserving a plurality of cell clusters of biological cells having the features of the independent claims. Advantageous embodiments and applications of the invention are set out in the dependent claims.

[0010] According to a first general aspect of the invention, the above-mentioned object is achieved by a method for cryopreserving a plurality of cell aggregates of biological cells, which method comprises the steps of fractionating the cell aggregates into at least two fractions depending on at least one predetermined property of the cell aggregates, collecting the fractions in different containers and cryopreserving the cell aggregates in the at least two fractions, wherein specific pretreatment methods and / or freezing methods are applied for each fraction.

[0011] According to a second general aspect of the invention, the above-mentioned object is achieved by a cryopreservation device which is designed for cryopreserving a plurality of cell aggregates of biological cells and comprises a fractionation device which is designed for fractionating the cell aggregates into at least two fractions depending on at least one predetermined property of the cell aggregates, a container device with at least two different containers, each arranged for collecting one of the fractions, and a freezing device which is designed for cryopreserving the cell aggregates in the at least two fractions, wherein the freezing device is designed for applying specific pretreatment methods and / or freezing methods for each of the fractions.Preferably, the cryopreservation device or one of its embodiments is adapted to carry out the cryopreservation method according to the first general aspect of the invention or an embodiment of the method.

[0012] According to the invention, the cell clusters are fractionated into at least two fractions depending on at least one predetermined property of the cell clusters. Typically, up to 5 or up to 10 fractions are formed. However, more, e.g., up to 20 or more fractions, can also be provided.

[0013] Advantageously, the fractionation of the cell clusters creates at least two homogeneous fractions, for each of which the cryopreservation process parameters can be optimized. The inventors have discovered that optimal cryopreservation process parameters depend not only on the cell type, but also on the properties of a cell cluster itself, such as the size of the cell cluster. The inventors have further discovered that, in conventional processes, different sizes of cell clusters within an inhomogeneous fraction result in different distributions of excipients and water within the cell cluster for each size, thus influencing freezing differently, which has a detrimental effect on the success and yield of cryopreservation. The creation of homogeneous fractions according to the invention overcomes the limitations of conventional processing of inhomogeneous fractions.

[0014] The provision of homogeneous fractions has a further advantage for applications involving cell aggregates, e.g., for research purposes or for implantation treatment, where there is interest in fractions containing cell aggregates at the same or similar stages of maturity or development. Such fractions can be formed by obtaining identical cell aggregates for a desired purpose through a cultivation process, subjecting them to cryopreservation, and storing them in a frozen state.

[0015] The term "cell cluster" refers to a coherent, preferably three-dimensional, group of living biological cells, such as a tissue (especially a tissue model), a spheroid, or an organoid. The cell cluster can consist exclusively of cells or, in addition to the cells, also contain extracellular matrix substances. Cell clusters are obtained, for example, by culturing biological cells and / or by extraction from an organism. The term "fraction" refers to a large number of cell clusters in a liquid environment.

[0016] Fractionation involves the separation (sorting, separation process) of cell clusters from an initially inhomogeneous sample into a predetermined number of fractions. The separation is performed in such a way that each of the fractions contains cell clusters, and the cell clusters in each of the fractions exhibit at least one identical property. This means that the cell clusters in each of the fractions are identical with respect to at least one property, or have such minor differences that they do not affect cryopreservation, in particular the selection of optimal parameters for the pretreatment and freezing processes.

[0017] Each of the fractions is homogeneous with respect to at least one of the properties under consideration. Fractionation is, in particular, a separation process that preferably does not alter the cell aggregates. In particular, the cell aggregates are preserved during fractionation, meaning the cell aggregates are not broken down into parts.

[0018] The fractionation device preferably comprises a separation device which is designed to receive a composition of the cell aggregates in an ambient medium, to separate the cell aggregates into the various fractions and to deliver the fractions into the various containers.

[0019] According to the invention, the fractions are collected in different containers, i.e., fractionation involves separation into different containers. Each container generally contains a receptacle for the fraction, which typically contains the same cell aggregates and a liquid surrounding medium, such as a nutrient medium. The receptacles of the different containers are separated from each other.

[0020] Preferably, the fractions are collected in the containers in which cryopreservation will subsequently take place. This advantageously simplifies the fractionation-cryopreservation process and the setup of the cryopreservation device, and prevents any undesirable effects on the cell aggregates after fractionation.

[0021] During cryopreservation, the separated fractions obtained during fractionation are frozen. Alternatively, the fractions are subjected to a medium exchange and / or enrichment of the cell aggregates in the medium prior to freezing.

[0022] The cryopreservation of cell clusters comprises a pretreatment process and a subsequent freezing process. The pretreatment (or incubation) of the cell clusters comprises preparing the cell clusters for freezing, whereby, for example, the composition of the liquid surrounding medium with excipients or CPA (the totality of all additives used to improve the preservation result), the volume of the fraction, the density of the cell clusters in the fraction and / or other pretreatment parameters are adjusted and / or the cell clusters are modified using physical and / or chemical processes, e.g. permeabilized. The pretreatment of the cell clusters is preferably carried out at a temperature at which the surrounding medium is liquid, in particular at room temperature. Freezing comprises lowering the temperature of the fraction below 0°C to a cryopreservation temperature, e.g. in the range of -80°C to -200°C. Freezing parameters for freezing include, for example:B. the time course of the temperature reduction and the set cryopreservation temperature.

[0023] The pretreatment and freezing processes for cryopreservation are carried out in a conventional manner. However, according to the invention, specific pretreatment and / or freezing processes are used for the cryopreservation of the cell clusters of the at least two fractions. Different process parameters, in particular pretreatment and / or freezing parameters, are provided for each fraction. Process parameters are selected for each fraction to optimize the cryopreservation of the cell clusters, in particular with maximum preservation of vitality and / or functionality.

[0024] The application of cryopreservation process parameters involves the adjustment of preselected pretreatment and freezing parameters. Optimal pretreatment and freezing parameters can be determined through test series with cell clusters and / or from reference experiments from the specialist literature. The inventive selection of sample-specific cryopreservation process parameters can advantageously improve the yield, effectiveness, and / or applicability of cryopreservation for different cell types.

[0025] After freezing to the cryopreservation temperature, the frozen fractions are transferred to a cryobank for storage, preferably without interrupting the cold chain. In the cryobank, storage takes place at a storage temperature that may differ from the cryopreservation temperature.

[0026] Advantageously, a variety of properties are available on the basis of which the fractionation of the cell clusters can be carried out. According to preferred embodiments of the invention, the fractionation of the cell clusters takes place depending on at least one of the properties, which include size, shape, mass, elasticity, hydraulic conductivity, permeability to cryoprotectants (CPA), resistance to cryoprotectants, chemical nature, and cell composition of the cell clusters. The fractionation device is accordingly preferably configured for fractionation based on at least one of these properties. The above physical and chemical properties have advantageously proven to be particularly well suited for effective fractionation and for the selection of optimized process parameters for cryopreservation.

[0027] The fractionation of the cell clusters can be performed depending on several properties, for example, size and CPA permeability. When fractionating according to several properties, a multi-stage fractionation is preferably provided, in which a first property, e.g., the size of the cell clusters, is tested in a first stage, and at least one further property, e.g., CPA permeability, is tested in at least one further stage.

[0028] Size-dependent fractionation is particularly preferred. Numerous gentle separation methods are available for size-dependent separation. The effectiveness of cryopreservation process parameters can be particularly sensitive to the size of a cell cluster. The size of a cell cluster includes, for example, its cross-sectional dimension, in particular diameter, or another characteristic geometric dimension of the cell cluster that affects mass transport and / or the freezing process. Alternatively or additionally, fractionation into fractions with specific shapes of the respective cell clusters is particularly preferred. The shape of a cell cluster is the geometric shape that the cell cluster at least approximately has in the surrounding medium, such as a spherical shape, an elongated cylindrical shape, or an irregular shape.

[0029] During cryopreservation, each fraction undergoes a pretreatment characterized by fraction-specific pretreatment parameters. According to further preferred embodiments of the invention, the pretreatment methods for the fractions differ with respect to at least one of the pretreatment parameters, which include a duration, a temperature, a pressure, a media composition, a gassing composition, permeabilization conditions, and a media movement of the pretreatment. These pretreatment parameters have advantageously proven to be particularly well-suited for preparing the cell aggregates for effective cryopreservation with high yield.

[0030] Advantageously, according to further modifications of the invention, the pretreatment methods for the fractions can differ with respect to the time course of at least one of the pretreatment parameters. The pretreatment methods can be characterized by different time dependencies of the pretreatment parameter. This time dependency advantageously provides an additional degree of freedom for optimizing the pretreatment.

[0031] According to further preferred variants of the invention, the freezing methods for the fractions differ with respect to at least one of the freezing parameters, which include a duration, in particular cooling rate, a temperature, a pressure, a media composition, a gassing composition, and a media movement during freezing. Accordingly, the freezing device is preferably configured to apply the freezing methods with at least one of the aforementioned freezing parameters. Advantageously, this provides a multitude of parameters that can be used to optimize cryopreservation for different fractions, each with the same cell clusters.

[0032] If, according to a further preferred embodiment of the invention, the fractionation of the cell aggregates comprises a fluidic fractionation in which the cell aggregates are separated in a fluidic environment, further advantages arise since the cell aggregates can be kept in a liquid ambient medium from their provision, in particular cultivation, until freezing and a temporary transition to a gaseous or vaporous environment is avoided.

[0033] According to an advantageous embodiment of the invention, fractionation and cryopreservation are automated. The cryopreservation device is configured for automated operation, in particular operation without intervention by an operator of the cryopreservation device. Automation offers advantages in terms of avoiding process errors, the reproducibility and accuracy of setting process parameters, and the possibility of performing automated high-throughput fractionation into separate fractions with the subsequent cryopreservation of the separated fractions at high speed and high throughput.

[0034] If the fractionation of the cell clusters according to a further modification of the invention comprises fractionation, in particular size fractionation, in a fluidic flow, the cell clusters are arranged at different positions in a flow profile of the fluidic flow under the effect of at least one of flow forces of the fluidic flow, dielectrophoretic forces in the fluidic flow, and sound waves in the fluidic flow. The flow profile of the fluidic flow comprises the location-dependent distribution of the flow velocity in the cross-section of the flow. The different positions in the flow profile introduce a separation of the cell clusters into different flow paths in the flow. The transfer to the different containers takes place by directing the individual parts of the flow profile via separate partial flows, e.g.Partial channels of a fluidic system, and / or with different directions into the containers.

[0035] Preferably, the fluidic system of the fractionation device is a fluidic microsystem comprising channels and fluidic elements, such as branches or intersections, with characteristic cross-sectional dimensions of less than 2 mm. The fluidic flow is particularly preferably a parallel, vortex-free flow, which advantageously improves the separation within the flow and allows the parts of the flow profile to be directed into the containers at a distance from the separation of the cell clusters to the various positions in the flow profile.

[0036] Fractionation using fluid flow forces is a form of size fractionation using passive fluidics. Cell clusters arrange themselves at different positions in the flow profile according to their size and can thus be separated. Passive fluidics has the following advantages: It is a contactless process that places minimal stress on the cell clusters and does not require size sensors. Inhomogeneous mixtures of cell clusters can be added in portions and separated via transit time differences (chromatographic principle, field flow fractionation). However, continuous processes such as pinched flow fractionation (PFF) are preferred, as they can be designed with simpler equipment and are more scalable. In PFF, for example, cell clusters of different sizes exit an outlet, particularly a nozzle section, at different angles.

[0037] Fractionation using dielectrophoretic forces in the fluidic flow is a size fractionation or a fractionation based on an electrical property of the cell clusters through active fluidics, such as a separation based on dielectric properties of the cell clusters, such as polarizability or surface charge. For these separation processes, the fractionation device is preferably equipped with an electrode device designed to exert dielectrophoretic forces in a fluidic flow. The electrode device comprises, for example, at least one electrode which, when subjected to an alternating voltage, generates a dielectrophoretic field barrier that forms a deflection angle (other than 0°) with the flow direction in the fluidic system. The height of the dielectrophoretic field barrier acting on a cell cluster depends on the size of the cell cluster.Dielectrophoretic forces acting on the cell clusters perpendicular to the flow direction are superimposed on flow forces within the flow. Depending on their size and / or dielectric properties and the flow forces, cell clusters can pass the electrodes at various positions and be positioned accordingly within the flow profile. This advantageously also provides a contactless process that does not require upstream sensors. However, the equipment required is higher than with passive fluidics.

[0038] Alternatively, fractionation using dielectrophoretic forces can be combined with a sensor system. A sensor device configured to detect at least one property of the cell clusters can be arranged upstream of the electrode device. The electrode device is controlled in accordance with an output signal from the sensor device such that the individual cell clusters are directed to different positions in the flow profile depending on the detected property.

[0039] If fractionation is performed using sound waves in the fluid flow, another variant of size fractionation is provided by active fluidics. Acoustic fields (standing and / or traveling sound waves) of suitable frequencies and the ability of inert bodies to gather at the field minima of the sound waves are used for fractionation. In this case, too, a contactless process is provided that does not require any upstream sensors. The size range to which acoustic fractionation can be applied is advantageously larger than that for dielectrophoretic fractionation. For this separation process, the fractionation device is preferably equipped with a sound source device designed to generate sound waves in the fluid flow.

[0040] According to a further preferred embodiment of the invention, the at least one property of the cell aggregates and / or at least one state variable of the at least two fractions are detected by sensors. Accordingly, the cryopreservation device is preferably equipped with a sensor device configured to detect the at least one property of the cell aggregates and / or at least one state variable of the at least two fractions. Particularly preferably, the at least one property of the cell aggregates is detected by sensors immediately before fractionation, and the at least one state variable of the fractions is detected by sensors immediately before cryopreservation. The detection of the at least one property of the cell aggregates before fractionation advantageously expands the group of properties of the cell aggregates on the basis of which fractionation takes place.Sensor technology for at least one state variable of the fractions prior to cryopreservation offers the advantage of further optimizing the cryopreservation process parameters depending on the state of the fraction. State variables of the fractions include, for example, the density or the size of the cell clusters.

[0041] Another particularly important advantage of the invention for the further use of cell clusters after cryopreservation is that the vitality-preserving thawing can also be carried out fraction-specifically. According to an advantageous variant of the invention, the thawing of the cell clusters of at least two fractions is carried out in such a way that specific thawing procedures are applied for each fraction. Thawing parameters, like the cryopreservation process parameters, are optimized separately for the individual fractionated fractions, which allows for an increase in the vitality rate of the thawed cell clusters.

[0042] In general, a method for the vitality-preserving thawing of at least two fractions obtained by fractionating cell aggregates depending on at least one property of the cell aggregates and frozen, wherein a specific thawing method is applied for each fraction, and a thawing device configured to carry out the method can be considered as further independent objects of the present invention.

[0043] The features disclosed in connection with the method for cryopreserving a plurality of cell aggregates of biological cells and its embodiments also represent preferred features of the cryopreservation device or its embodiments. The above-mentioned aspects and inventive and preferred features, in particular with regard to the method, therefore also apply to the cryopreservation device and its components.

[0044] Further details and advantages of the invention are described below with reference to the accompanying drawings. They show schematically: Fig. 1: a sequence of the method for cryopreserving a plurality of cell aggregates and components of a cryopreservation device with features according to preferred embodiments of the invention; and Fig. 2: a fractionation device configured for dielectrophoretic separation of cell aggregates according to an embodiment of the invention.

[0045] Features of preferred embodiments of the invention are described below with exemplary reference to the application of size fractionation of cell aggregates. It is emphasized that the practical implementation of the invention is not limited to size fractionation, but is alternatively or additionally possible with fractionation based on another property of the cell aggregates, as described below with further examples. Details of the cell aggregates and their provision, and the cryopreservation and / or thawing process parameters applied in specific examples, are selected as is known per se from the cryopreservation of biological materials.

[0046] Fig. Figure 1 shows steps S1 to S4 of the method for cryopreserving a plurality of cell aggregates 1, 2 and the cryopreservation device 100 used for this purpose, comprising a fractionation device 10, a container device 20, and a freezing device 30 according to preferred embodiments of the invention. In addition, Fig. 1 a step S0 of providing the cell clusters 1, 2 and a step S5 of storing the frozen cell clusters in a cryobank 40. With the in Fig. In the example shown in Figure 1, for example, an automated, fluidic size fractionation of the cell clusters 1, 2 is carried out.

[0047] In step S0, an inhomogeneous sample, e.g., a mixture of cell aggregates 1, 2 of different sizes and / or a mixture of cell aggregates 1, 2 with different sensitivities to CPA, is provided. The cell aggregates 1, 2 comprise, e.g., organoids formed from adult stem cells in a conventional manner by cultivation in a nutrient medium and with differentiation factors and, e.g., having cross-sectional dimensions in the range of 10 µm to 10 mm or larger. The cell aggregates 1, 2 are provided, e.g., in a culture vessel.

[0048] In step S1, the cell clusters 1, 2 are separated into individual fractions 4 (fractions) using the schematically shown fractionation device 10, each containing cell clusters of specific sizes. The fractionation device 10 is constructed, for example, as described below with reference to Fig. 2, and it is preferably operated automatically. The fractions 4 are transferred into containers 21 of the container device 20 in step S2. The containers 21 preferably comprise plastic tubes with a lid, in particular so-called PP tubes, as used in the subsequent cryopreservation in steps S3 and S4 (see illustration in step S5). Alternatively, the containers can comprise other receptacles, such as bags or microtiter plates. According to a further alternative, the containers 21 can be part of the incubation unit 31 of the freezing device 30. The individual fractions 4 are collected in the containers 21 provided for storage until a predetermined load quantity, in particular concentration (mass of cell aggregates per volume of the surrounding medium), is reached.

[0049] The freezing device 30 comprises an incubation unit 31 and a cooling unit 32. In the freezing device 30, the individual fractions 4 are subjected to a pretreatment and freezing protocol adapted to the respective size.

[0050] In incubation unit 31, the fractions are pretreated. This means that a completely individual incubation program runs for each fraction 4. At least one CPA (in particular, a cryoprotectant) is added to be loaded into the cell clusters. As the cell clusters grow in size, increasing concentrations of CPA and / or increasing incubation times are used. Suitable cryoprotectants and their concentrations can be determined through testing.

[0051] The incubation may further include a predetermined temperature T(t), gassing, and / or perfusion with predetermined cryoprotectant (CPA) concentration profiles C(t, CPA1, CPA2, ...). Alternatively or additionally, membrane-permeable and / or even toxic CPAs can be temporarily added if the cell aggregates tolerate them. Furthermore, ice nucleation (to reduce and control hypothermia), media circulation (to homogenize T and C), and / or permeabilization of the cell aggregates of at least one fraction (to load with non-membrane-permeable CPAs) may be part of the pretreatment process. Permeabilization can be achieved, for example, chemically (e.g., using DMSO), with sound waves (sonoporation), with electric fields (electroporation), using liposomal substances, and / or through thermomodulation via membrane phase transition.Furthermore, the pretreatment in the incubation unit 31 comprises pre-cooling of the fractions 4 to a temperature above the freezing point of the fractions 4.

[0052] The incubation unit 31 preferably has individual receptacles for the containers 21, e.g., individual cavities, or provides the containers via reservoirs, preferably for fractions of equal volumes. The incubation unit 31 contains a pumping device for supplying CPA (sequential addition and / or concentration increase) and / or for removing media from the containers. Furthermore, the incubation unit 31 is preferably equipped with a drive, such as an agitator, for moving the media during pretreatment in each container.

[0053] Alternatively or additionally, a pre-cooling unit can be provided, which is designed to supercool fractions. This supercooling can induce membrane changes in the cells of the cell clusters, which can influence the pretreatment, e.g., the uptake of CPAS. Alternatively or additionally, a sound source can be provided with which the cell clusters of the fractions can be subjected to ultrasonic treatment. The ultrasonic treatment can induce further membrane changes in the cells of the cell clusters, in particular permeabilization.

[0054] Further pretreatment parameters of the size-dependent incubation include, for example, concentrations of the individual CPAs, exposure times of the individual CPAs, temporal concentration profiles of individual CPAs, adapted temperature profiles (> 0°C), continuous changes in the media composition, e.g., by means of mixing devices in conjunction with the incubation unit 31 and a CPA reservoir, and / or a change of the ambient medium (perfusion).

[0055] Fractions 4 are then frozen in the cooling unit 32 (step S4). Depending on the properties of the cell aggregates in fractions 4, such as the size or other properties such as the hydraulic conductivity of the individual components of the cell aggregates, the proportion of membrane-permeable and osmotically active additives in the medium, and / or the degree of subcooling, the individual fractions 4 are frozen in a controlled manner at different cooling rates and / or cooling sequences. For example, a cooling rate equal to or less than -1°C / min is used. Cooling is carried out to a cryopreservation temperature of, for example, -80°C or below, such as -140°C or below.

[0056] Temperature profiles for freezing individual fractions can be selected, for example, to adjust to an equilibration rate, a controlled nucleation to reduce subcooling, and / or a homogeneous cooling rate across the fraction volume (if necessary with circulation of the medium and / or with the use of a form-fitting fit of the fraction containers in the heat exchanger of the cooling unit 32).

[0057] The cooling unit 32 comprises a cooling chamber for each fraction with at least one cooling element and a heat exchanger. The cooling element is, for example, a Peltier element, a Stirling cooler, or a coolant flow cooler that operates, for example, with liquid nitrogen or isopentane. The cooling element is designed to set a defined cooling rate. The heat exchanger comprises, for example, a receptacle for the container of the respective fraction, preferably with a form fit between the container and the receptacle. If the containers 21 are part of the incubation unit 31 of the freezing device 30, a transfer to cryogenic containers, such as the aforementioned PP tubes, takes place before freezing. The cooling unit 32 can optionally be equipped with a nucleation device, for example, a cold needle, with which controlled nucleation is induced in the container.

[0058] Finally, the containers are closed and the frozen fractions are stored at cryogenic temperatures (e.g., -140 °C) in the cryobank 40 (step S5). Transfer to the cryobank 40 occurs without interrupting the cold chain, e.g., using a refrigerated airlock or by directly coupling the freezing device 30 to the cryobank 40.

[0059] To defrost, the Fig. 1 is reversed with a thawing device (not shown). During thawing, similar to the size-adjusted process during freezing, the individual fractions are treated separately in different incubation units during thawing and / or in an initial recovery phase until the excipients are washed out. For example, a size-dependent thawing rate, a size-dependent incubation in a hyperosmolar thawing medium, and / or a size-dependent washout of the thawed fractions are provided. This allows, for example, the metabolism of large cell aggregates to be slowed by a cooled environment to ensure sufficient dilution of toxic, membrane-permeable CPAs, whereas this process can proceed more quickly with small cell aggregates.

[0060] After thawing, a portioning step can be provided in which the thawed fractions are subjected to a viability test and, if viability is determined, transferred to a predetermined, application-specific container format, such as microtiter plates or suspension bioreactors. Size fractionation can be retained or abandoned.

[0061] Thawing and / or portioning can be carried out, for example, using a fluidic device, in particular a fluidic microsystem.

[0062] Fig. Figure 2 shows, by way of example, a fractionation device 10 in the form of a fluidic device 11, in particular a fluidic microsystem, with a main channel 11A and branch channels 11B through which a suspension of a liquid ambient medium containing cell clusters 1, 2, 3 of various sizes flows in the direction of arrow A. Located in the main channel 11A are an electrode device 12 and a sensor device 14, which are connected to a control device 13. The main channel 11A branches into the branch channels 11B, each of which is connected to one of the containers 21 of the container device 20.

[0063] The electrode device 12 comprises two ribbon-shaped electrodes or electrode pairs, e.g., on the bottom and / or a cover plate of the main channel 11A. When the electrodes are subjected to alternating voltages from the control device 13, the electrode device 12 can generate a field barrier transverse to the flow A. The field barrier can be temporarily generated to match a cell cluster arriving with the flow. Through the interaction of the field barrier with the flow forces in the flow A, cell clusters can be directed onto a predetermined flow path leading to one of the branch channels 11B (see, e.g., dotted flow path B of cell cluster 1).

[0064] The sensor device 14 is, for example, an optical sensor, in particular a camera in conjunction with an image processing device. The sensor device 14 can detect the cell clusters 1, 2, 3 and their respective sizes. Information about the positions and sizes of the cell clusters 1, 2, 3 is supplied to the control device 13. The control device 13 assigns the cell clusters 1, 2, 3 to three predetermined sizes of the desired fractions and controls the electrode device 12 such that the cell clusters 1, 2, 3 are each directed into one of the branch channels 11B, and via this into one of the containers 21, depending on the size.

[0065] Alternatively to the embodiment in Fig. 2, the electrode device 12 can comprise a dielectrophoretic field cage, and the sensor device 14 can be configured to detect a cell cluster in the field cage. The cell clusters are sequentially detected by sensors in the field cage, assigned to one of several fractions depending on their properties, and guided into the respective fraction by releasing the field cage and, if necessary, further dielectrophoretic deflections.

[0066] Alternatively or in addition to size fractionation using dielectrophoretic forces, at least one of the following separation methods can be used for fractionation. Passive separation methods can include, for example, flow profile fractionation (e.g., PFF), density fractionation (e.g., sedimentation), and geometric fractionation (e.g., using sieves). Active separation methods can include, for example, acoustic separation (e.g., using standing ultrasonic waves) or optical separation (e.g., using optical tweezers).

[0067] As an alternative to optical sensors, an impedance measurement can be provided on the cell clusters (e.g. as in a Coulter Counter device) and the fractionation can be carried out depending on the result of the impedance measurement.

[0068] The with reference to the Fig. 1 and Fig.The size fractionation described in section 2 can be supplemented or replaced by fractionation based on other properties. For example, a test for the permeability of cell aggregates to cryoprotectants and / or resistance to cryoprotectants can be performed in a fluidics device of a fractionation device, e.g., in a field cage of the fluidics device. Depending on the test result, different fractions can be formed, which are subsequently subjected to cryopreservation using different process parameters. For example, cell aggregates with low CPA permeability are treated with a longer CPA incubation period than cell aggregates with increased CPA permeability.

[0069] The features of the invention disclosed in the above description, the drawings and the claims may be important both individually and in combination or sub-combination for the realization of the invention in its various forms.

Claims

[1] Method for cryopreserving a plurality of cell aggregates (1, 2, 3) of biological cells, comprising the steps: - fractionation of the cell aggregates (1, 2, 3) depending on at least one property of the cell aggregates (1, 2, 3) into at least two fractions (4), - Collection of the fractions (4) in different containers (21), and - cryopreservation of the cell aggregates (1, 2, 3) of at least two fractions (4), specific pretreatment and / or freezing procedures being applied for each fraction. [2] Method according to claim 1, in which - the fractionation of the cell aggregates (1, 2, 3) is carried out depending on at least one of the properties comprising a size, a shape, a mass, an elasticity, a hydraulic conductivity, a permeability to cryoprotectants (CPA), a resistance to cryoprotectants, a chemical nature and a cell composition of the cell aggregates (1, 2, 3). [3] Method according to one of the preceding claims, in which - the pretreatment processes for the fractions (4) differ with respect to at least one of the pretreatment parameters, which include a duration, a temperature, a pressure, a media composition, a gassing composition, permeabilization conditions and a media movement of the pretreatment. [4] Method according to claim 3, wherein - the pretreatment procedures for the fractions (4) differ with respect to the time course of at least one of the pretreatment parameters. [5] Method according to one of the preceding claims, in which - the freezing methods for the fractions (4) differ with respect to at least one of the freezing parameters, which include a duration, a temperature, a pressure, a media composition, a gassing composition and a media movement of the freezing. [6] Method according to one of the preceding claims, with at least one of the features - the fractionation of the cell aggregates (1, 2, 3) comprises a fluidic fractionation in which the cell aggregates (1, 2, 3) are separated in a fluidic environment, - the fractions (4) are collected in the containers (21) in which cryopreservation subsequently takes place, - the frozen fractions (4) are made available for storage in a cryobank (40) without interrupting the cold chain, and - fractionation and cryopreservation are automated. [7] Method according to one of the preceding claims, in which - the fractionation of the cell clusters (1, 2, 3) comprises a fractionation in a fluidic flow, wherein the cell clusters (1, 2, 3) are arranged at different positions in a flow profile of the fluidic flow under the effect of at least one of flow forces of the fluidic flow, dielectrophoretic forces in the fluidic flow, and sound waves in the fluidic flow. [8] Method according to one of the preceding claims, in which - at least one property of the cell aggregates (1, 2, 3) and / or at least one state variable of the at least two fractions (4) are detected by sensors. [9] Method according to one of the preceding claims, comprising the further step - Thawing the cell aggregates (1, 2, 3) in at least two fractions (4), using specific thawing procedures for each fraction. [10] Cryopreservation device (100) adapted for cryopreserving a plurality of cell aggregates (1, 2, 3) of biological cells, comprising: - a fractionation device (10) which is designed to fractionate the cell aggregates (1, 2, 3) into at least two fractions (4) depending on at least one property of the cell aggregates (1, 2, 3), - a container device (20) with at least two different containers (21), each arranged to collect one of the fractions (4), and - a freezing device (30) which is designed to cryopreserve the cell aggregates (1, 2, 3) in the at least two fractions (4), wherein the freezing device (30) is designed to apply specific pretreatment methods and / or freezing methods for each of the fractions (4). [11] Cryopreservation device according to claim 10, wherein - the fractionation device (10) is designed to fractionate the cell aggregates (1, 2, 3) depending on at least one of the properties comprising a size, a shape, a mass, an elasticity, a hydraulic conductivity, a permeability to cryoprotectants (CPA), a resistance to cryoprotectants, a chemical nature and a cell composition of the cell aggregates (1, 2, 3). [12] Cryopreservation device according to claim 10 or 11, wherein - the freezing device (30) is designed to apply the pretreatment methods which differ with respect to at least one of the pretreatment parameters and / or its time course, which include a duration, a temperature, a pressure, a media composition, a gassing composition, permeabilization conditions and a media movement during the pretreatment. [13] Cryopreservation device according to claim 10 to 12, wherein - the freezing device (30) is arranged to apply the freezing methods which differ with respect to at least one of the freezing parameters, which include a duration, a temperature, a pressure, a media composition, a gassing composition and a media movement during freezing. [14] Cryopreservation device according to claim 10 to 13, having at least one of the features - the fractionation device (10) comprises a fluidic device (11) which is designed to separate the cell clusters (1, 2, 3) in a fluidic environment, in particular in a fluidic flow, - the at least two containers (21) are part of the freezing device (30), and - the cryopreservation device (100) is designed for automated operation. [15] Cryopreservation device according to claim 10 to 14, wherein - the fractionation device (10) comprises an electrode device (12) which is designed to exert dielectrophoretic forces in a fluidic flow and / or a sound source device which is designed to generate sound waves in the fluidic flow. [16] Cryopreservation device according to claims 10 to 15, comprising - a sensor device (14) which is designed to detect at least one property of the cell aggregates (1, 2, 3) and / or at least one state variable of the at least two fractions (4).

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