Method for cryopreservation of multiple cell clusters formed from biological cells

Abstract

A method for cryopreserving a plurality of cell clusters (1, 2) formed of biological cells comprises the steps of fractionating the cell clusters (1, 2) into at least two fractions (4) according to at least one characteristic of the cell clusters (1, 2), collecting the fractions (4) in different containers (21), and cryopreserving the cell clusters (1, 2) of the at least two fractions (4), wherein a specific pretreatment method and / or freezing method is applied to each fraction. A cryopreservation device (100) for cryopreserving a plurality of cell clusters (1, 2) formed of biological cells is also described, comprising a fractionation mechanism.

Description

Technical Field

[0001] The present invention relates to a method and a cryopreservation device for cryopreserving a plurality of cell clusters (also referred to as cell clumps) formed by biological cells, such as cell tissues or organoids. The present invention has applications, for example, in biomedicine and / or biotechnology. Background Art

[0002] In the research of biotechnology / pharmacology and in the biomedicine such as transplant medicine, the application of the cell cluster formed by multiple biological cells is interested.Cell cluster especially provides the ability of the specific characteristics or function of the organoid of simulation organism, without the need to form a complete organ.Cell cluster includes for example biological primary tissue (cell matrix cluster of in vivo growth), spheroid (cell cluster of spheroid) or organoid (cell matrix cluster of in vitro artificial culture).The formation of cell cluster needs the cultivation of several days, several weeks or even several months, and wherein the developmental speed of each cell cluster is different.Therefore, as the result of culture method, usually produce and have in different maturation stages or developmental stage, especially the inhomogeneous sample of the cell cluster of different sizes.

[0003] Cryopreservation of biological materials such as cells, cell components and / or cell groups is a generally known method by which biological materials are frozen in a manner that maintains vitality. Freezing is carried out according to a predetermined freezing scheme, usually with the addition of a cryoprotectant (cryoprotectant, CPA), which is used to prevent or inhibit the formation of ice crystals during freezing. In particular, process parameters (pretreatment conditions and subsequent freezing conditions) are selected according to the characteristics of the biological material (see, for example, MA Taylor et al. in Transfus. Med. Hemother 46: 2019, 197-215, in the article "New Approaches to Cryopreservation of Cells, Tissues and Organs (a new method for deep cryopreservation of cells, tissues and organs)".

[0004] In order to effectively cryopreserve suspensions of individual cells with a high viability yield, it is necessary to optimize the process parameters, such as the composition of the auxiliary agents, the duration of action, and the cooling rate, for the cell type. This is very time-consuming and requires a great deal of experience. In the cryopreservation of cell clusters, the success of the preservation is even more sensitive to the selection of the process parameters, so that the effort required to select the optimized freezing parameters is also increased.

[0005] Although especially by slow freezing, individual cells in suspension are carried out to cryopreservation extensively, up to now, can only freeze the heterogeneous sample that is formed by the cell cluster of three-dimensional extension with different sizes, cell cluster such as tissue, spheroid or organoid by slow freezing under the situation of limited yield.For example, the necessary duration of action that causes the desired concentration of auxiliary agent inside cell cluster increases with the square of the diameter of cell cluster.Because above-mentioned heterogeneity, especially polydispersity usually occur in the common (expandable) production method of cell cluster, even in the preservation scheme that is best selected in view of cell type and certain size, also can expect the loss that causes due to duration of action and action condition that are not suitable for different sizes.Therefore, up to now, in the traditional cryopreservation of the polydisperse sample of cell cluster, always must accept compromise when selecting freezing parameters.

[0006] It is also known to preserve smaller cell clusters by vitrification (vitrification achieved by ultrafast cooling). However, vitrification has narrow technical limitations in order to provide very high concentrations of the additive and to ultrafastly reach temperatures below the glass point at every location in a three-dimensional tissue cluster. This is primarily related to the sample volume, which is limited upwards due to the limited thermal conductivity of the aqueous medium (λ = 0.56 W / km, α = 0.14 mm). 2 / s). However, the concentration of adjuvants and the duration of their action are also limited due to their cytotoxicity. In addition, the possibility of thermal stress cracking damage increases with increasing sample size. Therefore, vitrification is not suitable for routine storage of samples containing multiple cell clusters of different sizes.

[0007] In practice, the aforementioned limitations in the selection of freezing parameters do not only arise when cryopreserving samples with cell clusters of varying sizes. Sample inhomogeneities can also result from the presence of other different characteristics of the coexisting cell clusters, such as different shapes or elasticities. Summary of the Invention

[0008] The object of the present invention is to provide an improved method and an improved cryopreservation device for cryopreserving a plurality of cell clusters formed from biological cells, which avoid the disadvantages of conventional technologies. In particular, the cryopreservation of cell clusters should be improved with regard to 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 formed from biological cells having the features of the independent claims. Advantageous embodiments and uses of the invention are revealed in the dependent claims.

[0010] According to a first general aspect of the present invention, the above-mentioned object is achieved by a method for deep cryopreservation of cell clusters formed by biological cells, the method comprising the steps of: fractionating the cell clusters into at least two components according to at least one predetermined characteristic of the cell clusters, collecting the components in different containers, and deep cryopreserving the cell clusters into at least two components, wherein a specific pretreatment method and / or freezing method is applied to each component.

[0011] According to a second general aspect of the present invention, the above-mentioned object is achieved by a cryopreservation device configured for cryopreserving a plurality of cell clusters formed from biological cells, the cryopreservation device comprising a sorting mechanism, a container mechanism, and a freezing mechanism, the sorting mechanism being configured for sorting the cell clusters into at least two fractions based on at least one predetermined characteristic of the cell clusters, the container mechanism comprising at least two different containers, each of which is arranged to collect one of the fractions, the freezing mechanism being configured for cryopreserving the cell clusters into the at least two fractions, wherein the freezing mechanism is configured for applying a specific pretreatment method and / or freezing method to each of the fractions. Preferably, the cryopreservation device, or one of its embodiments, is configured for implementing the method for cryopreservation according to the first general aspect of the present invention, or an embodiment of the method.

[0012] According to the invention, the cell clusters are classified into at least two components according to at least one predetermined characteristic of the cell clusters. Typically, up to 5 or up to 10 components are formed. However, more components, for example up to 20 or more components, can also be provided.

[0013] Advantageously, by fractionating the cell clusters, at least two homogeneous components are obtained, and the process parameters for cryopreservation can be optimized for each of these components. The inventors have found that the optimal process parameters for cryopreservation are not only related to the cell type, but also to the characteristics of the cell cluster itself, such as the size of the cell cluster. The inventors have also found that in traditional methods, cell clusters of different sizes within the heterogeneous components cause the adjuvant and water to be distributed differently in the cell cluster at each size, and thus affect freezing differently, which has an adverse effect on the success and yield of cryopreservation. By providing a homogeneous component according to the present invention, the limitations of traditional processing of heterogeneous components are overcome.

[0014] Providing a homogeneous composition also facilitates the use of cell clusters, for example for research purposes or for implantation treatments, where a composition having cell clusters at the same or similar stage of maturation or development is of interest. Such a composition is formed by obtaining identical cell clusters for the desired purpose by a culture method, cryopreserving them, and storing them in a frozen state.

[0015] The term "cell cluster" refers to an associated, preferably three-dimensionally extended group of cells formed by living biological cells, such as a tissue (especially a tissue model), a spheroid, or an organoid. A cell cluster can be composed solely of cells, or can contain extracellular matrix materials in addition to cells. Cell clusters are provided, for example, by culturing biological cells and / or by extracting from an organism. The term "component" refers to a plurality of cell clusters in a liquid environmental medium.

[0016] Grading comprises separating (sorting, separation process) cell clusters into a predetermined number of components from an initially non-uniform sample. Separation is achieved so that each of the components contains cell clusters, and the cell clusters in each of the components have at least one identical feature. This means that the cell clusters in each of the components are identical or have very little difference in at least one feature, so that the difference does not affect cryopreservation, especially does not affect the selection of optimal parameters for pretreatment methods and freezing methods. Each of the components is similar in at least one feature considered. Grading is especially preferably not changing the separation method of the cell clusters. During grading, the cell clusters are especially maintained, that is, the cell clusters are not broken down into parts.

[0017] The fractionation device preferably comprises a separation device which is provided for receiving the components of the cell clusters in a surrounding medium, for separating the cell clusters into different components, and for discharging the components into different containers.

[0018] According to the present invention, the components are collected in different containers, i.e., fractionation involves separation into different containers. Each container generally includes a receptacle for the component, which typically contains the same cell clusters and a liquid environmental medium, such as a nutrient medium. The receptacles of the different containers are separated from each other.

[0019] The fractions are preferably collected in containers and subsequently cryopreserved in the containers. This advantageously simplifies the method comprising fractionation and cryopreservation, as well as the structure of the cryopreservation device, and avoids possible undesirable effects on the cell clusters after fractionation.

[0020] During cryopreservation, the separated fractions obtained during fractionation are frozen. Alternatively, the fractions are subjected to a change of the environmental medium and / or to enrichment of the cell clusters in the environmental medium before freezing.

[0021] The cryopreservation of cell clusters includes a pretreatment method and a subsequent freezing method. The pretreatment (or incubation) of the cell clusters includes preparing the cell clusters for freezing, wherein for example, the composition of the liquid environmental medium with an adjuvant or CPA (a collection of all additives for improving the preservation result), the volume of the components, the density of the cell clusters in the components and / or other pretreatment parameters are adjusted, and / or the cell clusters are changed by physical and / or chemical methods, such as osmosis. The pretreatment of the cell clusters is preferably carried out at a temperature such that the environmental medium is liquid, especially at room temperature. Freezing includes reducing the temperature of the components to below 0°C until the cryopreservation temperature, for example, in the range of -80°C to -200°C. The freezing parameters of freezing are, for example, the time course of temperature drop and the cryopreservation temperature set.

[0022] The cryopreservation pretreatment and freezing methods are carried out in a known manner. However, according to the present invention, a specific pretreatment and / or freezing method is applied to each of the at least two component cell clusters during cryopreservation. Further process parameters, in particular pretreatment parameters and / or freezing parameters, are set for each component. For each component, process parameters are selected to optimize the cryopreservation of the cell clusters, in particular by maximizing the preservation of viability and / or functionality.

[0023] The application of cryopreservation process parameters involves setting preselected pretreatment parameters and freezing parameters. Optimal pretreatment parameters and freezing parameters can be determined through a series of tests on cell clusters and / or reference experiments from the specialized literature. By selecting sample-specific cryopreservation process parameters according to the present invention, the yield, effectiveness, and / or applicability of cryopreservation for different cell types can be advantageously improved.

[0024] After freezing to the cryogenic storage temperature, the frozen components are preferably provided to a cryobank for storage without interrupting the cooling chain. Storage in the cryobank takes place at a storage temperature which may deviate from the cryogenic storage temperature.

[0025] Advantageously, a variety of features are available, and cell clusters can be graded based on these features. According to a preferred embodiment of the present invention, cell clusters are graded based on at least one of the features, including size, shape, mass, elasticity, hydraulic conductivity, permeability to cryoprotectants (CPA), resistance to cryoprotectants, chemical properties, and cellular components of the cell clusters. A grading mechanism is preferably configured to grade based on at least one of these features. The above-mentioned physical and chemical features have been shown to be particularly suitable for effective classification and for selecting optimized process parameters for cryopreservation.

[0026] The cell clusters can be classified according to a variety of characteristics, for example, size and CPA permeability. In the case of classification according to a variety of characteristics, a multi-stage classification is preferably provided, wherein a first characteristic, for example, the size of the cell clusters, is tested in a first stage, and at least one further characteristic, for example, CPA permeability, is tested in at least one further stage.

[0027] Particularly preferably, the classification relevant to size is stipulated.For the separation relevant to size, a large amount of economical separation methods are available.The effectiveness of the process parameters of deep cryopreservation can be particularly sensitively relevant with the size of the cell cluster.The size of the cell cluster for example comprises its cross-sectional dimensions, especially diameter, or the geometric dimensions of other characterizations of the cell cluster, and the geometric dimensions influence material transport and / or freezing process. Alternatively or additionally, particularly preferably, the corresponding cell cluster is classified into the component with a specific shape.The shape of the cell cluster is following geometric configuration, that is, the geometric configuration that the cell cluster in the ambient medium has at least approximately, for example spherical or elongated cylindrical or irregular shape.

[0028] During cryopreservation, each component is subjected to a pretreatment characterized by component-specific pretreatment parameters. According to another preferred embodiment of the present invention, the pretreatment methods for the components differ in at least one of the pretreatment parameters, including pretreatment duration, temperature, pressure, medium composition, gas supply composition, osmotic conditions, and medium movement. These pretreatment parameters have been shown to be particularly well-suited for preparing cell clusters for efficient cryopreservation with high yield.

[0029] According to other embodiments of the present invention, the pretreatment methods for the components can advantageously differ in the temporal course of at least one of the pretreatment parameters. The pretreatment methods can be characterized by different temporal dependencies of the pretreatment parameters. Utilizing the temporal dependencies advantageously allows for additional degrees of freedom in optimizing the pretreatment.

[0030] According to another preferred embodiment of the present invention, the freezing methods used for the components differ in at least one of the freezing parameters, including the duration of freezing, in particular the cooling rate, temperature, pressure, medium composition, gas supply composition, and medium movement. The freezing device is accordingly preferably configured to apply a freezing method having at least one of the aforementioned freezing parameters. This advantageously provides a variety of parameters that can be used to optimize the cryopreservation of different components having the same cell cluster.

[0031] When, according to another preferred embodiment of the present invention, the fractionation of cell clusters includes fluid fractionation, in which the cell clusters are separated in a fluid environment, there is an additional advantage, because the cell clusters can be kept in a liquid ambient medium from the time they are provided, in particular cultured, until they are frozen and temporary transfer to a gas or vapor environment is avoided.

[0032] According to an advantageous embodiment of the present invention, fractionation and cryopreservation are performed automatically. The cryopreservation device is configured for automatic operation, in particular without operator intervention in the cryopreservation device. Automation offers advantages in terms of avoiding process errors, reproducible and accurate setting of process parameters, and high-throughput mechanical fractionation into separate components and downstream cryopreservation of the separated components at high speed and throughput.

[0033] When the classification of the cell clusters according to another modification of the present invention includes classification in the flowing fluid, especially size classification, the cell clusters are arranged in the flow cross section of the flowing fluid under the action of at least one of the flow force of the flowing fluid, the dielectrophoretic force in the flowing fluid, and the acoustic wave in the flowing fluid. The flow profile of a fluid in a flow state includes a position-dependent distribution of flow velocities in a cross section of the fluid. By separating the cell clusters at different positions in the flow profile, different flow paths in the flow are introduced. Delivery to different containers is achieved by guiding the individual parts of the flow profile via separate partial fluids (e.g., partial channels of a fluid system) and / or guiding them into the containers in different directions.

[0034] Preferably, the fluid system of the fractionation mechanism is a fluid microsystem comprising channels and fluidic elements, such as branches or intersections, with characteristic cross-sectional dimensions of less than 2 mm. The fluid flow is particularly preferably parallel, vortex-free, which advantageously improves separation in the fluid and allows portions of the flow profile to be directed into the container at different locations in the flow profile while being separated from the cell clusters.

[0035] The classification carried out by the flow force of the fluid of flowing state is the size classification carried out by passive fluidics. Cell clusters are arranged on different positions in the flow profile according to their size and are separated in this way. Passive fluidics has the following advantages. It relates to a non-contact method, which only applies a very small load to the cell clusters and does not require a size sensor. The heterogeneous mixture of cell clusters can be transported in portions and separated by running time difference (stratification principle, field flow classification). However, it is preferred to adopt a continuous method, for example, pinched flow fractionation (PFF), which can be more easily designed and more easily expanded. In the PFF method, cell clusters of different sizes leave outlets, especially nozzle sections, at different angles.

[0036] The classification by the dielectrophoretic force in the fluid of flow state is the size classification or classification that is carried out according to the electrical characteristics of cell clusters by active fluidics, for example, the separation carried out according to the dielectric characteristics (for example polarizability or surface charge) of cell clusters.For these separation methods, the classification mechanism is preferably equipped with an electrode mechanism, and the electrode mechanism is arranged to apply the dielectrophoretic force in the fluid of flow state. The electrode mechanism for example comprises at least one electrode, and at least one electrode produces the field barrier of dielectrophoresis when applying alternating voltage, and this field barrier forms a deflection angle (not equal to 0 °) with the flow direction in the fluid system. The height of the dielectrophoretic field barrier acting on the cell cluster is relevant with the size of the cell cluster. The dielectrophoretic force acting on the cell cluster transversely to the flow direction is superimposed with the flow force in the flow. The cell cluster can be positioned according to its size and / or dielectric characteristics and flow force by the electrodes in different positions and accordingly in the flow profile. Thus, advantageously, a contactless method is also provided, and this method does not require a pre-placed sensing device. However, the equipment complexity is higher than that in the case of passive fluidics.

[0037] Alternatively, fractionation can be combined with a sensor system using dielectrophoretic forces. A sensor system can be arranged upstream of the electrode system, configured to detect at least one characteristic of the cell clusters. The electrode system is controlled based on the output signal of the sensor system, such that individual cell clusters are directed to different locations in the flow profile based on the detected characteristic.

[0038] Another variant of size classification by active fluidics is provided by classification using sound waves in the flowing fluid. An acoustic field of suitable frequency (standing and / or traveling waves) and the nature of inertial objects converging at the minimum field of the sound waves are used for classification. In this case, there are also contactless methods that do not require a pre-placed sensor device. The size range in which acoustic classification can be applied is advantageously greater than that of dielectrophoretic classification. For this separation method, the classification mechanism is preferably equipped with a sound source mechanism that is configured to generate sound waves in the flowing fluid.

[0039] According to another preferred embodiment of the present invention, at least one characteristic of the cell cluster and / or at least one state parameter of at least two components is sensed. Accordingly, the cryopreservation device is preferably equipped with a sensor mechanism, which is configured to detect at least one characteristic of the cell cluster and / or at least one state parameter of at least two components. It is particularly preferred that the at least one characteristic of the cell cluster is sensed immediately before grading, and the at least one state parameter of the component is sensed immediately before cryopreservation. The sensing of at least one characteristic of the cell cluster before grading advantageously expands the group of characteristics of the cell cluster, based on which grading is performed. The sensing of at least one state parameter of the component before cryopreservation provides the advantage of further optimizing the process parameters of cryopreservation according to the state of the component. The state parameter of the component is, for example, the density or size of the cell cluster.

[0040] Another particularly important advantage of the present invention regarding the continued use of cell clusters after cryopreservation is that component-specific thawing with preserved viability is also possible. According to an advantageous embodiment of the present invention, cell clusters of at least two components are thawed, so that a specific thawing method is applied to each component. Thawing parameters, like the cryopreservation process parameters for each fractionated component, are individually optimized, which allows for an increased viability of the thawed cell clusters.

[0041] In general, a method for thawing at least two components while preserving their viability, wherein the at least two components are obtained by fractionating cell clusters according to at least one characteristic of the cell clusters and are frozen, wherein a specific thawing method is applied to each component, and a thawing device constructed for carrying out the method can be considered as further independent subjects of the present invention.

[0042] The features disclosed in the context of the method for cryopreservation of a plurality of cell clusters formed from biological cells and embodiments thereof also constitute preferred features of the cryopreservation device or embodiments thereof. Therefore, in particular, the aforementioned aspects and inventions with respect to the method and preferred features also apply to the cryopreservation device and its components. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Further details and advantages of the present invention will be described below with reference to the accompanying drawings.

[0044] Figure 1 shows the process of a method for cryopreserving a plurality of cell clusters and components of a cryopreservation device having features according to a preferred embodiment of the present invention; and

[0045] Figure 2 A fractionation mechanism configured for dielectrophoretic separation of cell clusters is shown according to an embodiment of the present invention. DETAILED DESCRIPTION

[0046] The features of a preferred embodiment of the present invention are described below, exemplarily, with respect to its application to size fractionation of cell clusters. It should be emphasized that practical implementation of the present invention is not limited to size fractionation but is also possible, alternatively or additionally, with fractionation based on other characteristics of the cell clusters, as described below in conjunction with further examples. The details of the cell clusters and their provision, as well as the cryopreservation process parameters and / or thawing parameters used in the specific examples, are selected as is known from the cryopreservation of biological materials.

[0047] Figure 1 Steps S1 to S4 of a method for cryopreserving a plurality of cell clusters 1 and 2 according to a preferred embodiment of the present invention and a cryopreservation apparatus 100 for this method are shown. The cryopreservation apparatus has a grading mechanism 10 , a container mechanism 20 , and a freezing mechanism 30 . Figure 1 Additionally shown are step S0 of preparing cell clusters 1 and 2 and step S5 of storing the frozen cell clusters in the cryogenic storage 40. Figure 1 In the example shown, for example, an automated fluid size fractionation of cell clusters 1 , 2 is performed.

[0048] In step S0, a non-homogeneous sample is prepared, for example, a mixture of cell clusters 1, 2 of different sizes and / or a mixture of cell clusters 1, 2 with different sensitivities to CPA. Cell clusters 1, 2, for example, include organoids, which are formed from adult stem cells in a manner known per se by culturing in a nutrient medium and using differentiating factors, and for example have a cross-sectional dimension ranging from 10 μm to 10 mm or larger. Cell clusters 1, 2 are prepared, for example, in a culture vessel.

[0049] In step S1, the cell clusters 1, 2 are separated into individual fractions 4 (fractions) by means of a schematically shown fractionation mechanism 10, each fraction containing cell clusters of a specific size. The fractionation mechanism 10 is, for example, as described below with reference to Figure 2 Component 4 is transferred to the container 21 of container mechanism 20 in step S2. Container 21 preferably comprises a plastic tube with a lid, especially a so-called PP tube, as used in subsequent deep cryopreservation in step S3 and S4 (see the description in step S5). Alternatively, container can comprise other receptacles, such as bags or microtiter plates. According to another alternative, container 21 can be a part for the incubation unit 31 of freezing mechanism 30. Each component 4 is collected in the container 21 provided for storage, until a predetermined loading is reached, especially concentration (the quality of the cell cluster of the environmental medium per volume) is reached.

[0050] The freezing mechanism 30 includes an incubation unit 31 and a cooling unit 32. In the freezing mechanism 30, each component 4 is subjected to a pretreatment protocol and a freezing protocol that match the corresponding size.

[0051] In the incubation unit 31, the components are pretreated. This means that a complete, unique incubation program is carried out for each component 4. At least one CPA (especially a cryoprotectant) is supplied with which the cell clusters are to be loaded. As the size of the cell clusters increases, for example, an increasing concentration of CPA and / or an increased incubation time are used. Suitable cryoprotectants and their concentrations can be determined through testing.

[0052] Furthermore, incubation can include a predetermined temperature control T(t), filling with a predetermined cryoprotectant (CPA) concentration profile C(t, CPA1, CPA2, ...), and / or gassing. Alternatively or additionally, if the cell cluster can tolerate it, a membrane-permeable and / or even toxic CPA can be temporarily supplied. Furthermore, ice nucleation (to reduce and control supercooling), medium circulation (to homogenize T and C), and / or infiltration of the cell cluster with at least one component (to load the membrane-impermeable CPA) can be part of the pretreatment method. Infiltration can be achieved, for example, chemically (e.g., with DMSO), using sound waves (sonoporation), using an electric field (electroporation), using liposomes, and / or through thermal modulation via changes in membrane state. Furthermore, pretreatment in incubation unit 31 includes precooling component 4 to a temperature above its freezing point.

[0053] The incubation unit 31 preferably has a separate receptacle for the container 21, such as a separate cavity, or provides a container via a reservoir, preferably for components of the same volume. The incubation unit 31 comprises a pump mechanism for supplying CPA (adding and / or increasing the concentration sequentially) and / or for extracting the medium from the container. In addition, the incubation unit 31 is preferably equipped with a drive, such as an agitator, for moving the medium in each container during pretreatment. Alternatively or additionally, a precooling unit designed for supercooling the components can be provided. Supercooling can cause membrane changes in the cells of the cell cluster, thereby affecting the pretreatment, such as affecting the absorption of CPAS. Alternatively or additionally, a sound source can also be provided, by which the cell clusters of the components can be ultrasonically treated. By this ultrasonic treatment, other membrane changes, especially permeation, can be induced in the cells of the cell cluster.

[0054] Other pretreatment parameters of the size-dependent incubation include, for example, the concentration of the individual CPAs, the action time of the individual CPAs, the concentration distribution of the individual CPAs over time, a suitable temperature profile (>0°C), continuous change of the medium composition (for example by means of a mixing mechanism connected to the incubation unit 31 and the CPA reservoir) and / or replacement (filling) of the ambient medium.

[0055] Next, component 4 is frozen in cooling unit 32 (step S4). Depending on the characteristics of the cell clusters of component 4, such as size or other characteristics, such as the hydraulic conductivity of each component of the cell cluster, the fraction of additional components in the medium that actively permeate the membrane, and / or supercooling, each component 4 is controlled and frozen at different cooling rates and / or cooling processes. For example, a cooling rate of equal to or less than -1 K / min is used. Cooling is performed to a cryopreservation temperature of, for example, -80°C or lower, for example, -140°C or lower.

[0056] For example, the temperature curve for the freezing of the individual components can be selected in order to adjust the adaptability to the equilibrium speed, controlled nucleation for reducing supercooling and / or a uniform cooling rate with respect to the component volume (if necessary by means of circulation of the medium and / or using form-fitting adaptation of the component containers in the heat exchanger of the cooling unit 32).

[0057] For each component, the cooling unit 32 comprises a cooling chamber having at least one cooling element and a heat exchanger. The cooling element is, for example, a Peltier element, a Stirling cooler (Stirling-Kühler) or a coolant flow cooler, which, for example, works with liquid nitrogen or isopentane. The cooling element is designed to adjust a defined cooling rate. The heat exchanger, for example, comprises a receptacle for a container of the corresponding component, the receptacle preferably having a form fit between the container and the receptacle. If the container 21 is part of the incubation unit 31 of the freezing mechanism 30, it is transferred to a low-temperature container, for example a so-called PP tube, before freezing. The cooling unit 32 can optionally be equipped with a nucleation mechanism, for example a cold needle, with which a controlled nucleation is induced in the container.

[0058] Finally, the container is closed and the frozen components are stored at a cryogenic temperature (e.g., -140° C.) in a cryogenic store 40 (step S5). The transfer to the cryogenic store 40 is carried out without interrupting the cooling chain, for example, by means of a cooled isolation chamber or by direct connection of the refrigeration unit 30 to the cryogenic store 40.

[0059] To defrost, turn the container upside down using a defrosting mechanism (not shown). Figure 1 During thawing, similar to the size-adaptation process during freezing, the individual components are also processed separately in different incubation units during thawing and / or in the first recovery phase until the adjuvants have matured. For example, a size-dependent thawing rate, a size-dependent incubation period in a highly osmotic thawing medium, and / or a size-dependent maturation of the thawed components can be set. This allows, for example, the cooling of the environment to slow down the metabolism of large cell clusters, ensuring sufficient dilution of toxic membrane-permeating CPAs, while this can occur more quickly in the case of small cell clusters.

[0060] After thawing, a distribution step can be provided in which the thawed components are tested for vitality and, if vitality is detected, transferred to predetermined, application-related container formats, such as microtiter plates or suspension bioreactors. Size fractionation can be maintained or omitted.

[0061] For example, the thawing and / or dispensing can be carried out using fluidic means, in particular fluidic microsystems.

[0062] Figure 2 As an example, a staging mechanism 10 configured as a fluidic mechanism 11, in particular as a fluidic microsystem, is shown. The staging mechanism has a main channel 11A and branch channels 11B, through which a suspension of a liquid ambient medium containing cell clusters 1, 2, and 3 of different sizes flows in the direction of arrow A. An electrode mechanism 12 and a sensor mechanism 14 are located in the main channel 11A and are connected to a control mechanism 13. The main channel 11A branches into branch channels 11B, each of which is connected to one of the containers 21 of the container mechanism 20.

[0063] The electrode mechanism 12 includes, for example, two strip-shaped electrodes or electrode pairs on the bottom and / or cover of the main channel 11A. When an AC voltage of the control mechanism 13 is applied to the electrodes, the electrode mechanism 12 can be used to generate a field barrier transverse to the fluid A. The field barrier can be temporarily created to match the cell clusters arriving through the fluid. Through the interaction of the field barrier with the flow force in the fluid A, the cell clusters can be guided to a predetermined flow path that leads to one of the branch channels 11B (see, for example, the dotted flow path B of the cell cluster 1).

[0064] The sensor mechanism 14 is, for example, an optical sensor, in particular a camera coupled to an image processing mechanism. The sensor mechanism 14 can detect the cell clusters 1, 2, and 3 and their respective sizes. Information regarding the positions and sizes of the cell clusters 1, 2, and 3 is provided to the control mechanism 13. The control mechanism 13 assigns the cell clusters 1, 2, and 3 to three predetermined sizes of desired components and controls the electrode mechanism 12 so that the cell clusters 1, 2, and 3 are each guided into one of the branch channels 11B adapted to their size and, via the branch channels, into one of the containers 21.

[0065] Alternative Figure 2 In one embodiment, the electrode arrangement 12 may include a dielectrophoretic field cage, and the sensor arrangement 14 may be configured to detect cell clusters in the field cage. The cell clusters are sequentially detected in the field cage by the sensor, assigned to one of a plurality of components based on their characteristics, and directed to the corresponding component by releasing the field cage and, if necessary, further dielectrophoretic deflection.

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

[0067] As an alternative to optical sensor systems, it can be provided, for example, that an impedance measurement of the cell clusters is performed (for example, as in a “Coulter Counter” instrument), and a classification can be carried out based on the results of the impedance measurement.

[0068] refer to Figure 1 and Figure 2 The size classification described can be supplemented or replaced by classifications based on other characteristics. For example, a test of the permeability of the cells of the cell cluster to a cryoprotectant and / or a test of their resistance to a cryoprotectant can be performed in a fluidic system of the classification system, such as in a field cage of the fluidic system. Based on the test results, different fractions can be formed and subsequently cryopreserved using different process parameters. For example, cell clusters with low cell CPA permeability can be treated with a longer CPA incubation time than cell clusters with increased cell CPA permeability.

[0069] The features of the invention disclosed in the above description, in the drawings and in the claims can be essential for realizing the invention in its various embodiments, both individually and in combination or subcombination.

Claims

1. A method for cryopreserving a plurality of cell clusters (1, 2, 3) formed of biological cells, each of the cell clusters (1, 2, 3) comprising a group of associated, three-dimensionally extended living biological cells, the method comprising the steps of: - fractionating the cell clusters (1, 2, 3) into at least two components (4) based on at least one characteristic of the cell clusters (1, 2, 3), wherein the fractionating of the cell clusters (1, 2, 3) comprises fractionating of a fluid, wherein the cell clusters (1, 2, 3) are separated in a fluid in a flowing state, and the cell clusters (1, 2, 3) are arranged at different positions in a flow profile of the fluid in a flowing state under the action of at least one of dielectrophoretic forces in the fluid in the flowing state and acoustic waves in the fluid in the flowing state, wherein the cell clusters (1, 2, 3) are kept intact during the fractionating, - collecting the components (4) in different containers (21), and - cryopreserving said cell clusters (1, 2, 3) of said at least two components (4), wherein Specific pretreatment methods and freezing methods with different process parameters are applied for each component.

2. The method according to claim 1, wherein - performing a classification of the cell clusters (1, 2, 3) according to at least one of the characteristics, including size, shape, mass, elasticity, hydraulic conductivity, permeability to cryoprotectants (CPA), resistance to cryoprotectants, chemical properties and cellular composition of the cell clusters (1, 2, 3).

3. The method according to claim 1, wherein - the pretreatment methods used for the components (4) differ in at least one of the pretreatment parameters including duration of pretreatment, temperature, pressure, medium composition, composition of feed gas, permeation conditions and medium movement.

4. The method according to claim 3, wherein The pretreatment methods for the components (4) differ with respect to the time course of at least one of the pretreatment parameters.

5. The method according to any one of claims 1 to 4, wherein - the freezing methods used for the components (4) differ in at least one of the freezing parameters, including duration of freezing, temperature, pressure, medium composition, composition of the supply gas and medium movement.

6. The method according to claim 5, having at least one of the following features: - collecting said components (4) in said container (21) and subsequently subjecting said components (4) thereto to said cryopreservation, - providing the components (4) frozen to a cryogenic store (40) for storage without interrupting the cooling chain, and - said fractionation and said cryopreservation are performed automatically.

7. The method according to claim 1, wherein - said fractionation of the cell clusters (1, 2, 3) comprises performing said fractionation in a fluid in said flow state, wherein, The cell clusters (1, 2, 3) are further arranged at different positions in the flow cross section of the fluid in the flow state under the action of the flow force of the fluid in the flow state.

8. The method according to claim 1, wherein - sensory detection of the at least one characteristic of the cell cluster (1, 2, 3) and / or at least one state variable of the at least two components (4).

9. The method according to claim 1 , further comprising the steps of: - Thawing the cell clusters (1, 2, 3) into the at least two components (4), wherein, A specific thawing method with different thawing parameters was applied for each component.

10. A cryopreservation device (100) configured to cryopreserve a plurality of cell clusters (1, 2, 3) formed of biological cells, each of the cell clusters (1, 2, 3) comprising an associated, three-dimensionally extended group of living biological cells, the cryopreservation device (100) comprising: - a classification device (10) configured to classify the cell clusters (1, 2, 3) into at least two components (4) according to at least one characteristic of the cell clusters (1, 2, 3); wherein - the fractionation mechanism (10) comprises a fluid mechanism (11) configured to separate the cell clusters (1, 2, 3) in a fluid in a flowing state, and wherein - the classification mechanism (10) comprises at least one electrode mechanism (12) and a sound source mechanism, the at least one electrode mechanism (12) being configured to exert a dielectrophoretic force in the fluid in the flowing state, and the sound source mechanism being configured to generate sound waves in the fluid in the flowing state, so that the cell clusters (1, 2, 3) can be arranged at different positions in the flow cross section of the fluid in the flowing state under the action of at least one of the dielectrophoretic force in the fluid in the flowing state and the sound waves in the fluid in the flowing state, wherein the cell clusters (1, 2, 3) are kept intact during the classification, a container arrangement (20) having at least two different containers (21), each of which is arranged to collect one of the components (4); and - A freezing device (30) configured to cryopreserve the cell clusters (1, 2, 3) into the at least two components (4), wherein the freezing device (30) is configured to apply a specific pretreatment method and freezing method with different process parameters to each of the components (4).

11. The cryopreservation device according to claim 10, wherein - The grading mechanism (10) is configured to grade the cell clusters (1, 2, 3) according to at least one of their characteristics, including size, shape, mass, elasticity, hydraulic conductivity, permeability to cryoprotectants (CPA), resistance to cryoprotectants, chemical properties and cellular composition of the cell clusters (1, 2, 3).

12. The cryopreservation device according to claim 10, wherein - The freezing device (30) is configured to apply a pretreatment method that differs in at least one of the pretreatment parameters and / or their time course, the pretreatment parameters including duration, temperature, pressure, medium composition, composition of the supply gas, osmotic conditions and medium movement during the pretreatment period.

13. The cryopreservation device according to any one of claims 10 to 12, wherein The freezing mechanism (30) is configured to apply freezing methods that differ in at least one of the freezing parameters, including duration of the freezing period, temperature, pressure, medium composition, composition of the supply gas, and medium movement.

14. The cryopreservation device according to claim 10, having at least one of the following characteristics: - said at least two containers (21) are part of said freezing mechanism (30), and - The cryopreservation device (100) is configured to operate automatically.

15. The cryopreservation device according to claim 10, comprising A sensor device (14) is provided for detecting the at least one characteristic of the cell cluster (1, 2, 3) and / or at least one state variable of the at least two components (4).

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