METHOD AND CORRESPONDING DEVICE FOR CRYOPERATIVES FOR THE CRYOPERATIVE OF A MULTIPLE CELL ASSOCIATIONS OF BIOLOGICAL CELLS

Description

[0001] The invention relates to a method and a cryopreservation device for cryopreserving a plurality of cell aggregates of biological cells (also referred to as cell aggregates), e.g., of 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 applications of cell aggregates composed of a variety of biological cells. Cell aggregates offer, in particular, the ability to replicate specific properties or functions of organs of a biological organism without forming a complete organ. Cell aggregates include, for example, biological native tissues (cell-matrix aggregates grown in vivo), spheroids (spherical cell clusters), or organoids (artificially grown cell-matrix aggregates in vitro). The formation of cell aggregates can require cultivation periods of days, weeks, or even months, with individual cell aggregates developing at different rates. As a result of a cultivation procedure, inhomogeneous samples with cell aggregates at various stages of maturation or development, especially in size, are typically generated.

[0003] Cryopreservation of biological materials, such as cells, cell components, and / or cell groups, is a well-established method for freezing biological materials while preserving their viability. Freezing is performed according to predetermined freezing protocols, typically with the addition of a cryoprotectant (CPA) to prevent or suppress ice crystal formation during freezing. Process parameters (pretreatment and subsequent freezing conditions) are selected based on the specific properties of the biological materials (see, for example, the review article by M.A. Taylor et al., "New Approaches to Cryopreservation of Cells, Tissues and Organs," in "Transfus. Med. Hemother." 46: 2019, 197–215).

[0004] For the effective cryopreservation of single-cell suspensions with a high viability yield, cell-type-specific process parameters, such as excipient composition, exposure time, and cooling rate, are optimized. This can be very time-consuming and require considerable experience. When cryopreserving cell clusters, the success of the preservation is even more dependent on the choice of process parameters, thus increasing the effort required to select optimized freezing parameters.

[0005] While the cryopreservation of single cells in suspension, particularly by slow freezing, is widely used, inhomogeneous samples from extended, three-dimensional cell aggregates, such as tissues, spheroids, or organoids of varying sizes, can currently only be frozen slowly with limited yield. For example, the required exposure time of excipients to achieve a desired concentration within the cell aggregate increases quadratically with its diameter. Since the aforementioned inhomogeneity, especially polydispersity, typically occurs in conventional (scalable) cell aggregate production methods, even with a preservation protocol optimally chosen for a specific cell type and size, losses would be expected due to exposure times and conditions that are unsuitable for different sizes.Therefore, in the conventional cryopreservation of a polydisperse sample of cell clusters, compromises in the choice of freezing parameters must always be accepted.

[0006] It is also known to preserve smaller cell clusters by vitrification (vitrification by ultra-rapid cooling). However, vitrification has strict technical limitations when it comes to providing very high concentrations of excipients and achieving sub-glass-point temperatures at every point within the three-dimensional tissue structure. 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 mm² / s). Furthermore, the concentrations and exposure times of excipients are also limited due to their cytotoxicity. Additionally, the probability of damage from thermal stress cracks increases with increasing sample size. Therefore, vitrification is unsuitable for the routine preservation of samples containing many cell clusters of varying sizes.

[0007] The aforementioned limitations in the selection of freezing parameters do not only arise in practice when cryopreserving samples with cell clusters of different sizes. Sample inhomogeneity can also result from the presence of other differing properties, such as varying shapes or elasticities, among the cell clusters provided together.

[0008] WO 2016 / 064896 A1 discloses a processing method for a tissue sample and a corresponding device comprising a microfluidic fractionation unit and a cryopreservation device. In the fractionation unit, materials in the tissue sample are separated into different fractions, for example according to size, and collected in appropriate containers.

[0009] The object of the invention is to provide an improved method and an improved cryopreservation device for the cryopreservation of a variety of cell aggregates from biological cells, thereby avoiding 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.

[0010] This problem is solved by a method and a cryopreservation device for cryopreserving a large number of cell clusters from biological cells with the features of the independent claims. Advantageous embodiments and applications of the invention are described in the dependent claims.

[0011] According to a first general aspect of the invention, the above-mentioned problem is solved by a cryopreservation method according to claim 1.

[0012] According to a second general aspect of the invention, the above-mentioned problem is solved by a cryopreservation device according to claim 7.

[0013] Preferably, the cryopreservation device or one of its embodiments is configured to carry out the cryopreservation method according to the first general aspect of the invention or an embodiment of the method.

[0014] According to the invention, the cell aggregates are fractionated into at least two fractions depending on at least one predetermined property of the cell aggregates. Typically, up to 5 or up to 10 fractions are formed. However, more are also possible, e.g., up to 20 or more fractions.

[0015] Advantageously, fractionating the cell aggregates creates at least two homogeneous fractions, for each of which the cryopreservation process parameters can be optimized. The inventors have determined that optimal cryopreservation process parameters depend not only on the cell type but also on the properties of the cell aggregate itself, such as its size. Furthermore, the inventors have found that in conventional methods, different cell aggregate sizes within an inhomogeneous fraction result in the uneven distribution of excipients and water within the cell aggregate, thus affecting the freezing process differently and negatively impacting the success and yield of the cryopreservation. The creation of homogeneous fractions according to the invention overcomes the limitations of conventional processing of inhomogeneous fractions.

[0016] Providing homogeneous fractions offers a further advantage for cell aggregate applications, such as research or implantation treatments, where fractions containing cell aggregates at the same or similar stages of maturation or development are desired. Such fractions can be generated by culturing identical cell aggregates for a specific purpose, subjecting them to cryopreservation, and storing them frozen.

[0017] The term "cell cluster" refers to a coherent, preferably three-dimensionally extended group of living biological cells, such as a tissue (especially a tissue model), a spheroid, or an organoid. The cell cluster may consist exclusively of cells or may also contain extracellular matrix substances. Cell clusters are generated, for example, by culturing biological cells and / or by extraction from an organism. The term "fraction" refers to a multitude of cell clusters in a liquid environment.

[0018] Fractionation involves separating (sorting, separation process) cell clusters from an initially inhomogeneous sample into a predetermined number of fractions. The separation is carried out such that each fraction contains cell clusters, and the cell clusters in each fraction exhibit at least one identical property. This means that the cell clusters in each fraction are identical with respect to this at least one property, or differ so slightly that these differences do not affect cryopreservation, particularly the selection of optimal parameters for the pretreatment and freezing processes. Each fraction is homogeneous with respect to the at least one property under consideration. Fractionation is, in particular, a separation process that preferably does not alter the cell clusters. Specifically, the cell clusters are preserved during fractionation; that is, they are not broken down into smaller parts.

[0019] The fractionation device preferably comprises a separation device which is designed to receive a composition of cell clusters in an environmental medium, to separate the cell clusters into the different fractions and to discharge the fractions into the different containers.

[0020] According to the invention, the fractions are collected in different containers; that is, the fractionation comprises separation into different containers. Each container generally comprises a receptacle of the fraction, which typically includes the same cell clusters and a liquid surrounding medium, such as a nutrient medium. The receptacles of the different containers are separated from each other.

[0021] Preferably, the fractions are collected in the containers in which cryopreservation subsequently takes place. This advantageously simplifies the fractionation and cryopreservation process and the design of the cryopreservation device, and avoids any undesirable influences on the cell clusters after fractionation.

[0022] In cryopreservation, the separated fractions obtained during fractionation are frozen. Alternatively, prior to freezing, the fractions undergo an exchange of the surrounding medium and / or an enrichment of the cell clusters in the surrounding medium.

[0023] The cryopreservation of cell aggregates comprises a pretreatment process followed by a freezing process. The pretreatment (or incubation) of the cell aggregates includes preparing them for freezing, for example, by adjusting the composition of the liquid environment with additives or CPA (the sum of all additives used to improve the preservation result), the volume of the fraction, the density of the cell aggregates within the fraction, and / or other pretreatment parameters, and / or modifying the cell aggregates using physical and / or chemical processes, such as permeabilization. Pretreatment of the cell aggregates preferably takes place at a temperature where the environment is liquid, particularly at room temperature. Freezing involves lowering the temperature of the fraction below 0°C to a cryopreservation temperature, for example, in the range of -80°C to -200°C. Freezing parameters include, for example,the time course of the temperature reduction and the set cryopreservation temperature.

[0024] The pretreatment and freezing procedures for cryopreservation are carried out in a manner known per se, but according to the invention, specific pretreatment and / or freezing procedures are applied to the cryopreservation of the cell aggregates of the at least two fractions. Different process parameters, in particular pretreatment and / or freezing parameters, are provided for each fraction. For each fraction, such process parameters are selected that the cryopreservation of the cell aggregates is optimized, in particular with maximum preservation of viability and / or functionality.

[0025] The application of cryopreservation process parameters includes the setting of pre-selected pretreatment and freezing parameters. Optimal pretreatment and freezing parameters can be determined through test series with cell clusters and / or from reference experiments in the literature. By selecting sample-specific cryopreservation process parameters according to the invention, the yield, effectiveness, and / or applicability of cryopreservation for various cell types can advantageously be improved.

[0026] After freezing to the cryopreservation temperature, the frozen fractions are preferably transferred to a cryobank for storage without interrupting the cold chain. Storage in the cryobank takes place at a temperature that may differ from the cryopreservation temperature.

[0027] Advantageously, a multitude of properties are available on the basis of which the fractionation of cell aggregates can be carried out. According to preferred embodiments of the invention, the fractionation of the cell aggregates is carried out depending on at least one of the properties, which include size, shape, mass, elasticity, hydraulic conductivity, permeability to cryoprotectants (CPA), resistance to cryoprotectants, chemical properties, and cell composition of the cell aggregates. The fractionation device is 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 selecting optimized process parameters for cryopreservation.

[0028] The fractionation of cell aggregates can be performed based on several properties, for example, size and CPA permeability. When fractionating according to multiple properties, a multi-stage fractionation process is preferably used, in which a first property, e.g., the size of the cell aggregates, is tested in a first stage, and at least one further property, e.g., CPA permeability, is tested in at least one further stage.

[0029] 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, especially its 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 form that the cell cluster exhibits, at least approximately, in the surrounding medium, such as a spherical shape, an elongated cylindrical shape, or an irregular shape.

[0030] Each fraction undergoes pretreatment during cryopreservation, characterized by fraction-specific pretreatment parameters. According to the invention, the pretreatment procedures for the fractions differ with respect to at least one of the pretreatment parameters, which include duration, temperature, pressure, media composition, gassing composition, permeabilization conditions, and media agitation. These pretreatment parameters have advantageously proven to be particularly well-suited for preparing cell aggregates for effective cryopreservation with high yield.

[0031] Advantageously, according to further modifications of the invention, the pretreatment processes for the fractions can differ with respect to the time profile of at least one of the pretreatment parameters. The pretreatment processes 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.

[0032] According to the invention, the freezing processes for the fractions differ with respect to at least one of the freezing parameters, which include duration, in particular cooling rate, temperature, pressure, media composition, gassing composition, and media agitation during freezing. The freezing device is preferably configured to apply the freezing processes 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 composition.

[0033] The fractionation of cell aggregates involves fluidic fractionation, in which the cell aggregates are separated in a fluidic environment. This offers further advantages, as the cell aggregates can be kept in a liquid medium from their preparation, particularly cultivation, until freezing, thus avoiding a temporary transition to a gaseous or vaporous environment.

[0034] According to an advantageous embodiment of the invention, fractionation and cryopreservation are automated. The cryopreservation device is designed for automated operation, in particular operation without intervention by an operator. Automation offers advantages in terms of avoiding process errors, reproducibility and accuracy of setting process parameters, and the possibility of performing high-throughput, automated fractionation into separate fractions followed by high-speed, high-throughput cryopreservation of the separated fractions.

[0035] In the fractionation process according to the invention, particularly size fractionation, in a fluidic flow, the cell clusters are arranged at different positions within a flow profile of the fluidic flow under the influence of at least one of the following: fluidic flow forces, 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 across the cross-section of the flow. The different positions within the flow profile cause the cell clusters to separate onto different flow paths. Transfer to the various containers is achieved by directing the individual parts of the flow profile through separate partial flows, e.g., partial channels of a fluidic system, and / or by directing them into the containers in different directions.

[0036] 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, irrotational flow, which advantageously improves separation within the flow and allows the portions of the flow profile to be directed into the containers at various positions within the flow profile, at a distance from the separation point of the cell clusters.

[0037] Fractionation using fluidic flow forces is a size fractionation method using passive fluidics. Cell clusters align themselves according to their size at different positions in the flow profile and can thus be separated. Passive fluidics offers the following advantages: It is a contactless process that places minimal stress on the cell clusters and requires no size sensors. The inhomogeneous mixtures of cell clusters can be introduced in portions and separated based on flow time differences (chromatographic principle, field-flow fractionation). However, continuous methods, such as pinched flow fractionation (PFF), are preferred, as they are simpler in terms of equipment and more scalable. In PFF, for example, cell clusters of different sizes exit a single outlet, particularly a nozzle section, at different angles.

[0038] Fractionation using dielectrophoretic forces in a fluidic flow is either size fractionation or fractionation based on an electrical property of the cell assemblies using active fluidics, such as separation according to dielectric properties of the cell assemblies, like polarizability or surface charge. For these separation methods, the fractionation device is preferably equipped with an electrode assembly configured to exert dielectrophoretic forces in a fluidic flow. The electrode assembly comprises, for example, at least one electrode which, when subjected to an alternating voltage, generates a dielectrophoretic field barrier that forms a deflection angle (not equal to 0°) with the flow direction in the fluidic system. The height of the dielectrophoretic field barrier acting on a cell assembly depends on the size of the cell assembly.Dielectrophoretic forces acting perpendicular to the flow direction on the cell arrays are superimposed with flow forces within the flow. Depending on their size and / or dielectric properties and the flow forces, cell arrays can pass the electrodes at different positions and thus be positioned accordingly within the flow profile. Advantageously, this also provides a contactless method that does not require upstream sensors. However, the equipment required is more complex than in passive fluidics.

[0039] Alternatively, fractionation using dielectrophoretic forces can be combined with a sensor system. A sensor system can be arranged upstream of the electrode array, designed to detect at least one property of the cell clusters. The electrode array is controlled by an output signal from the sensor system such that the individual cell clusters are directed to different positions in the flow profile depending on the detected property.

[0040] When fractionation is performed using sound waves in the fluidic flow, a further variant of size fractionation is provided by active fluidics. Acoustic fields (standing and / or traveling sound waves) of a suitable frequency and the ability of inertial bodies to accumulate 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 configured to generate sound waves in the fluidic flow.

[0041] According to a further preferred embodiment of the invention, at least one property of the cell assemblies 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 array configured to detect at least one property of the cell assemblies and / or at least one state variable of the at least two fractions. Particularly preferably, the sensory detection of the at least one property of the cell assemblies and the sensory detection of the at least one state variable of the fractions are performed immediately before cryopreservation. The sensory detection of the at least one property of the cell assemblies before fractionation advantageously expands the group of properties of the cell assemblies on which the fractionation is based.The sensory analysis of at least one state parameter of the fractions prior to cryopreservation offers the advantage of further optimizing the cryopreservation process parameters depending on the state of the fraction. State parameters of the fractions include, for example, density or cell size.

[0042] Another particularly important advantage of the invention for the further use of cell aggregates after cryopreservation is that the viability-preserving thawing can also be carried out fraction-specifically. According to an advantageous embodiment of the invention, the cell aggregates of the at least two fractions are thawed such that specific thawing procedures are applied for each fraction. Thawing parameters, like the process parameters of cryopreservation, are optimized separately for the individual fractionated fractions, which allows for an increase in the viability rate of the thawed cell aggregates.

[0043] In general, a method for thawing while preserving vitality at least two fractions obtained and frozen by fractionating cell assemblies depending on at least one property of the cell assemblies, 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 subject matter of the present invention.

[0044] The features disclosed in connection with the method for cryopreserving a large number of cell clusters from biological cells and its embodiments also constitute preferred features of the cryopreservation device or its embodiments. The aforementioned aspects and inventive and preferred features, particularly with regard to the method, therefore also apply to the cryopreservation device and its components.

[0045] Further details and advantages of the invention are described below with reference to the accompanying drawings. These schematically show: Figure 1: a process for cryopreserving a plurality of cell clusters and components of a cryopreservation device with features according to preferred embodiments of the invention; and Figure 2: a fractionation device configured for the dielectrophoretic separation of cell clusters according to an embodiment of the invention.

[0046] 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 further examples are described below. Details of the cell aggregates and their preparation, as well as the cryopreservation and / or thawing process parameters used in specific examples, are selected as is known from the cryopreservation of biological materials per se.

[0047] Figure 1Figure 1 shows steps S1 to S4 of the method for cryopreserving a plurality of cell clusters 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. Figure 2 further shows Figure 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 Figure 1 In the example shown, an automated, fluidic size fractionation of the cell clusters 1, 2 is performed.

[0048] In step S0, an inhomogeneous sample is provided, e.g., a mixture of cell clusters 1, 2 of different sizes and / or a mixture of cell clusters 1, 2 with different sensitivities to CPA. The cell clusters 1, 2 comprise, for example, organoids that were formed from adult stem cells in a known manner by cultivation in a nutrient medium with differentiation factors and have, for example, cross-sectional dimensions ranging from 10 µm to 10 mm or larger. The cell clusters 1, 2 are provided, for example, in a culture vessel.

[0049] In step S1, the cell clusters 1, 2 are separated into individual fractions 4 (fractions) by the fractionation device 10 shown schematically, each fraction containing cell clusters of specific sizes. The fractionation device 10 is constructed, for example, as shown below with reference to Figure 2The process is described and is preferably automated. In step S2, the fractions 4 are transferred to container 21 of the container system 20. 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 another 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 loading quantity, in particular concentration (mass of cell clusters per volume of the surrounding medium), is reached.

[0050] The freezing unit 30 comprises an incubation unit 31 and a cooling unit 32. In the freezing unit 30, the individual fractions 4 are subjected to a pretreatment and freezing protocol adapted to their respective size.

[0051] In incubation unit 31, the fractions undergo pretreatment. This means that a completely individualized incubation program is run for each fraction 4. At least one cryoprotectant (CPA) is added to the cell clusters. As the size of the cell clusters increases, for example, increasing concentrations of CPA and / or increasing incubation times are used. Suitable cryoprotectants and their concentrations can be determined through testing.

[0052] Incubation can further include predetermined temperature control T(t), gassing, and / or perfusion with predetermined cryoprotectant (CPA) concentration profiles C(t, CPA1, CPA2, ...). Alternatively or additionally, membrane-penetrating and / or even toxic CPAs can be temporarily introduced if the cell clusters tolerate them. Furthermore, ice nucleation (to reduce and control hypothermia), media recirculation (to homogenize T and C), and / or permeabilization of the cell clusters of at least one fraction (for loading with non-membrane-penetrating CPAs) can be part of the pretreatment procedure. Permeabilization can be achieved, for example, chemically (e.g., using DMSO), with sound waves (sonoporation), with electric fields (electroporation), using liposomal substances, and / or by thermomodulation via membrane phase change.Furthermore, the pretreatment in the incubation unit 31 includes pre-cooling the fractions 4 to a temperature above the freezing point of the fractions 4.

[0053] The incubation unit 31 preferably has individual compartments for the containers 21, e.g., individual cavities, or provides the containers via reservoirs, preferably for fractions of equal volume. The incubation unit 31 includes 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. Alternatively or additionally, a precooling unit can be provided, which is configured for subcooling fractions. Subcooling can induce membrane changes in the cells of the cell clusters, thereby influencing the pretreatment, e.g., the uptake of CPAS.Alternatively or additionally, a sound source can be provided to subject the cell clusters of the fractions to ultrasound treatment. This ultrasound treatment can induce further membrane changes in the cells of the cell clusters, in particular permeabilization.

[0054] Further pretreatment parameters for 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] The fractions 4 are then frozen in cooling unit 32 (step S4). Depending on the properties of the cell assemblies of fractions 4, such as their size or other characteristics like the hydraulic conductivity of the individual components of the cell assemblies, the proportion of membrane-permeable and osmotically active additives in the medium, and / or the degree of subcooling, the individual fractions 4 are frozen under controlled conditions at different cooling rates and / or cooling profiles. For example, a cooling rate of -1 K / min or less is applied. 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 adapt to an equilibration rate, controlled nucleation to reduce subcooling, and / or a homogeneous cooling rate across the fraction volume (possibly with recirculation of the medium and / or with the use of a form-fitting connection between the fraction containers and the heat exchanger of the cooling unit 32).

[0057] The cooling unit 32 comprises a cooling chamber for each fraction, containing at least one cooling element and a heat exchanger. The cooling element is, for example, a Peltier element, a Stirling condenser, or a flow-through coolant condenser, operating, for instance, 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 unit 30, they are transferred to cryogenic containers, such as the aforementioned PP tubes, prior to 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 cryobank 40 (step S5). The transfer to cryobank 40 takes place without interrupting the cold chain, e.g., using a refrigerated airlock or by directly connecting the freezing unit 30 to cryobank 40.

[0059] To thaw, the in Figure 1The process shown is reversed using a thawing unit (not shown). During thawing, similar to the size-adjusted process during freezing, the individual fractions are treated separately in different incubation units, both during thawing and / or in an initial recovery phase until the excipients are washed out. For example, a size-dependent thawing rate, size-dependent incubation in a hyperosmolar thawing medium, and / or size-dependent washing of the thawed fractions are implemented. This allows, for example, the metabolism of large cell clusters to be slowed by a cooled environment to ensure sufficient dilution of toxic, membrane-penetrating CPAs, while this process can be accelerated for small cell clusters.

[0060] After thawing, a portioning step may be included in which the thawed fractions undergo a viability test and, if viability is confirmed, are transferred to a predetermined, application-dependent container format, such as microtiter plates or suspension bioreactors. The size fractionation can be maintained or abandoned at this stage.

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

[0062] Figure 2Figure 10 shows an example of 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 with cell clusters 1, 2, 3 of different sizes flows in the direction of arrow A. An electrode device 12 and a sensor device 14, connected to a control device 13, are located in the main channel 11A. 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 assembly 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 unit 13, the electrode assembly 12 can generate a field barrier transverse to the flow A. The field barrier can be temporarily generated to match a cell assembly arriving with the flow. Through the interaction of the field barrier with the flow forces in the flow A, cell assemblies can be guided onto a predetermined flow path leading to one of the branch channels 11B (see, e.g., the dotted flow path B of the cell assembly 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 so that the cell clusters 1, 2, 3 are directed, according to their sizes, into one of the branch channels 11B and from there into one of the containers 21.

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

[0066] Alternatively or additionally to size fractionation using dielectrophoretic forces, at least one of the following separation methods can be employed. 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., with standing ultrasound waves) or optical separation (e.g., with optical tweezers).

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

[0068] The reference to the Figures 1 and 2The described size fractionation can be supplemented or replaced by fractionation based on other properties. For example, in a fluidic unit of a fractionation system, such as a field cage, a test of the permeability of cells in cell clusters to cryoprotectants and / or resistance to cryoprotectants can be performed. Depending on the test results, different fractions can be formed, which are subsequently subjected to cryopreservation with different process parameters. For example, cell clusters with low CPA permeability are treated with a longer CPA incubation time than cell clusters with increased CPA permeability.

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

Claims

1. A method for cryopreserving a plurality of cell clusters (1, 2, 3) of biological cells selected from cell-matrix clusters grown in vivo, spherical clusters of cells and cell-matrix clusters grown artificially in vitro, comprising the steps of: - fractionating the cell clusters (1, 2, 3) into at least two fractions (4) in dependency on at least one property of the cell clusters (1, 2, 3), wherein the fractionation of the cell clusters (1, 2, 3) comprises fluidic fractionation, in which the cell clusters (1, 2, 3) are separated in a fluid environment, and the fractionation of the cell clusters (1, 2, 3) comprises fractionation in a fluid flow, wherein the cell clusters (1, 2, 3) are arranged at different positions in a flow profile of the fluid flow under the effect of at least one of dielectrophoretic forces in the fluid flow, and sound waves in the fluid flow, - collecting the fractions (4) in different containers (21), and - cryopreserving the cell clusters (1, 2, 3) of the at least two fractions (4), wherein specific pretreatment methods and / or freezing methods are used for each fraction, wherein the pretreatment methods for the fractions (4) differ in terms of at least one of the pretreatment parameters including a duration, a temperature, a pressure, a medium composition, a gas supply composition, permeabilisation conditions and a movement of the medium of the pretreatment and the freezing methods for the fractions (4) differ in terms of at least one of the freezing parameters including a duration, a temperature, a pressure, a medium composition, a gas supply composition, and a movement of the medium of the freezing.

2. The method according to claim 1, wherein - the fractionation of the cell clusters (1, 2, 3) takes place on the basis of at least one of the properties including a size, a shape, a mass, an elasticity, a hydraulic conductivity, a cryoprotectant (CPA) permeability, a resistance to cryoprotectants, a chemical constitution and a cell composition of the cell clusters (1, 2, 3).

3. The method according to claim 1 or 2, wherein - the pretreatment methods for the fractions (4) differ in terms of the time profile of at least one of the pretreatment parameters.

4. The method according to any one of the preceding claims, comprising at least one of the following features: - the fractions (4) are collected in the containers (21) in which the cryopreservation subsequently takes place, - the frozen fractions (4) are provided for storage in a cryobank (40) without interrupting the cold chain, and - the fractionation and cryopreservation are carried out in an automated manner.

5. The method according to any one of the preceding claims, wherein - the at least one property of the cell clusters (1, 2, 3) and / or at least one state variable of the at least two fractions (4) are detected by means of sensing.

6. The method according to any one of the preceding claims, comprising the additional step of - thawing the cell clusters (1, 2, 3) in the at least two fractions (4), wherein specific thawing methods are used for each fraction.

7. A cryopreservation apparatus (100), which is adapted for cryopreserving a plurality of cell clusters (1, 2, 3) of biological cells selected from cell-matrix clusters grown in vivo, spherical clusters of cells and cell-matrix clusters grown artificially in vitro, comprising: - a fractionation device (10), which is adapted for fractionating the cell clusters (1, 2, 3) into at least two fractions (4) in dependency on at least one property of the cell clusters (1, 2, 3), wherein the fractionation device (10) comprises a fluidics device (11) which is adapted for separating the cell clusters (1, 2, 3) in a fluid environment, in particular in a fluid flow, and the fractionation device (10) comprises an electrode device (12) which is adapted to apply dielectrophoretic forces in a fluid flow, and / or comprises a sound source device which is adapted to generate sound waves in the fluid flow, - a container device (20) having at least two different containers (21), each of which is arranged for collecting one of the fractions (4), and - a freezing device (30), which is adapted for automated cryopreserving the cell clusters (1, 2, 3) in the at least two fractions (4), wherein the freezing device (30) is adapted for using specific pretreatment methods and / or freezing methods for each of the fractions (4) such that it comprises an incubation unit (31) for pretreating the fractions (4) and a cooling unit (32) for freezing the fractions (4), wherein the freezing device (30) is adapted to apply the pretreatment methods which differ in terms of at least one of the pretreatment parameters and / or the time profile of said parameter, said parameters including a duration, a temperature, a pressure, a medium composition, a gas supply composition, permeabilisation conditions and a movement of the medium during the pretreatment, and the freezing device (30) is adapted to apply the freezing methods which differ in terms of at least one of the freezing parameters, said parameters including a duration, a temperature, a pressure, a medium composition, a gas supply composition, and a movement of the medium during the freezing.

8. The cryopreservation apparatus according to claim 7, wherein - the fractionation device (10) is adapted for fractionating the cell clusters (1, 2, 3) on the basis of at least one of the properties including a size, a shape, a mass, an elasticity, a hydraulic conductivity, a cryoprotectant (CPA) permeability, a resistance to cryoprotectants, a chemical constitution and a cell composition of the cell clusters (1, 2, 3).

9. The cryopreservation apparatus according to claim 7 or 8, comprising at least one of the following features: - the at least two containers (21) are part of the freezing device (30), and - the cryopreservation apparatus (100) is adapted for automated operation.

10. The cryopreservation apparatus according to any one of claims 7 to 9, comprising - a sensor device (14) which is adapted for detecting the at least one property of the cell clusters (1, 2, 3) and / or at least one state variable of the at least two fractions (4).