METHOD FOR CRYOPRESERVING A PLURALITY OF CELL CLUSTERS OF BIOLOGICAL CELLS

Abstract

A method for cryopreserving a plurality of cell clusters (1, 2) of biological cells includes the steps of fractionating the cell clusters (1, 2) into at least two fractions (4) in dependency on at least one property 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 specific pretreatment methods and/or freezing methods are used for each fraction. A cryopreservation apparatus (100), for cryopreserving a plurality of cell clusters (1, 2) of biological cells, having a fractionation device, is also described.

Claims

1. A method for cryopreserving a plurality of cell clusters of biological cells, comprising the steps of: fractionating the cell clusters into at least two fractions in dependency on at least one property of the cell clusters, collecting the fractions in different containers, and cryopreserving the cell clusters of the at least two fractions, wherein at least one of specific pretreatment methods and specific freezing methods are used for each fraction.

2. The method according to claim 1, wherein the fractionating of the cell clusters takes place on a basis of at least one property selected from the group consisting of a size, a shape, a mass, an elasticity, a hydraulic conductivity, a cryoprotectant permeability, a resistance to cryoprotectants, a chemical constitution and a cell composition of the cell clusters.

3. The method according to claim 1, wherein the pretreatment methods for the fractions differ in terms of at least one pretreatment parameter selected from the group consisting of a duration, a temperature, a pressure, a medium composition, a gas supply composition, permeabilization conditions and a movement of a medium of the pretreatment.

4. The method according to claim 3, wherein the pretreatment methods for the fractions differ in terms of a time profile of at least one of the pretreatment parameters.

5. The method according to claim 1, wherein the freezing methods for the fractions 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.

6. The method according to claim 1, comprising at least one of the features: the fractionating of the cell clusters comprises fluidic fractionation, in which the cell clusters are separated in a fluid environment, the fractions are collected in the containers in which the cryopreserving subsequently takes place to provide frozen fractions, the frozen fractions are provided for storage in a cryobank without interrupting a cold chain, and the fractionating and cryopreserving are carried out in an automated manner.

7. The method according to claim 1, wherein the fractionating of the cell clusters comprises fractionation in a fluid flow, wherein the cell clusters are arranged at different positions in a flow profile of the fluid flow under an effect of at least one of flow forces of the fluid flow, dielectrophoretic forces in the fluid flow, and sound waves in the fluid flow.

8. The method according to claim 1, wherein at least one of the at least one property of the cell clusters and at least one state variable of the at least two fractions are detected by sensing.

9. The mMethod according to claim 1, further comprising the additional step of thawing the cell clusters in the at least two fractions, wherein specific thawing methods are used for each fraction.

10. A cryopreservation apparatus, which is configured for cryopreserving a plurality of cell clusters of biological cells, comprising: a fractionation device, which is configured for fractionating the cell clusters into at least two fractions in dependency on at least one property of the cell clusters, a container device having at least two different containers, each of which is arranged for collecting one of the fractions, and a freezing device, which is configured for cryopreserving the cell clusters in the at least two fractions, wherein the freezing device is configured for using at least one of specific pretreatment methods and specific freezing methods for each of the fractions.

11. The cryopreservation apparatus according to claim 10, wherein the fractionation device is configured for fractionating the cell clusters on a basis of at least one property selected from the group consisting of a size, a shape, a mass, an elasticity, a hydraulic conductivity, a cryoprotectant permeability, a resistance to cryoprotectants, a chemical constitution and a cell composition of the cell clusters.

12. The cryopreservation apparatus according to claim 10, wherein the freezing device is configured to apply the pretreatment methods which differ in terms of at least one of: (a) a pretreatment parameters; and (b) a time profile of said pretreatment parameter, wherein said pretreatment parameter is selected from the group consisting of a duration, a temperature, a pressure, a medium composition, a gas supply composition, permeabilization conditions and a movement of a medium during the pretreatment.

13. The cryopreservation apparatus according to claim 10, wherein the freezing device is configured to apply the freezing methods which differ in terms of at least one freezing parameter selected from the group consisting of a duration, a temperature, a pressure, a medium composition, a gas supply composition, and a movement of a medium during the freezing.

14. The cryopreservation apparatus according to claim 10, comprising at least one of the features the fractionation device comprises a fluidics device which is configured for separating the cell clusters in a fluid environment, the at least two containers are part of the freezing device, and the cryopreservation apparatus is configured for automated operation.

15. The cryopreservation apparatus according to claim 10, wherein the fractionation device comprises at least one of an electrode device which is configured to apply dielectrophoretic forces in a fluid flow, and a sound source device which is configured to generate sound waves in the fluid flow.

16. The cryopreservation apparatus according to claim 10, comprising a sensor device which is configured for detecting at least one of the at least one property of the cell clusters and at least one state variable of the at least two fractions.

17. The cryopreservation apparatus according to claim 14, comprising the fluidics device, which is configured for separating the cell clusters in a fluid flow.

Description

[0042] Further details and advantages of the invention will be described below with reference to the appended drawings. The drawings show, schematically:

[0043] FIG. 1: a sequence of the method for cryopreserving a plurality of cell clusters and components of a cryopreservation apparatus having features according to preferred embodiments of the invention; and

[0044] FIG. 2: a fractionation device which is adapted for the dielectrophoretic separation of cell clusters, according to an embodiment of the invention.

[0045] Features of preferred embodiments of the invention are described below by means of exemplary reference to the use of size-based fractionation of cell clusters. It is emphasized that, in practice, implementation of the invention is not limited to size-based fractionation but rather is also possible, alternatively or additionally, with fractionation based on another property of the cell clusters, as is described below with further examples. Details of the cell clusters and their preparation, and process parameters for cryopreservation and/or thawing parameters used in concrete examples are selected as is known per se from the cryopreservation of biological materials.

[0046] FIG. 1 shows steps S1 to S4 of the method for cryopreserving a plurality of cell clusters 1, 2, and the cryopreservation apparatus 100 used to this end, having a fractionation device 10, a container device 20 and a freezing device 30 according to preferred embodiments of the invention. FIG. 1 additionally shows a step S0 of preparing the cell clusters 1, 2 and a step S5 of storing the frozen cell clusters in a cryobank 40. The example shown in FIG. 1 is used to carry out for example an automated, fluidic size-based fractionation of the cell clusters 1, 2.

[0047] With step S0, an inhomogeneous sample is prepared, for example a mixture of cell clusters 1, 2 of different sizes and/or a mixture of cell clusters 1, 2 having different sensitivities to CPA. The cell clusters 1, 2 comprise for example organoids which were formed in a known way by culturing in a nutrient medium and using differentiation factors from adult stem cells and which for example have cross-sectional dimensions in the range from 10 .Math.m to 10 mm or greater. The cell clusters 1, 2 are prepared for example in a culture vessel.

[0048] With step S1, the cell clusters 1, 2 are separated into individual fractions 4 (fractions), each of which contains cell clusters of specific sizes, using the schematically depicted fractionation device 10. The fractionation device 10 is for example constructed as described below with reference to FIG. 2, and it is preferably operated in an automated manner. The fractions 4 are transferred into container 21 of the container device 20 in step S2. The containers 21 preferably comprise plastic tubes with a lid, in particular what are referred to as PP tubes, as are used in the subsequent cryopreservation in steps S3 and S4 (see illustration in step S5). Alternatively, the containers may comprise other receptacles, for example pouches or microtiter plates. According to a further alternative, the containers 21 may 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 particularly concentration (mass of cell clusters per unit volume of surrounding medium), is reached.

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

[0050] Pretreatment of the fractions takes place in the incubation unit 31. This means that a complete and individual incubation program is run for each fraction 4. In the process, at least a CPA (in particular a cryoprotectant) is added, with which the cell clusters are intended to be loaded. As the size of the cell clusters increases, for example increasing concentrations of CPA and/or increasing incubation times are used. Suitable cryoprotectants and the concentrations thereof can be determined using tests.

[0051] The incubation can furthermore comprise a predetermined temperature conditioning T(t), supply of gas and/or perfusion with predetermined cryoprotectant (CPA) concentration profiles C(t, CPA1, CPA2, ...). Alternatively or additionally, temporarily membrane-permeable and/or even toxic CPA can be supplied, if the cell clusters tolerate same. Furthermore, ice nucleation (for reducing and controlling supercooling), medium circulation (for homogenizing T and C), and/or permeabilization of the cell clusters of at least one fraction (for loading with non-membrane-permeable CPA) may be part of the pretreatment method. Permeabilization can take place for example chemically (for example by means of DMSO), using sound waves (sonoporation), using electric fields (electroporation), by means of liposomal substances and/or by thermomodulation via membrane phase conversion. Furthermore, the pretreatment in the incubation unit 31 comprises pre-cooling of the fractions 4 to a temperature above the freezing point of the fractions 4.

[0052] The incubation unit 31 preferably has individual receptacles for the containers 21, for example individual cavities, or provides the containers by reservoirs, preferably for fractions of the same volume. The incubation unit 31 includes a pump device for supplying CPA (sequential addition and/or concentration increase) and/or for discharging media from the containers. Furthermore, the incubation unit 31 is preferably provided with a drive, for example a stirring mechanism, for moving the media during the pretreatment in each container. Alternatively or additionally, a pre-cooling unit can be provided, which is adapted for supercooling fractions. Supercooling can induce membrane changes in the cells of the cell clusters, as a result of which the pretreatment, for example the uptake of CPAs, can be influenced. Alternatively or additionally, a sound source can furthermore be provided, by means of which the cell clusters of the fractions can be subjected to an ultrasonic treatment. The ultrasonic treatment can induce further membrane changes in the cells of the cell clusters, in particular permeabilization.

[0053] Further pretreatment parameters of the size-dependent incubation include for example concentrations of the individual CPAs, durations of action of the individual CPAs, concentration profiles over time of individual CPAs, adapted temperature profiles (> 0° C.), continual changes in the medium composition, for example by means of mixing devices in conjunction with the incubation unit 31 and a CPA reservoir, and/or a change in the surrounding medium (perfusion).

[0054] Subsequently, the fractions 4 are frozen in the cooling unit 32 (step S4). Depending on the properties of the cell clusters of the fractions 4, for example the size or other properties, such as the hydraulic conductivity of the individual components of the cell clusters, the proportion of membrane-permeable and osmotically-active additives in the medium and/or the supercooling, the individual fractions 4 are frozen in a controlled manner at different cooling rates and/or with different cooling profiles. For example, a cooling rate which is equal to or less than -1 K/min is used. The cooling takes place down to a cryopreservation temperature of for example -80° C. or lower, for example -140° C. or lower.

[0055] Temperature profiles for the freezing of individual fractions can be selected for example in order to set an adaptation to an equilibration rate, a controlled nucleation for reducing supercooling, and/or a homogeneous cooling rate, via the fraction volume (optionally with circulation of the medium and/or with use of a form-fit of the fraction container in the heat exchanger of the cooling unit 32).

[0056] For each fraction, 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 or a coolant flow cooler, operating for example with liquid nitrogen or isopentane. The cooling element is adapted for setting a defined cooling rate. The heat exchanger comprises for example a receptacle for the container of each respective fraction, preferably with a form-fit between the container and the receptacle. If the containers 21 are part of the incubation unit 31 of the freezing device 30, transfer into cryocontainers, for example said PP tubes, takes place before the freezing. The cooling unit 32 can optionally be provided with a nucleation apparatus, for example a cold needle, by means of which controlled nucleation can be induced in the container.

[0057] Finally, the containers are closed and the frozen fractions are stored in the cryobank 40 at cryogenic temperatures (for example -140° C.) (step S5). The transfer to the cryobank 40 takes place without interrupting the cold chain, for example using a cooled sluice or by directly coupling the freezing device 30 to the cryobank 40.

[0058] For thawing, the process shown in FIG. 1 is reversed using a thawing device (not shown). During thawing, like the size-adapted process for freezing, the individual fractions are also treated separately in different incubation units during the thawing and/or in a first recovery phase until the auxiliary agents are washed away. For example, a size-dependent thawing rate, a size-dependent incubation in a hyperosmolar thawing medium and/or a size-dependent washing out of the thawed fractions is provided. As a result, for example, the metabolism of relatively large cell clusters can be slowed by a cooled environment in order to ensure sufficient dilution of toxic, membrane-permeable CPAs, while this process can take place more quickly in the case of small cell clusters.

[0059] After the thawing, a portionation step can be provided in which the thawed fractions are subjected to a vitality test and, when vitality is detected, are transferred to a predetermined usage-dependent container format, for example microtiter plates or suspension bioreactors. In this case, the size-based fractionation can be maintained or dispensed with.

[0060] The thawing and/or portionation can be carried out for example using a fluidics device, in particular a fluidic microsystem.

[0061] FIG. 2 shows by way of example a fractionation device 10 in the form of a fluidics device 11, in particular a fluidic microsystem, having a main channel 11A and branching channels 11B, through which a suspension of a liquid surrounding medium having cell clusters 1, 2, 3 of different sizes flows, in the direction of arrow A. An electrode device 12 and a sensor device 14, which are connected to a control device 13, are located in the main channel 11A. The main channel 11A branches into the branching channels 11B, each of which are connected to a container 21 of the container device 20.

[0062] The electrode device 12 comprises two strip-like electrodes or electrode pairs, for example at the bottom and/or on a cover plate of the main channel 11A. When AC voltages from the control device 13 are applied to the electrodes, the electrode device 12 can produce a field barrier at right angles to the flow A. The field barrier can be generated temporarily to match a cell cluster arriving with the flow. By interaction of the field barrier with the flow forces in flow A, cell clusters can be guided onto a predetermined flow path which leads to one of the branching channels 11B (see for example the dotted flow path B of cell cluster 1).

[0063] The sensor device 14 is for example an optical sensor, in particular a camera connected to an image processing device. The cell clusters 1, 2, 3 and their respective sizes can be detected by means of the sensor device 14. Information regarding the positions and sizes of the cell clusters 1, 2, 3 is delivered to the control device 13. The control device 13 assigns the cell clusters 1, 2, 3 to three predetermined sizes of the desired fractions and controls the electrode device 12 such that the cell clusters 1, 2, 3 are each guided based on their sizes into one of the branching channels 11B and via same into one of the containers 21.

[0064] As an alternative to the embodiment in FIG. 2, the electrode device 12 can comprise a dielectrophoretic field cage and the sensor device 14 can be adapted to detect a cell cluster in the field cage. The cell clusters are detected one after the other in the field cage by means of sensors, assigned to one of a plurality of fractions on the basis of their properties, and guided into the fraction in question by releasing the field cage and optional further dielectrophoretic deflection.

[0065] Alternatively or additionally to the size-based fractionation using dielectrophoretic forces, at least one of the following separation methods may be provided for fractionation. Passive separation methods can include for example fractionation in the flow profile (for example PFF), density-based fractionation (for example sedimentation), and geometric fractionation (for example using screens). Active separation methods can include for example acoustic separation (for example with standing ultrasound waves) or optical separation (for example using optical tweezers).

[0066] Alternatively to optical sensors, for example an impedance measurement at the cell clusters may be provided (for example as in a “Coulter Counter” device), and the fractionation can be carried out on the basis of the result of the impedance measurement.

[0067] The size-based fractionation described with reference to FIGS. 1 and 2 can be supplemented by fractionation in relation to other properties, or replaced thereby. For example, in a fluidics device of a fractionation device, for example in a field cage of the fluidics device, a test of the permeability of the cells of cell clusters to cryoprotectants and/or a resistance to cryoprotectants can be carried out. In dependency on the test result, different fractions can be formed, which are subsequently subjected to cryopreservation using different process parameters. For example, cell clusters with low CPA permeability of the cells are treated using a longer CPA incubation duration than cell clusters with increased CPA permeability of the cells.

[0068] The features of the invention disclosed in the preceding description and in the drawings and claims may be significant, both individually and in combination or in subcombinations, for implementing the invention in the various embodiments thereof.