Device and method for manipulating biological cells, and method for manufacturing the device

Abstract

The present invention relates to a device (10) for manipulating biological cells, said device (10) comprising at least one container (22) for culturing biological cells, said container (22) having an internal space (11) and at least one electrode (14) for manipulating said biological cells, said device (10) comprising at least one separator layer (20) arranged at least between said at least one electrode (14) and said internal space (11) such that said at least one electrode (14) is arranged outside said internal space (11). The present invention further relates to a method (300) for manipulating biological cells. The present invention provides a device (10) and a method (300) for manipulating biological cells, which avoids deterioration of cells in the container (22) during manipulation of cells by an electric field.

Description

[Technical field]

[0001] The present invention relates to devices and methods for manipulating biological cells and methods for making the devices. [Background technology]

[0002] For the production of organoids and surface tissue systems, biological cells need to be arranged in a defined proximity, grouped, maintained in a well-maintained stable formation, or contacted with a surface in a defined manner in a reproducible and highly parallel arrangement.In automated systems or lines, 96, 384 and 1536 well microwell plates with well volumes of 100μl-300μl, 30μl-100μl and 5μl-15μl are used for the growth and culture of biological cells in stochastic arrangements.

[0003] For cell engineering and medical applications, cell densities are typically around 10 5 pieces / ml (i.e. 10 2 The cell numbers vary widely from tens of thousands in a well of a 96-well microwell plate to as few as 1000 in a well of a 1536-well microwell plate. These cells may distribute stochastically in the nutrient solution in the well and settle to the bottom of the well.

[0004] According to G. Fuhr and T. Schnelle, Dielektrische Mikrofeldkafige, Physikalische Blatter 57 (2001) Nr. 1, pages 49 to 52, it is known to generate defined proximity relationships for these cells or aggregates of these cells in the wells of a microwell plate by means of a microelectrode system. The microelectrode system can generate an electromagnetic field cage for contactlessly manipulating the biological cells in the wells. The cells can then form aggregates. Furthermore, the cells can also be positioned with micrometer precision and / or suspended in a nutrient solution.

[0005] However, electrode processes occurring in the vicinity of the microelectrodes cause metal ions to dissolve from the electrodes into solution, resulting in alkalized and acidified regions in the solution, as well as temperature gradients in the range of 3°C to 10°C that are highest near the electrode surface and decrease toward the electric field minimum between the electrodes. These processes cause mammalian or human cells to deteriorate after 30 minutes of continuous use of an electric field.

[0006] Therefore, there is a need for a device and method for manipulating biological cells, and a method for manufacturing the device, that can avoid degradation of the cells in the wells during manipulation of the cells by electric fields.

[0007] The problem of the invention is solved by the subject matter of the independent claims. Further embodiments are incorporated in the dependent claims.

[0008] The present invention provides a device for manipulating biological cells, the device comprising at least one container for culturing biological cells, the container having an internal space and at least one electrode for manipulating the biological cells, the device comprising at least one separator layer, the at least one separator layer being disposed at least between the at least one electrode and the internal space such that the at least one electrode is disposed outside the internal space.

[0009] The present invention provides a separator layer between at least one electrode for manipulating biological cells and the interior space of at least one container. The at least one electrode is assigned to the at least one container, so that each container of the device has its own electrode. Moreover, the at least one electrode is disposed outside the interior space of the container and has no direct contact with the interior space. Thus, the separator layer shields the interior space from the electrode material, so that the electrode does not physically contact with the suspension (containing the nutrient solution and cells) that may be disposed in the interior space of the at least one container. Thus, the at least one electrode may be referred to as a dry electrode. The at least one electrode capacitively couples an electric field into the liquid disposed in the interior space. In other words, the electrode couples the electric field to the liquid indirectly, i.e., without charge transfer or flow between the electrode and the liquid. As a result, the pure displacement current in the electrode without the involvement of an ohmic contact is sufficient to provide a sufficiently strong electric field to manipulate the biological cells in the container. In this specification, the term cell refers to a biological cell. During this particular application of the electric field, electrode processes are avoided. The lack of cell degradation places no limit on the amount of time that at least one electrode can apply an electric field to a cell.

[0010] By way of example, the at least one electrode may be a microelectrode.Further by way of example, the at least one reservoir may be a well of a microwell of a microwell plate.

[0011] As an example, at least one bottom element of at least one container may comprise a separator layer and at least one electrode may be arranged outside the interior space in the bottom element.

[0012] At least one container bottom element can be attached to the container wall to close the container bottom in a liquid-tight manner. Furthermore, the bottom element can only comprise a separator layer, which is the bottom element. And, the at least one electrode is provided below the bottom element. One side of the bottom element can face the interior space, and the opposite side of the bottom element can face the at least one electrode. This arrangement can effectively shield the interior space from the electrode material.

[0013] In another embodiment, the at least one bottom element has a thickness, at least in said at least one electrode, of 200 μm or less, preferably 100 μm or less, more preferably 25 μm or less.

[0014] Also, reducing the thickness of the bottom element reduces relaxation of the electric field of the at least one electrode in the bottom element.

[0015] In a further example, at least one electrode may be disposed between the bottom element and the electrically insulating layer.

[0016] The electrically insulating layer can shield the at least one electrode from a user or from further devices below the device for manipulating biological cells. Additionally, the insulating layer can stabilize the bottom element and the device for manipulating biological cells.

[0017] Furthermore, the at least one electrode may, for example, be at least partially disposed within the at least one container and covered, at least within the container, with a separator layer.

[0018] A compartment with at least one electrode may be disposed within the container. Further, the entire surface of the electrode disposed within the container may be covered with a separator layer, i.e., the separator layer separates the electrode from the interior space. The surface of the electrode does not directly contact the nutrient solution or the cells disposed within the interior space.

[0019] In another example, the separator layer may contain a material having a relative dielectric constant in the range of 10 to 10,000, preferably 10 to 1,000, and more preferably 20 to 500, for a voltage / current waveform having a frequency in the range of 1 kHz to 10 MHz, and preferably has ferromagnetic properties.

[0020] The term dielectric constant is also known as the dielectric constant. The higher the dielectric constant of the separator layer, the higher the electric field in the interior space that is generated by the at least one electrode. Furthermore, the higher the dielectric constant, the thicker the separator layer can be without changing the strength of the electric field in the interior space. A high dielectric constant also allows a reduction in the voltage applied to the at least one electrode to generate the electric field.

[0021] The separator layer may contain, for example, particles having a crystalline structure, preferably microcrystalline, with a uniform size in the range of 100 nm to 1000 nm, preferably 300 nm, or particles with different sizes in the range of 100 nm to 1000 nm, preferably 300 nm and 700 nm.

[0022] The particles may have a high relative dielectric constant, for example, greater than 100 and not greater than 10,000, and the particles are embedded in the main material of the separator layer, which has a relative dielectric constant of about 10 or more. In this way, the particles can increase the overall relative dielectric constant of the separator layer.

[0023] In a further example, the particles may be arranged in a columnar manner within the separator layer between two opposing faces of the separator layer.

[0024] If the particles have a higher dielectric constant than the primary material of the separator layer, such an arrangement of particles within the separator layer may further increase the dielectric constant of the separator layer.

[0025] In another example, the separator layer is made of an alkaline earth metal titanate, preferably CaTiO3, SrTiO 3、 BaTiO 3、 Ba1-x Sr x TiO3 and / or combinations thereof may be included, preferably in an amount of 10% to 60% by volume, more preferably 30% to 50% by volume, and most preferably 40% or less by volume.

[0026] The alkaline earth metal titanate may be, for example, microcrystalline as described above. These titanates have a high dielectric constant. The titanate ratio allows for a separator with a high dielectric constant and for the separator material to remain optically transparent. Since the separator material is located at the bottom of the container, the optical transparency allows for the use of optical means to observe the cells within the container.

[0027] By way of example, the separator layer may contain at least one polymer, preferably a cyanoresin, more preferably cyanoethyl pullulan (CRS), cyanoethyl poly(vinyl alcohol) (CRV) and / or cyanoresin type M (CRM), and even more preferably a polymer having a dielectric constant greater than 10.

[0028] Such at least one polymer simplifies the manufacture of the separator layer. At the beginning of the manufacturing process, the polymer is liquid. Particles for increasing the overall dielectric constant may be mixed with the liquid polymer. Curing of the polymer (e.g., by UV irradiation) can fix the orientation and position of the particles within the polymer.

[0029] The separator layer may have an overall relative dielectric constant in the range of 10 to 200, preferably 16 to 120, and more preferably 20 to 120, for a voltage-current waveform having a frequency in the range of, for example, 1 kHz to 10 MHz.

[0030] Also, the electric field lines from at least one electrode can pass through the separator layer without significant attenuation. This simplifies the manipulation of cell or cell group manipulation in the container. In the context of this specification, the terms cell group, cell aggregate, cell mass and organoid are used synonymously. Furthermore, the high dielectric constant of the separator layer can reduce the voltage required to manipulate the electric field to a range that is not dangerous to the user.

[0031] In a further example, the separator layer may include at least one curved region in the interior space.

[0032] Furthermore, the at least one curved region may, for example, include at least one convex portion and / or at least one concave portion.

[0033] The surface region between the separator layer and the interior space is a material boundary between materials with different dielectric constants. The electric field lines from at least one electrode refract at this material boundary. The shape / geometry of the surface region can be configured to obtain a desired electric field pattern within the vessel. For example, a curved region can cause the electric field lines in the interior space to have a different shape than a flat surface region. This can simplify and / or support the formation and / or manipulation of cell groups.

[0034] In another example, the separator layer may include at least two regions, each of the at least two regions having a different dielectric constant, and the at least two regions preferably having different materials and / or different material mix ratios.

[0035] The different regions can shape the electric field lines in the interior space to simplify and / or support the formation and manipulation of cells or cell groups. In combination with regions having curved surfaces, the simplification and / or support effect is further increased. In particular, cells or cell groups with different distribution patterns can be formed in this manner.

[0036] At least one electrode may comprise, for example, a base metal, especially aluminum or nickel; an alloy of a base metal; or at least one plotter-writable conductive ink or paste.

[0037] Since the at least one electrode does not come into contact with the inner space, especially with the nutrient solution of the cells, the material of the electrode does not need to have the properties of a precious metal. Moreover, alloys or toxic metals may also be used in the manufacture of the electrode. That is, the at least one electrode can be manufactured from cheaper materials. The manufacture of the electrode can be simplified by using a plotter-writable conductive ink or paste.

[0038] In a further example, the device may comprise at least two electrodes, preferably at least four electrodes in a quadrupole configuration, or at least eight electrodes in an octupole configuration.

[0039] The electric fields from these electrodes can simplify and / or support the manipulation of cells or groups of cells. The use of quadrupole or octopole configurations allows for the use of electromagnetic field cages. These cages can be used to collect, shape, position and suspend cells or groups of cells within a container in a contactless manner, typically to form a desired cell distribution pattern.

[0040] Furthermore, the electrodes may be, for example, electrically connected to different phases of a multi-phase voltage supply or electrically connected in pairs to one phase of a multi-phase voltage supply.

[0041] When the electrodes are connected in pairs of electrical connections to one phase of a multi-phase voltage supply, each pair may be connected to a different phase of the multi-phase voltage supply. The paired or singular connection of electrodes to different phases simplifies the manipulation, particularly the positioning and levitation, of cells or cell groups.

[0042] In another example, at least one electrode has at least one segment, which is cross-shaped or Y-shaped.

[0043] In a further example, the at least one electrode comprises an end piece having a circular, triangular, square, or T-shaped cross-sectional area.

[0044] Further, for example, the at least one electrode may be straight or zigzag and / or have a plurality of, preferably triangular shaped, protrusions extending along the base element.

[0045] The shape of the electrodes and / or the end pieces of the electrodes influences the electric field generated in the interior space. Thus, the shape of the electrodes and / or the end pieces may allow for simplification of the manipulation of cells or groups of cells. The above mentioned shapes may be combined in any reasonable combination.

[0046] As an example, the device may comprise a number of reservoirs and a number of electrodes, at least one of the electrodes being disposed in each of the reservoirs, the device preferably being formed as a microwell plate.

[0047] The device is capable of manipulating cells and / or groups of cells in all of the vessels simultaneously, and therefore may monitor multiple different conditions in multiple different cells and / or vessels.

[0048] In a further example, a first group of the plurality of electrodes may be electrically connected to a first phase of a multi-phase voltage supply via a first wire and a second group of the plurality of electrodes may be electrically connected to a second phase of the multi-phase voltage supply via a second wire, with at least one electrode of each group disposed on each of the containers.

[0049] As a result, the electrodes can generate the same electric field in each container. If the separator layers in each container have the same properties, i.e., the same surface shape, the same thickness, and the same dielectric constant, etc., the electric field in the interior space of the containers will be the same.

[0050] The device may further include at least one energy storage device and at least one electronic circuit for generating a voltage having a frequency, for example, in a range of at least 1 kHz to 10 MHz, where the at least one electronic circuit electrically couples the at least one electrode to the at least one energy storage device.

[0051] This creates an alternating electric field in the interior space, which allows the movement of cells or groups of cells within the container by positive or negative dielectrophoresis, depending on the frequency of the applied electric field and the physiological conductivity of the cells within the interior space.

[0052] Frequencies in the range of 1 kHz to 10 MHz are also called radiofrequency or high frequency in the context of this specification. These high frequencies avoid the induction of thermal particles in the suspension. Furthermore, the high frequency of the alternating electric field reduces the stress on the living cells caused by the application of the electric field.

[0053] In another example, the at least one electrode may be disposed on a first module and the at least one electronic circuit may be disposed on a second module that is removable from the first module, and the at least one electrode is electrically connected to the at least one electronic circuit via removable electrical contacts disposed between the first module and the second module.

[0054] The arrangement of detachable electrical contacts between the at least one electrode and the at least one electronic circuit can facilitate the manufacture of the device. Furthermore, the first module is detachable from the second module. This allows for flexible arrangement of the electronic circuitry relative to the container.

[0055] The removable electrical contacts may include a gold material. Additionally, the removable electrical contacts may include spring contacts.

[0056] Additionally, the electrical contacts may include miniature digital electronic components.

[0057] For example, the device may include at least one conductive or insulating island element disposed in electrical isolation from at least one electrode on the container, where a separator layer separates the conductive or insulating island element from the interior space.

[0058] Conductive islands can concentrate the electric field lines. Thus, when a material exhibiting positive dielectrophoresis is included in the nutrient solution, this material will gather at the concentrated electric field lines (regions of higher or highest electric field line density). Insulating islands defocus the electric field lines. Thus, materials exhibiting negative dielectrophoresis will gather at the defocused electric field lines (regions of lower or lowest electric field line density). This further simplifies the manipulation of cells or cell groups.

[0059] In other examples, the at least one conductive or insulating island element may have a length in the range of 1 μm to 200 μm.

[0060] Further, by way of example, the interior space may comprise at least one bead, tube and / or wire.

[0061] The cells may aggregate around the beads, tubes and / or wires. Thus, as the cell aggregates grow, the beads, tubes and / or wires may provide channels for the nutrient solution into the cell aggregates. As a result, the cells in the center of the cell aggregates remain in contact with the nutrient solution. The beads, tubes and / or wires may contain, for example, natural or artificial semi-permeable materials.

[0062] In a further example, at least a portion of the at least one electrically conductive insulating island element includes a biofunctional surface that promotes aggregation of cells on the island in a nutrient solution.

[0063] The biological cells may for example be stem cells, preferably induced pluripotent stem cells.

[0064] Such cells can be used to grow tissue in a container, and the device can simplify and control the growth of tissue from these cells.

[0065] According to another aspect of the present invention, there is provided a method for manipulating biological cells using the device described above, the method comprising the steps of: introducing a suspension containing at least one biological cell into at least one container; applying an electrical signal to at least one electrode to generate a variable electric field; and using the variable electric field to move the at least one biological cell to a predetermined position within the container, thereby manipulating the at least one biological cell.

[0066] An electric signal (e.g., voltage) applied to the at least one electrode generates an electric field that extends through the separator layer of the device and ultimately into the interior space of the container. The variable electric field can exert a force on at least one biological cell in the interior space by positive or negative dielectrophoresis. The force can move the at least one biological cell in the interior space. The movement can occur in any direction in the suspension depending on the strength, direction, and frequency of the variable electric field at the location of the at least one biological cell. Depending on the presence or absence of positive or negative dielectrophoresis, the cell moves toward a maxima or minima of the electric field. Thus, the positioning of the maxima or minima of the electric field can purposefully and precisely position the cell at a specific location in the interior space. Furthermore, multiple cells can be collected at the location to form a cell group. Since the separator layer separates at least the electrodes that generate the variable electric field from the interior space (i.e., the cells and the nutrient solution), deterioration of the cells is avoided.

[0067] As an example, the electrical signal may have a voltage peak-to-peak value in the range of greater than 4V to less than 100V, preferably greater than 10V to less than 100V, and more preferably greater than 10V to less than 50V.

[0068] In another example, the at least one electrical signal may be applied continuously or discontinuously, preferably in a pulsed manner, thereby reducing heat generation at the electrodes, which in turn reduces heating of the device.

[0069] In a further example, the device comprises at least two electrodes and the electrical signal can be configured such that the at least two electrodes can generate an electric field with a stable temporal pattern of minima and maxima within the container. The generation of an electric field with a stable temporal pattern can result in a force being exerted on cells or particles in suspension, further simplifying the manipulation of cells or groups of cells.

[0070] The electric field may, for example, exert a force in the range of 1 pN to 1000 pN on cells within the container at a distance of 10 μm to 5 mm from the proximal surface of at least one electrode.

[0071] Additionally, the at least one cell may, for example, migrate to at least a minimum of the electric field.

[0072] For example, the at least one cell can be polarized by an electric field.

[0073] Polarization of the at least one cell results in positive or negative dielectrophoresis, causing the at least one cell to move toward the maximum or minimum of the electric field lines, respectively.

[0074] In one example, the suspension includes at least two cells, and the cells in the suspension form at least one cell aggregate by the electric field.

[0075] Furthermore, at least one cell aggregate may be precipitated, for example, to the bottom of the container, by the reduction of the strength of the electric field. Thus, cell aggregates may be formed when the cells are suspended. The formed cell aggregate may be precipitated to position the cell aggregate at the bottom of the container.

[0076] In a further example, the electrical signal is switched off as soon as at least one cell aggregate has settled, such that no electric field is present during further growth of the biological cells of the cell aggregate.

[0077] In another example, the electric field may exert a collecting force on at least one precipitated cell aggregate, thus holding the cells of the cell aggregate together during cell growth.

[0078] According to another example, the electric signal may be applied constantly or intermittently with an overall short application time of the electric field. The short application time may correspond to a few hours, preferably the time the cells are maintained in the well. The short application time of the electric field may have a switch-on time in the range of 1 ms to 1000 ms and a switch-off time in the range of 1 ms to 60 minutes.

[0079] As an example, after the step of introducing the suspension into the container, the suspension may be conditioned with at least one group of cells, which may enhance the chemical reactions of the suspension to optimize cell growth.

[0080] Furthermore, after adjusting the suspension, the at least one cell group may be separated and aggregated, for example, by an electric field in the container. In this example, at least one additional cell is introduced into the suspension, and the at least one additional cell is moved to the minimum of the electric field and held there for a predetermined time. The at least one cell is positioned at the maximum of the electric field, so that the at least one cell and the at least one additional cell are separated from each other. This allows the formation of more than one cell group in one container for co-culture.

[0081] As an example, at least one cell can be observed under a microscope in a container and the behavior and growth of the cell can be recorded.

[0082] In another example, the method further comprises the following steps prior to the step of introducing the suspension: generating positive or negative dielectrophoresis for at least one biological cell by selecting a suspension based on a desired electrical conductivity and selecting a frequency of a variable electrical signal.

[0083] As an example, at least one cell may be lifted and moved within the container by negative dielectrophoresis.

[0084] Furthermore, the method may further comprise, after the step of applying an electrical signal to the at least one electrode, for example at least one of the following steps: cooling the at least one electrode by direct contact with a coolant during frozen storage of the suspension; and / or heating the at least one electrode when thawing the frozen suspension by supplying at least one electrical signal with a frequency in the range of 1 kHz to 10 MHz to the at least one electrode.

[0085] The separator layer may be formed to have a high thermal conductivity so that the suspension can be cooled quickly, thus simplifying frozen storage of the suspension.

[0086] In other examples, at least one cell may be electrically and / or dielectrically measured by induced polarization due to an electric field.

[0087] The measuring may further comprise generating an electric field sufficient to induce polarization of the at least one cell. After switching of the electric field, the at least one electrode may measure the decay of the induced polarization.

[0088] Furthermore, the biological cells may be stem cells, preferably induced pluripotent stem cells.

[0089] According to another aspect of the present invention, there is provided a method of manufacturing a separator layer of the device described above, the method comprising the steps of: providing at least one liquid polymer having a first dielectric constant; mixing a plurality of particles, preferably crystallites, having a second dielectric constant with the at least one liquid polymer to obtain a separator layer mixture; and curing the separator material mixture to obtain the separator layer.

[0090] The plurality of particles and at least one liquid polymer have different dielectric constants, the polymer having a lower dielectric constant than the particles. The particles can be introduced into the polymer to obtain a mixture having a dielectric constant between the first and second dielectric constants, i.e., the mixture has a third dielectric constant higher than the first dielectric constant. The cured separator material mixture relaxes / attenuates the electric field less than the polymer. Overall, the method provides a device that avoids cell degradation during manipulation of the cells by an electric field.

[0091] For example, an electric field may be applied to the separator material mixture during at least a portion of its curing process to align, aggregate, and chain form particles within the liquid polymer to produce ordered columnarly aligned particle domains and / or particle agglomerates within the separator layer. The ordered columnarly aligned particle domains and / or particle agglomerates further increase the dielectric constant of the separator layer compared to a homogenous mixture of particles and liquid polymer. The electric field may be an alternating electric field.

[0092] In another example, in the step of mixing the at least one liquid polymer with the plurality of particles, the particles have a uniform size and preferably a volume ratio of 40% or less to the liquid polymer. When an electric field is applied during curing of the separator layer material, the uniformly sized particles produce columns with a uniform shape. The uniformly sized particles form chains that follow the electric field lines, producing the columns.

[0093] In a further example, in the step of mixing the particles with at least one liquid polymer, the particles may have at least two different sizes. When the two particle fractions form chains along the electric field lines, the particles form a non-uniform column. During the application of the electric field, the smaller particles move between the larger particles, thereby forming a column with a non-uniform shape. As a result, rigid pre-polarization of the cells can be avoided.

[0094] According to one example, the separator material mixture is cured to form a separator layer, and an electric field is applied perpendicular to the separator material mixture during curing, causing columns of particles to extend perpendicular to the separator layer, and in the device, one end of the columns may face an electrode and an opposite end of the columns may face an interior space.

[0095] As an example, prior to curing of the separator material mixture, the separator material mixture is applied onto at least one electrode in a sealing layer, the application method being preferably sputtering, spin coating, screen printing and / or a sol-gel process.

[0096] The sealing layer covers a surface portion of the electrode disposed within the container, and after the layer is complete, the electrode is isolated from the interior space of the container, thereby behaving as if it were disposed outside the interior space of the container.

[0097] In yet another aspect of the invention, there is provided a method of manufacturing the device as described above, the method comprising the steps of providing a vessel for culturing biological cells, providing a layer of a separator layer, and providing at least one electrode for forming and manipulating the biological cells, the electrode being isolated from an interior space of the vessel by the separator layer.

[0098] The advantages and effects of the method for manufacturing the device as well as further developments are obtained by the advantages and effects of the method for manufacturing the device as described above as well as further developments, therefore, reference is made to the previous part of the description.

[0099] As an example, based on the above description, the separator layer can be manufactured based on the method for manufacturing the separator layer of the device.

[0100] In yet another aspect of the invention there is also provided the use of a device according to the above description for the manipulation of living cells.

[0101] Additionally, in yet another aspect of the present invention, there is provided the use of a variable electric field capacitively coupled from outside a container containing a suspension containing at least one biological cell to manipulate the biological cells in the suspension.

[0102] The use of the above devices to manipulate biological cells and / or variable electric fields opens up a variety of applications not possible with prior art devices. For example, the period of electric field application can be extended indefinitely while the electric field is applied, avoiding degradation of mammalian or human cells. This can aggregate and shape cells in ways that were not possible before.

[0103] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. [Brief description of the drawings]

[0104] [Figure 1a-b] 1a and 1b are schematic diagrams of a device having a reservoir and for manipulating biological cells. [Figure 2a-f] 2a-f are schematic diagrams of examples of at least one electrode. [Figure 3a-b] 3a and 3b are diagrams of the electrophoretic and device reservoirs. [Figure 4a-f] 4a-f are schematic diagrams of the examples of FIG. 2a-f including cell aggregates. [Figure 5a-f] 5a-f are schematic diagrams of the examples of FIG. 2a-f including cell aggregates with positive electrophoresis. [Figure 6a-f] 6a-f are schematic diagrams of at least further examples of electrodes. [Figure 7]FIG. 7 is a schematic diagram of an example of an electrode connecting multiple containers. [Figure 8] FIG. 8 is a schematic diagram of another example of an electrode connecting multiple containers. [Figure 9] FIG. 9 is a schematic diagram of one example of electrical contacts for electrodes on a device. [Figure 10a-b] 10a and 10b are schematic diagrams of an example of a first and second module of a device. [Figure 11a-b] 11a and 11b are schematic diagrams of an example of a device having electronic circuitry. [Figure 12] 12a-d are schematic diagrams of an example of the internal structure of a separator layer. [Figure 13] 13a-c are schematic illustrations of the formation of the columnar structures of FIGS. 12b and 12d. [Figure 14a-c] 14a-c are schematic illustrations of the formation of cell aggregates within the reservoirs of the device. [Figure 15] 15a-d are schematic illustrations of different states of manipulation of cells within a vessel. [Figure 16] 16a-c are schematic diagrams of vessels with islet elements for generating and engineering vascular organoids from cells. [Figure 17] 17a-d are schematic diagrams of the preparation and execution of cryopreservation of cell aggregates. [Figure 18] 18a-c are schematic illustrations of the use of capture particles for the formation of organoids from cells. [Figure 19] 19a-d are schematic diagrams of examples of separator layers with surfaces in the interior space. [Figure 20a-d] 20a-d are schematic diagrams of examples of different ratios of the dielectric constants of separator layers and materials within the interior space. [Figure 21] FIG. 21 is a schematic diagram of an example with a separator layer and materials within the interior space having the same dielectric constant. [Figure 22a-b] 22a, 22b are schematic illustrations of the levitation effect of the device on cells (b) compared to the prior art (a). [Figure 23a-b]23a and 23b are schematic diagrams of examples of separator layers and isolation elements between electrodes. [Figure 24a-b] 24a and 24b are schematic diagrams of further examples of separator layers and isolation elements between electrodes. [Figure 25a-b] 25a and 25b are schematic diagrams of examples of the effect of the shape of the separator layer and the isolation elements between the electrodes on cell aggregates. [Figure 26] 26a and 26b are schematic diagrams showing examples of the effect on cells of four electrodes operated at reservoir voltages. [Figure 27a-b] 27a and 27b are schematic diagrams of examples of containers with a central trap. [Figure 28] FIG. 28 is a schematic diagram of an example device having a standoff layer between the electrodes and the separator layer. [Figure 29] FIG. 29 is a schematic diagram of a dielectric anisotropy switchable separator layer. [Diagram 30] FIG. 30 shows an example of a method for engineering living cells. [Diagram 31] FIG. 31 shows an example of a method for fabricating the separator layer of the device. [Diagram 32] FIG. 32 shows an example of a method for fabricating a device.

[0105] In the following description, the reference numeral 10 generally refers to a device for manipulating biological cells.

[0106] 1a is a top view of an example of a device 10. The device 10 in this example comprises a microwell plate in which 96 vessels 22 are arranged as wells. The number of vessels 22 is exemplary. Thus, the number of vessels 22 can be, for example, 6, 12, 24, 48, 384, 1536 or 3456 or any other number.

[0107] The containers 22 are attached to the base element 21. Each container 22 comprises an interior space 11 capable of containing a nutrient solution 15 and a cell suspension containing biological cells.

[0108] As shown in Fig. 1b, separator layer 20 of bottom element 21 comprises an upper surface 12 facing interior space 11. Cells may settle on upper surface 12. Lower surface 13 of separator layer 20 faces away from interior space 11. Furthermore, lower surface 13 may be in contact with the atmosphere outside device 10.

[0109] The separator layer 20 has a high relative dielectric constant, especially at the position of the electrode 14. The relative dielectric constant may be 10 to 10,000, preferably 10 to 1,000, and more preferably 20 to 500, for a voltage-current waveform having a frequency in the range of 1 kHz to 10 MHz. The separator layer 11 may contain a material having ferromagnetic properties.

[0110] The separator layer 20 may contain particles having a high dielectric constant. These particles may be, for example, alkaline earth metal titanates, preferably CaTiO3, SrTiO3, BaTiO3, Ba 1-x Sr x It may contain TiO3 and / or a combination thereof. The material mixture of the separator layer 20 may contain these particles in a ratio of 10% to 60% by volume, more preferably 30% to 50% by volume, and most preferably 40% or less by volume.

[0111] Table 1 below shows the relative dielectric constants of the titanate family.

[0112] [Table 1]

[0113] These particles may be embedded in the polymer of the material mixture. The polymers used for the bottom elements 21 of the container 22 have a low dielectric constant, i.e., they attenuate the electric field that propagates through them.

[0114] Particles embedded in a polymer and having a high dielectric constant can increase the overall dielectric constant of the material mixture, which therefore reduces relaxation of the electric field extending through the bottom element.

[0115] In this example, the container 22 has four electrodes 14. The electrodes 14 are not disposed within the interior space 11 of the container 22. Instead, the electrodes 14 are formed on the lower surface 13 of the separator layer 20. That is, the separator layer 20 is disposed between the electrodes 14 and the interior space 11.

[0116] In another example, electrode 14 may be disposed on top surface 12. Separator layer 20 may also cover the entire surface portion of electrode 14 adjacent to interior space 11. That is, in this example, separator layer 20 is also disposed between interior space 11 and electrode 14, such that electrode 14 is disposed outside interior space 11.

[0117] In the four-electrode example of FIG. 1b, two electrodes 14 may form a pair. For example, two electrodes 14 on opposite sides of the container 22 may be paired. An alternating potential Φ1, Φ2 may be applied to each pair of electrodes 14, such that these pairs of electrodes 14 generate two alternating electric fields. The frequency of the alternating electric fields may be in the radio frequency spectrum, which is a radio frequency electric field sometimes also called a short wave electric field. The alternating potentials of each electrode 14 in a pair of electrodes 14 may have the same phase. The phase difference between Φ1 and Φ2 may be, for example, 180°.

[0118] The container 11 may be filled with a cell suspension 15. The dashed oval represents the meniscus of the suspension 15. The suspension 15 fills half of the interior space 11.

[0119] Radio frequency electric fields can be achieved by varying the frequency, field strength and electrode configuration, where the resulting electric field polarizes biological cells in suspension, and polarization occurs by the electric field exerting a force on the cells, which acts without physical contact.

[0120] These forces cause the cells to form aggregates 16, 17, 18. In this example, the cell aggregates 17 float at a height h that corresponds approximately to the distance between the electrodes 14 in the center of the container 11. The height h can be adjusted between 1 μm and several mm by adjusting the electric field strength.

[0121] Cellular aggregates 16 may accumulate at the bottom of the vessel 11 between the electrodes 14. Cellular aggregates 18 may be attracted to and above the electrodes or into the narrowest gaps.

[0122] For contactless, non-mechanical generation of forces acting on the cells, metal electrodes with widths of 10 μm to 1 mm and corresponding supply lines may be fabricated on the lower surface 13 of the separator layer 20. Fabrication can be performed, for example, by semiconductor techniques, screen printing, spotting, etc.

[0123] The total height of the microwell plate may be in the range of, for example, 5 mm to 20 mm. The depth of the internal space 11 may be in the range of, for example, 1 mm to 15 mm. The diameter of the internal space 11 may be in the range of, for example, 0.5 mm to 10 mm.

[0124] 2a-2f are plan views showing various electrode shapes relative to the lower surface 13. The ring shows the position of the wall of the container 22, e.g. a cylinder, relative to the electrode 14. The electrode 14 has an end 26 in the center of the container 22.

[0125] Figure 2a shows a linear shape of the electrode 14. The electrode 14 may be operated with more than two phases Φ1, Φ2, Φ3, Φ4 as shown in Figure 2a. Or, the electrode 14 may be operated with a pure alternating electric field with two phases Φ1, Φ2 as shown in Figure 2b. The electrode 14 shown in Figure 2b has a cross shape. The electrode 14 in Figure 2c has a Y shape.

[0126] 2d-2f, the end 26 may have a number of circular, T-shaped, and / or triangular shaped protrusions extending along the bottom element 21. These protrusions may shape the electric field generated by the electrode 14.

[0127] The generated pattern of electromagnetic field maxima and minima of a high frequency voltage-current waveform having a frequency in the range of 1 kHz to 10 MHz can be predetermined by shape and / or surface area.

[0128] Depending on the induced polarization of the cells, choosing the frequency of the electric field and / or the conductivity of the suspension (i.e., cell medium) results in positive or negative dielectrophoresis, which describes the type of force imparted to the cells via the electric field and the movement of the cells.

[0129] Figure 3a is a diagram of a cell spectrum showing the force as a function of frequency, which is plotted logarithmically, in which three curves a), b) and c) show different electrical conductivities of the suspension 15. Curve a) shows the suspension 15 with the lowest electrical conductance and curve c) shows the suspension 15 with the highest electrical conductance.

[0130] The polarization of the cells depends inter alia on the conductivity of the suspension and on the frequency of the electric field. The polarization in the region of arrow 31 is the opposite of the polarization in the region of arrow 32. That is to say, in curves a) and b) the polarization of the cells may switch depending on the frequency of the applied electric field.

[0131] Furthermore, polarization of cells in the region of arrow 31 exhibits positive dielectrophoresis. Polarization of cells in the region of arrow 32 exhibits negative dielectrophoresis. Thus, if suspension 15 has an appropriate conductivity, as shown for example in curve a) or b), the applied electric field can result in positive or negative dielectrophoresis.

[0132] In the kHz range from 1 kHz to 100 kHz, negative dielectrophoresis occurs without exception. At lower conductivities a) and b), polarization of the cells changes polarity between 100 kHz and 50 MHz, resulting in positive dielectrophoresis. Above 50 MHz, negative dielectrophoresis occurs again.

[0133] By choosing a high electrical conductivity, such as that of common cell culture media, negative dielectrophoresis can be achieved over the entire frequency range. Negative dielectrophoresis is favorable for cell manipulation because cells move to areas of low electric field strength, and exposure to electric fields is also minimized. High frequencies are also favorable because rapid changes reduce the displacement of charge carriers (e.g., ions). This further reduces irreversible processes.

[0134] The dielectric properties of the bottom element determine the cut-off frequency. Frequencies up to 100 kHz are feasible. As the dielectric constant of the bottom element decreases with increasing frequency, the coupling of the electric field gets worse at higher frequencies. This happens because the average relaxation time of the charge carriers / dipoles is exceeded, in other words, they can no longer follow the high frequency changes of the electric field.

[0135] When positive dielectrophoresis begins, cells are attracted in the direction of the electromagnetic field maximum. They are always located close to the electrodes 14, especially at the edges of the electrodes and at the closest distance between the electrodes 14. Thus, the geometry of the electrodes 14 can be used to determine with high precision the placement of dense cell groups, lines or even clumps, as shown for example in Figures 2a-2f.

[0136] When negative dielectrophoresis begins, the cells are polarized in the opposite direction to positive dielectrophoresis: they move in the direction of the electric field minimum, or congregate or lift off onto the surface where no electrodes are present.

[0137] Figure 3b shows a spatial depiction of the different cell behaviors and the effect of the induced forces. The electrodes 14 are arranged as quadrupoles and controlled via two phases Φ1, Φ2. In positive dielectrophoresis, the cells aggregate at the location 36 of highest electric field strength and are attracted to the bottom of the container 22. In this example, the applied electric field creates an electric field minimum in the center 33 of the container 22. The electric field minimum spreads into a funnel shape 34.

[0138] In the case of negative dielectrophoresis, at low voltage, the cells collect at the bottom in the center 33 of the container 22. As the voltage is increased, the electrical forces acting on the cells from the electric field exceed the sedimentation forces acting on the cells. The electrical forces cause the cell aggregates 17 to suspend; these electrical forces may thus be referred to as levitation forces.

[0139] The buoyancy force causes the cell aggregates 17 to float in free suspension in the force funnel 34 at a height where the buoyancy force is exactly equal to the sedimentation force. Furthermore, the buoyancy force holds the cell aggregates 17 on their central axis above the electrode 14, as shown in Figure 3b.

[0140] If the vessel 22 contains different types of cells with different dielectrophoretic spectra, then at certain frequencies of the alternating electric field, positive and negative dielectrophoresis will occur, which is one condition for co-culture in the device.

[0141] 4a-4f show the arrangement of electrodes 14 shown in Figs. 2a-2f for cells with negative dielectrophoresis. Cell aggregates 41, 42 may be formed while negative dielectrophoresis is occurring. Cell aggregate 42 is positioned at the center of the bottom of container 22, and cell aggregate 41 is spaced from the center of the bottom of container 22 by the space between electrodes 14. Cell aggregate 41 and cell aggregate 42 may contain different types of cells in view of their dielectrophoretic spectrum.

[0142] The cell aggregates 42 may be exposed to an alternating electric field of any of two to four phases.

[0143] The cell aggregates 41 are always exposed to only one of the two phases of the alternating electric field from the nearby electrodes 14, ie, from the electrodes 14 adjacent to the space occupied by the cell aggregates 41.

[0144] Thus, the central cells and cell aggregates 42 can be rotated at a slow speed, for example, 5 times / second to 0.001 times / second. Furthermore, these cell aggregates 42 may float, which allows them to be easily removed while the cell aggregates 41 remain at the bottom of the container 22.

[0145] That is, the cells and cell aggregates 41, 42 may be arranged in a pattern in the array of dry electrodes 14. These arrays allow the cells to be co-cultured without barriers. For example, the cell type of the cell aggregate 41 may be a nurse culture that prepares a nutrient solution for the cell type of the cell aggregate 42.

[0146] 4c and 4f show examples of parallel formation, growth and culture of 13 organoids or other tissue formations in one container 22, as required in biotechnology, medicine and pharmacy. These examples thus allow the generation of highly ordered organoid / tissue / cell masses in a 96-well microwell plate, i.e. 96×13=1,248 organoids in a 96-well microwell plate.

[0147] Figures 5a-5f show the arrangement of electrodes 14 of Figures 2a-2f for cells with positive dielectrophoresis. During positive dielectrophoresis, cell aggregates 51 collect above the electrodes 14 at the bottom of the container 22. The alternating electric field cannot lift these cell aggregates 51. Therefore, the number of phases used is of secondary importance, since only the alternating electric field of the electrodes directly below these cells applies a force to the cell aggregates 51.

[0148] The device 10 may be operated just at the turning point of the dielectrophoresis from positive to negative, which may be 100 kHz to 1 MHz according to Figure 3. In this case, the suspension may contain at least two cells with different types of dielectrophoresis.

[0149] That is, the combination of Figures 4a-4f and 5a-5f shows that one cell type is attracted to the electrode 14 and the other cell type is repelled away from the electrode 14.

[0150] After changing the frequency to values ​​where both show negative dielectrophoresis, the cell groups 42, 51 may be disentangled from different cell types to constitute, for example, organoids, which are of great importance in, for example, angiogenesis.

[0151] Figures 6a-6f show other examples of electrodes 14. These examples include electrodes 14 for two-phase operation. One group of electrodes 14 is connected to a first strip electrode 61, and another group of electrodes 14 is connected to a second strip electrode 62. The strip electrodes 61, 62 have different electrical phases. The strip electrodes 61, 62 may be connected to electrodes of multiple containers 22. In this way, the number of lines is minimized. The structures of electrodes 14 shown in Figures 6d, 6e and 6f allow the generation of more than 20 electric field minima. That is, these structures can provide more than 20 cell aggregates / organoids per container 22.

[0152] Figure 7 shows an example of multiple reservoirs 22 with the electrode configuration of Figure 6d. Strip electrode 70 is a zero conductor that connects to one group of electrodes 14 in each reservoir 22 shown in this example. Strip electrode 61 operated at phase Φ1 connects to a group of electrodes 14 in the bottom row of reservoirs 22. Strip electrode 62 operated at phase Φ2 connects to a group of electrodes 14 in the top row of reservoirs 22. This example shows a mirrored combination of an array of electrodes 14 across a row of device 10, such as a microwell plate. In this two-phase operation, multiple supply lines are not required.

[0153] For the generation of a rotating or moving multiphase electric field, the crossing of supply lines is necessary, which can be manufactured in a multilevel process. The manufacturing of the electrodes 14 can be simplified if these levels of electrodes 14 are located outside the interior space on the underside 13 of the separator layer 20.

[0154] Figure 8 shows a further example of multiple receptacles 22 with the electrode configuration of Figure 6f. This example also shows a mirror image combination of the arrangement of electrodes 14 across the row of the device 10. This example includes two groups of strip electrodes 61, 62. Each receptacle 22 comprises electrodes 14 that connect to a first group of strip electrodes 61 of phase Φ1 or to a second group of strip electrodes 62 of phase Φ2. The first group of strip electrodes 61 connects to a first contact element 81. The second group of strip electrodes 62 connects to a second contact element 82. The first and second contact elements 81, 82 may be connection pads.

[0155] Figure 9 shows a device 10 with a 96-well microwell plate as the reservoir 22. Electrical connections by two contact elements 81, 82 show a two-phase system in all reservoirs 22. The electrodes 14 have the configuration of figure 6a.

[0156] 10a and 10b are cross-sectional views of a device 10 comprising a micro-well plate 103 with a reservoir 22. Contact elements 81, 82 are arranged on the lower surface of the separator layer 20. The device 10 further comprises a receptacle 101 for the micro-well plate 103. The receptacle 101 comprises two spring-loaded contact pins 102 which can be electrically connected to a radio-frequency generator (not shown). A lid 105 can cover the micro-well plate 103.

[0157] In FIG. 10 a , the micro-well plate 103 is separated from the container 101 .

[0158] Figure 10b shows the accommodation of a micro-well plate 103 in the accommodation container 101. Contact pins 102 connect to contact elements 81, 82 to form contacts. Of course, many other contacts are possible, including removable and fixed contacts.

[0159] Since the electrode 14 of the present invention is placed outside the inner space, i.e., outside the nutrient solution, the electrode 14 capacitively couples in the suspension. Compared with the prior art electrodes that directly contact the nutrient solution, the electrode 14 of the present invention reduces ohmic losses when generating an alternating electric field. A large number of containers 22 enhances the effect of reducing ohmic losses in the device 10. That is, according to the present invention, the huge ohmic loss factor of the electrode that directly couples to the suspension 15 compared to capacitive coupling can be avoided. A higher current may compensate for the ohmic loss factor of the electrode that directly couples in the suspension 15. However, this leads to the electrode process described above.

[0160] Figures 11a and 11b show a 96-well microwell plate 103. Figure 11a is a top view of the microwell plate 103, and Figure 11b is an AA cross-sectional view of the microwell plate 103.

[0161] In Fig. 11a, the device 10 comprises an energy storage device 111, e.g. a battery, and electronic circuitry 112, for generating an alternating electric field at the electrodes 14. The energy storage device 111 and electronic circuitry 112 may be integrated into the micro-well plate 103. This allows the device 10 to be operated automatically without peripheral electronic devices. Miniature digital electronic components can be used with low currents and with the use of rectangular voltage waveforms optimized for the purpose of inducing dielectrophoresis. Alternatively, the electronic circuitry may be housed in the lid 105, e.g. in a very flat design, and electrically connected via plug sockets or spring-loaded contacts on a metal surface, etc.

[0162] 12 shows an example of the internal structure of the separating layer 20. The separating layer 20 has a high relative dielectric constant, which allows coupling of the electromagnetic field with sufficient force acting on the cells in the suspension 15.

[0163] All commercially available polymer, resin, and plastic materials have unsuitable dielectric constants, ε, between 2 and 4. relThis results in a 20- to 40-fold reduction in the effective electric field strength compared to direct coupling from the electrode to the suspension.

[0164] The force acting on a cell is proportional to the square of the electric field strength. To reduce the attenuation of the electric field strength by a factor of four compared to direct binding in suspension, the separator layer 20 requires an overall dielectric constant of at least 20.

[0165] 10V for direct coupling in suspension pp A voltage less than or equal to (voltage peak-to-peak) is used; otherwise the applied voltage will heat the electrodes. As a result, the electrodes heat the suspension, which causes bubbles to form and electrolysis occurs.

[0166] According to the present invention, the electrodes 14 are disposed outside the interior space 11, and the separator layer 20 shields the heat from the electrodes 14 from the suspension 15. Thus, the voltage amplitude can be increased to a maximum of 40V. pp and can be increased even higher with proper insulation of the underside 13.

[0167] The reduction in the field strength of the electric field generated by electrode 14 is compensated for by the four times higher amplitude of the voltage. If separator layer 20 has a dielectric constant greater than 30, the same forces act on the cells as in the case of conventional direct binding in suspension.

[0168] The thickness of the separator layer 20 weakens the electric field and reduces the desired gradient of the electric field, therefore the thickness of the separator layer 20 must be much less than the distance between the electrodes 14.

[0169] When the electrode spacing is 100 μm to several mm and the thickness is less than 100 μm, preferably 25 μm or less, relaxation of the electric field is reduced.

[0170] Such materials and films are not commercially available. They can be produced by mixing the particles with cyanoresin 121, which has a dielectric constant of about 10, and the particles have ferromagnetic properties and a high dielectric constant ε of 100-1000.rel and preferably in a crystalline form, for example barium titanate (BaTiO3)122 or barium strontium titanate (Ba 1-x Sr x TiO3)123, etc.

[0171] A mixture of cyanoresin and more than 40% by volume of crystals loses transparency, so when observing the cells in the container 22 with an inverted microscope, the amount of crystals should be kept below 40% by volume.

[0172] 12a and 12c show typical mixtures obtained by mixing crystals 122, 123 with high dielectric constants at less than 40% by volume, resulting in an overall dielectric constant of less than 25.

[0173] FIG. 12a shows a mixture of crystals 122 of uniform size, for example between 200 nm and 1000 nm, preferably 300 nm.

[0174] FIG. 12c shows a mixture of small crystals 122 and large crystals 123 with different sizes, for example 300 nm and 700 nm.

[0175] 12b and 12d show a preferred arrangement of crystals 122, 123 within cyanoresin 121. The columnar arrangement of crystals 122, 123 can further increase the dielectric constant of separator layer 20. Separator layer 20 can achieve an overall dielectric constant of, for example, greater than 40 by mixing less than 40% by volume of crystals 122, 123.

[0176] 13a-13c show how the desired configuration of crystals 122, 123 is obtained.

[0177] FIG. 13a shows the stochastic distribution of crystals 123 within the cyanoresin 121 after mixing.

[0178] According to Fig. 13b, during curing of the separator layer 20, e.g. annealing or UV light exposure, an electric field is applied vertically through the material mixture. The electric field is preferably an alternating high frequency electric field. At an appropriate frequency, between kHz and MHz, the electric field aligns the dipoles of the crystals 123, causing them to arrange in a chain shape by dielectrophoresis as shown in Fig. 13b. If the crystals 122, 123 have different sizes, the electric field forms pillars with the shape shown in Fig. 13c.

[0179] The alignment in the pillar shape may disappear or may be very precise, depending on the time between switching the electric field on and off during curing. For example, if the electric field is on until curing is complete, the crystals 122, 123 will align very precisely.

[0180] To avoid fixed pre-polarization that can occur with precise alignment of the crystals 122, 123, it is preferable to form rather irregularly arranged columns of crystals 122, 123, i.e., the electric field is turned off prior to curing, allowing positional changes of the crystals 122, 123 due to local temperature fluctuations.

[0181] 14a-14c show another example of the effect of dielectrophoresis in device 10. FIG.

[0182] Figure 14a is a cross-sectional view of two containers 22 with cells 146 and nutrient solution 15. The transparent bottom element 21 comprises a separator layer 20 and an insulating layer 144 which insulates the electrodes 14 from the outside. The advantage of adding the insulating layer 144 is that the thickness of the insulating layer 144 does not affect the electric field. The insulating layer 144 stabilizes the bottom element 21 even if the separator layer 20 has a thickness in the μm range.

[0183] Figure 14b shows the effect of turning on the radio frequency electric field under conditions such that negative dielectrophoresis occurs. Cell aggregates 147 are formed, which are required to advance the formation of organoids or tissues. In the left vessel 22, the electric field strength is low, so the cell aggregates 147 remain at the bottom.

[0184] In the right vessel 22, the electric field has a higher field strength than the left vessel 22. The force resulting from the electric field lifts the cell aggregates 148 and keeps them floating freely within the electric field funnel. In this position, depending on the control of the electric field generated by the electrodes 14, the cell aggregates 148 can rotate and deform.

[0185] The cell aggregates 147 may be fabricated to improve cryopreservation, for example, since cooling and heating occurs primarily through the bottom element 21 of the microwell plate. Both the electrodes 14 and the thin separator layer 20 with good thermal conductivity allow for rapid temperature changes in the organoids or cell aggregates 147.

[0186] For a temperature change in the cell aggregates 148, the temperature of the surrounding nutrient solution must first change, so to achieve cryopreservation, the electric field must be turned off or the frequency must be switched to positive dielectrophoresis to cause the cell aggregates 148 to precipitate into the bottom element 21.

[0187] Figure 14c shows a microscopic image of the formation of such particles, centered between the outer four electrodes 14 of a separator layer with a total relative dielectric constant of about 15, under alternating electric field excitation at the phase position shown for 100 kHz quadrupole excitation. Negative dielectrophoresis occurs. The surface portion of the bottom element 21 around the electrodes 14 is completely free of particles. Cells 149, which are part of a cell aggregate 147, gather between the electrodes 14.

[0188] 15a-15d show example operational sequences representative of a variety of such cell-particle operations.

[0189] According to figure 15a, a container 22 contains a cell suspension 15. The electric field is switched off.

[0190] After turning on the radio frequency electric field and using negative dielectrophoresis, cell aggregates 151 are formed in the solution, as shown in FIG. 15b.

[0191] According to FIG. 15 c , a decrease in the electric field strength causes the cell aggregates 151 to sink to the bottom of the container 22 .

[0192] In adherent cell growth, the cells spread and grow on the bottom element 21, as shown in Figure 15d.

[0193] For example, when additional cells are added, the electric field may be insufficient to detach the adherent cells, causing an additional cell layer to deposit on top of the existing cell layer.

[0194] The procedure illustrated in Figures 15a-15d may be used immediately prior to initiating cryopreservation of an entire microwell plate.

[0195] 16a-16c show a schematic of how vascularized organoids are generated.

[0196] 16a is a schematic top view of an electrode 14 connected to a radio frequency source for generating an alternating electric field, and a conductive island element 162 not connected to the radio frequency source. The island element 162 may be made of, for example, an electrode material. The island element 162 may be disposed on the same side as the electrode 14 with respect to the separator layer 20.

[0197] The island elements 162 may be non-conductive, however, the following examples describe conductive island elements 162.

[0198] 16b, a natural or artificial semi-conductor tube 163 may be added to the solution. Then, an electric field is alternately switched on with a frequency v, bunching the electric field lines above the electrodes 14 and the conductive island elements 162.

[0199] If the tubes 163 exhibit positive dielectrophoresis, they will be collected above the conductive island elements 162 and at the electrode 14 .

[0200] Because the cells in the cell aggregates 164, 165 have different dielectric properties than the tube 163, cells added to the solution after the tube 162 is collected will exhibit negative dielectrophoresis at frequency v. The cells will be collected in the electric field minimum between the electrode 14 and the island element 162.

[0201] After switching off the alternating electric field, as the cells grow, the two cell aggregates 164, 165 move and grow towards each other to form the organoid 166 and envelop the tube 163, which causes the tube to grow within the forming organoid 166. Even in larger organoids 166, nutrients can reach the interior of the organoid via the tube 163, which is necessary to avoid cell death in the center of the organoid 166.

[0202] According to Fig. 16c, the organoid 166 has grown enough to be considered as a three-dimensional shape. The organoid 166 may then be lifted as shown by the arrow when the alternating electric field is turned on again. The electric field force increases as the cube of the radius of this organoid 166, so the organoid 166 can be easily transported or removed.

[0203] Figures 17a-17d show the principles behind cryopreservation.

[0204] In FIG. 17 a , cells 146 are shown in suspension 15 .

[0205] Figure 17b shows the formation of initial cell aggregates 173 in suspension 15. According to Figure 17c, the size of the cell aggregates 173 increases.

[0206] Figure 17d shows cooling during cryopreservation. In particular, the electrodes 14 and separator layer 20 greatly improve thermal conductivity.

[0207] FIG. 18 shows an example of the design of organoids using artificial or biological capture particles 181. The capture particles 181 can be designed as lossy dielectrics, similar to cells. For example, the capture particles 181 can be functionalized Sephadex particles ranging from 1 μm to 300 μm, preferably 50 μm. For the design of organoids, in this example, the capture particles 181 can be captured at the electric field minimum when the electric field is turned on with four phase rotations. The rotating electric field causes the capture particles 181 to levitate above the bottom of the container 22 at a height h.

[0208] According to Fig. 18b, cells 182 are added at a low concentration. The rotating electric field polarizes the cells 182, which causes the cells 182 to move to the center. In the vicinity of the capture particle 181, dipole-dipole interactions occur between the cells 182 and the capture particle 181, via higher order interactions, e.g. quadrupole or octupole. These interactions cause the cells 182 to encounter the capture particle 181. The cells 182 are pressed against the surface of the capture particle 181 so that the cells 182 adhere well.

[0209] Due to the rotation of the electric field, unlike a stationary alternating electric field, the surface of the captured particle 181 is uniformly covered by the cells 182. This is an important goal in such organoid technology.

[0210] 18b and 18c may be repeated with further cells so that cell layer upon cell layer can be deposited on the overgrown captured particle 181. When the rotating electric field is changed to an alternating electric field, for example by application of two phases to the electrodes 14, the cells loaded onto the captured particle 181 become inhomogeneous, i.e. the round bodies generated by the rotating electric field then have cell protrusions.

[0211] At least one electrode 14 can be used not only for excitation and force generation but also for measuring the induced cell polarization. The excit-echo technique known from ultrasound may be used, i.e. the electric field is switched off very quickly after excitation and the decay of the induced polarization in the cell is recorded as a current-voltage waveform at the same electrode 14. This requires very high amplification, with gains of more than 100-1000, and a large signal-to-noise ratio.

[0212] 19a-19d show cross-sectional views of an insulating layer 144, which may be a glass substrate. Flat microelectrodes 14 with a thickness of less than 1 μm can be fabricated on the glass substrate. Fabrication of the electrodes 14 can be performed, for example, by etching methods.

[0213] On top of the electrode 14 and the insulating layer 144 is a separator layer 20 with a thickness of about 50 μm and a high first relative dielectric constant ε1 and low electrical conductivity. The separator layer 20 is covered with an aqueous cell suspension 15, cells not shown. The suspension 15 has a second relative dielectric constant ε2 in the range of about 80. The relative dielectric constant ε2 cannot be changed significantly due to biological reasons of the cells. The electrode 14 is completely isolated from the suspension 15 by the separator layer 20.

[0214] Figures 19a-19d further show electric field lines 194 for different geometries of separator layer 20 as a function of the ratio of the first and second dielectric constants. In Figures 19a and 19c, the second dielectric constant ε2 is greater than the first dielectric constant ε1. In Figures 19b and 19d, the second dielectric constant ε2 is less than the first dielectric constant ε1.

[0215] In Figures 19a-19d, the separator layer 20 has a curved surface portion 192. Figures 19a and 19b have a concave surface portion 192. Figures 19c and 19d show a convex surface portion 192. The electric field lines 194 are refracted at the curved surface portion 192 of the separator layer 20 adjacent to the suspension 15. This can affect the electric field line characteristics and therefore the action on the cells in the suspension 15. The change in the electric field lines 194 depends not only on the change in the dielectric constant on the curved surface portion 192 but also on the shape of the curved surface portion 192 of the separator layer 20. The electric field is compressed or distorted in the solution. The cell aggregates adapt their shape to the characteristics of the electric field lines 194, for example by forming a sphere, a disk or a spindle shape. This can be a means to construct organoids.

[0216] The cases of Figures 19a and 19c correspond exemplarily to distributions of relative permittivities of ε1=40 and ε2=80, while the cases of Figures 19b and 19d correspond to distributions of relative permittivities of ε1=120 and ε2=80.

[0217] In order to obtain the curved surface portion, the thickness of the separator layer 20 can be changed within a range of 0.5 μm to 100 μm. As a result, the relative dielectric constant of the separator layer 20 can vary within a range of 10 to over 100.

[0218] 20a-20d show two further examples of the geometry of the surface 201 of the separator layer 20. FIG.

[0219] 20a and 20b show a separator layer 20 having a flat surface 201, the separator layer 20 having a thickness in the range of 1 μm to 100 μm.

[0220] 20c and 20d show a separator layer 20 with openings 202 between the electrodes 14. The electrodes 14 completely cover the separator layer 20, i.e., the electrodes 14 are provided outside the interior space 11 and are separated from the suspension 15. In particular, the design of the openings 202 can greatly change the change in the electric field lines 194. The ratio of the relative dielectric constants can calibrate the electric field lines 194. The calibration of the electric field lines 194 can be achieved by changing the mixing ratio of the separator layer materials so that the suspension 15 is not affected. For example, the calibration can be determined by adding barium titanate to the separator layer materials.

[0221] 21 shows a further example of a separator layer 20. In this example, the separator layer 20 has the same relative dielectric constant as the suspension 14. In this case, the electric field lines 194 run across the boundary layer between the separator layer 20 and the suspension 15, almost undisturbed. The advantage of this combination is that the shape and surface topography of the separator layer 20 do not affect the characteristics of the electric field lines 194. Furthermore, the separator layer 20 still isolates the electrode 14 from the interior space 11 and the suspension 15, and the electric field can couple with the suspension 15 without attenuation.

[0222] For the suspension of cells to form organoids, a certain distance from the surface is preferred, because associated multiple cells tend to adhere to the surface and cannot form non-three-dimensional organoids. Contact of cells with surfaces, especially with artificial materials, should be avoided as it affects the physiology and differentiation of cells. That is, not only the collection of cells in the lateral direction, but also the vertical force F z Lifting via the electric field is also necessary. Because cells exhibit negative dielectrophoresis at the frequencies used, in the range of 1 kHz to 10 MHz, the cells are pushed out of the electric field and lifted up. With an arrangement of four electrodes 14, the cells can be held within a force funnel.

[0223] Figure 22a shows a conventional electrode chamber according to the prior art. The electrodes 224 are in direct contact with the cell suspension 15. A cell 221 resting on the bottom of the container 22 experiences strong forces in the lateral direction, but small forces in the z-direction.

[0224] FIG. 22b shows an example with a separator layer 20 with a relative permittivity ε1. This ensures that the forces acting on the cells 221 are not only lateral but also strongly in the z-direction. Thus, the cells 221 resting at the bottom of the container 22 are also lifted and kept free-floating. Compared to the prior art configuration shown in FIG. 22a, covering the electrodes 14 with a separator layer 20 with a thickness in the range of 10 μm to 100 μm reduces the electric field losses, avoids electrolytic processes on the metallic surface, and even allows biological functionalization of the surface 223.

[0225] The device 10 can be further improved by covering the electrodes 14 and the gaps between the electrodes 14 with an insulating material 224 having a low dielectric constant, which reduces current leakage and capacitive overcoupling.

[0226] Figures 23a and 23b illustrate this approach. Figure 23a shows a flat separator layer 20 with rounded edges. Only in a small area does the electrode 14 make a direct connection with the separator layer 20. The main part of the electric field lines originate from there. The other area between the electrode 14 and the separator layer 20 comprises an insulating material 224, e.g. silicon oxide, silicon nitride, aluminum oxide, etc., with a thickness of 1 μm.

[0227] The rounded edges of the separator layer 20 keep the cells 234 located there away from the central region. This is interesting, for example, when a co-culture with conditioning cells is performed, where the conditioning cells do not integrate into the central organoids 222, but only condition the suspension 15 in a cellular physiological direction.

[0228] Figure 23b shows an example with a thicker insulating layer 231 than the example of Figure 23a. The insulating layer 231 can improve the concentration of the electric field lines as they run around the insulating layer 231. That is, the insulating layer 231 shifts the electric field lines that would otherwise run at the location of the insulating layer 231.

[0229] Figures 24a and 24b show further embodiments of this type. According to Figure 24a, the separator layer 20 comprises a considerable height and a depression 241 on the upper surface. Thus, the separator layer 20 defines an isolated area in the container 22. The organoids 222 are suspended in the depression 241. This configuration simplifies the deflection of the cells 243 located at the edge of the separator layer 20. Furthermore, the shape of the separator layer 20 avoids the mixing of the co-cultured cells 244 and the cells 242 to be added to the organoids 222 cells.

[0230] 24b shows another example with a separator layer 20 having a curved surface. The curved surface can deflect cells 243 that should not be added to the organoids 222.

[0231] 25a-b show examples of different geometric designs of the insulator layer 224 and the separator layer 20. The geometric designs of the insulator layers 224, 231 and the separator layer 20 can interact with the location and shape of the cell aggregates 222.

[0232] 25a shows an example with a thick central insulating layer 231 and a flat separator layer 20 sandwiched between electrodes 14. The resulting electric field lines can induce weak lateral forces on the organoids 222.

[0233] Figure 25b shows an example with a thin central insulating layer 231, where the separator layer 20 has a bulky end facing the central insulating layer 231. This configuration creates electric field lines that induce high lateral forces on the organoids 222. Thus, the organoids 222 are more laterally compressed than the organoids 222 in Figure 25a.

[0234] Figures 26a and 26b show two examples of the effect of four flat electrodes 14 on particles / cells. The electrodes 14 are operated with an AC voltage in the high kHz range. Furthermore, the electrodes 14 are covered with a layer with a high dielectric constant.

[0235] According to FIG. 26a, the separator layer 20 comprises an oxyphene film having a thickness of 22 μm. The relative dielectric constant of the oxyphene film was increased to a value of about 30 through holes filled with water and having a diameter of 400 nm. The frequency of the electric field in this example is 100 kHz and the amplitude is V pp The particles have a size of 20 μm and are disposed in an aqueous electrolyte solution.

[0236] The pores penetrate the oxyphene membrane, so that this type of layer is still not satisfactory for longer periods, since the pores reduce the separation between the electrode 14 and the suspension 15 and electrode-electrolyte processes can be initiated.

[0237] According to FIG. 26b, the separator layer 20 has a thickness of 70 μm. The separator layer 20 contains a cured cyanoresin CR5 mixed with 30% by volume of barium titanate nanoparticles. The separator layer 20 forms a completely insulating coating on the electrode 14 and has a relative dielectric constant of about 40. The frequency of the electric field in this example is 400 kHz and the amplitude is 19 V. pp The particles have a size of 20 μm and are disposed in an aqueous electrolyte solution 15.

[0238] In Figure 26a, the membrane is transparent enough to see the electrodes 14. In Figure 26b, the locations of the four electrodes 14 are indicated by white diagonal rectangles toward the center of the image. In both cases, large cell aggregates 222 form, becoming spherical in the central region and floating three-dimensionally.

[0239] The number of trapped cells can be adjusted from one to several thousand by the initial concentration of the cell suspension. To produce heterogeneous organoids, additional cells need to be added later. They are attracted onto the existing organoids, where they come into direct surface contact via dipole-dipole and quadrupole-dipole interactions, and can thus form molecular bonds.

[0240] In this way, complex cell groups, from cell clusters to organoids with hundreds and thousands of cells, can be generated and suspended freely in the solution. Cells 262 that are not in the central region are kept away from the electrodes 14. They interact with the organoid cells 222 by the fluid of the suspension 15, but cannot reach the organoid cells 222. This is important for co-cultures and when the ratio of certain cell types in the organoid should be kept constant.

[0241] Figures 27a and 27b show the structure shown in Figure 25b.

[0242] According to Fig. 27a, bodies 271 with diameters ranging from a few µm to 100 µm are added to the suspension. The material of the bodies 271 may be a mixture of cyanoresin and barium titanate. Thus, the bodies 271 have low electrical conductivity and strong polarizability in an electric field. Moreover, the electric field keeps the bodies 271 very reliably suspended.

[0243] In this polarized state, the body 271 acts on the much smaller cell 272 through dipole-dipole interactions of the polarization induced on all objects, including the central trap. The cell 272 moves from a wide area around the body 271 toward the body 271, as indicated by the arrows. The cell 272 cannot leave the surface of the body 271.

[0244] For organoids, the shape of the trapping body 271 can be adapted to the desired shape of the cell formation, such as a stellate, ellipsoid, or cylinder. The material of the body 271 can be designed to have holes or channels so that nutrient solutions can flow through the structure of the body 271. This allows for larger organoids and, consequently, vascularization. Thus, the body promotes cell aggregation, especially for small cells in the μm range, and the formation of organoids.

[0245] To measure organoid growth and size without microscopy and non-destructively, one alternative is to use an electric field generated via a flat electrode 14 once and applied along with a measurement signal.

[0246] In another alternative, as shown in Fig. 27b, the trapping bodies 271 may be attached to microelectrodes 273 and guided into the electric field region from above. The microelectrodes 273 may be formed as beads. The material of the trapping bodies 271 has a relatively high dielectric constant combined with low electrical conductivity, covering the metallic surface of the microelectrodes 273. If the device 10 comprises multiple containers 22, these electrode bead systems may be introduced into each container 22 through the lid 105 and easily removed when the lid 105 is opened.

[0247] Instead of the microelectrode 273, an optical fiber may be inserted, either alone or in addition to the microelectrode 273, for optically measuring the contents of the container 22. In the case of the electrode 273, the measurement signal is an impedance signal. In the case of optical detection, for example, scattered light may be detected.

[0248] 28 shows another example of the device 10. The electrodes 14 are arranged very close to each other. To avoid power supply losses through stray capacitance, a section 282 of each electrode 14 is covered with an insulating layer 281. The insulating layer 281 is arranged between the electrode 14 and the separator layer 20. Thus, the insulating layer 281 isolates the section 282 from the separator layer 20.

[0249] Fig. 29 shows an example of providing a dielectric anisotropic layer from a mixture of polymer and alkaline earth titanate as separator layer 20, which is switchable. Electrodes 291 generate a transverse electric field in the material of separator layer 20. This transverse electric field prevents the movement and displacement of the dipoles 292 of the titanate particles during the polarization change in the high-frequency electric field generated by electrodes 14. As a result, a displacement current through separator layer 20 can be influenced, which leads to a change in the high-frequency electric field in suspension 15 in interior space 11.

[0250] In another example, the Peltier element can cool the electrode or a bottom element that includes the electrode.

[0251] Additionally, in other examples, amperometric measurements may be performed to record the pH value and size of the organoids.

[0252] In other examples, dielectrically different beads / microbodies may be placed at different heights or used at different frequencies. The beads / microbodies may be influenced by different types of cells. Furthermore, these beads / microbodies can manipulate cells in an electric field, for example, by alternating alternating electric fields, keeping the cells in a dispersed suspended state and organizing them into aggregates.

[0253] In a further example, electrodes can be formed by doping a mixture of a polymer and an alkaline earth metal titanate to form conductive pathways in the mixture, which can be, for example, barium titanate and strontium titanate plates.

[0254] Furthermore, by way of example, radiofrequency fields can induce high frequency membrane potential changes in cells, which can affect the uptake and / or delivery of substances into cells, affect cell differentiation, synchronize cardiomyocytes or neuronal cells, etc., or affect cellular mechanotransduction.

[0255] The operating temperature of the device 10 may be, for example, in the range of −5° C. to 100° C., preferably in the range of 20° C. to 37° C. Furthermore, the pH value of the suspension may be, for example, in the range of 6-8.

[0256] The devices described above can be used to manipulate living cells.

[0257] Furthermore, a variable electric field capacitively coupled to the interior or exterior of a container comprising a suspension with at least one biological cell can be used for the manipulation of said biological cells in suspension.

[0258] 30 is a flow chart illustrating a method 300 of manipulating biological cells with a device based on the above description. The device may comprise at least two electrodes, and the electrical signal is configured such that the at least two electrodes generate an electric field with a stable temporal pattern of minima and maxima within the container.

[0259] The method 300 may comprise a first optional step 313. In step 313, the suspension is selected according to a desired electrical conductivity and the frequency of the variable electric signal is selected to generate a positive or negative dielectrophoretic motion for at least one biological cell. In this way, the suspension can be conditioned in preparation for the manipulation of the cells to optimize and simplify the subsequent manipulation process. Furthermore, the frequency of the alternating electric signal may be selected such that the dielectrophoretic force matches the requirements of the cell manipulation. The frequency may be selected depending on the frequency of the electric field, for example based on known characteristics of the polarization of the cells, as shown in FIG. 3a.

[0260] In step 301 of method 300, a suspension comprising at least one biological cell is introduced into at least one container of the device. If the device comprises multiple containers, such as a microwell plate having 1536 wells as the container, each container can receive the suspension. Furthermore, the suspension may be different in each container, e.g., the number or type of cells may differ between each container, and the mixture of nutrient solution in the suspension may differ between each container.

[0261] The biological cells may be stem cells, preferably induced pluripotent stem cells.

[0262] In a further optional step 314 of method 300, at least one cell is lifted and moved within the container by negative dielectrophoresis. Thus, in step 313, the suspension and frequency are selected such that negative dielectrophoresis occurs in the manipulated cells.

[0263] In a further optional step 310, the suspension may be adjusted with at least one cell group that may influence the nutrient solution of the suspension to be optimal for the growth of that cell type, or other cell types.

[0264] After optional step 310, method 300 may include a further optional step 311. In optional step 311, an electric field within the container is used to separate or aggregate at least one group of cells. This can create space within the suspension for additional cells that may be introduced into the suspension. These additional cells can grow and form organoids.

[0265] Such additional cells may be introduced into the suspension in a further optional step 312 of method 300. At least one additional cell may be moved to the electric field minimum and held there for a predetermined time to allow organoid formation and time for cell growth.

[0266] In step 302 of method 300, an electrical signal is applied to at least one electrode of the device to generate a variable electric field, sometimes referred to as an alternating electric field. The variable electric field may have a frequency that is selected in optional step 513.

[0267] The electrical signals may have a voltage peak-to-peak value in the range of 4 V to 100 V, preferably 10 V to 100 V, more preferably 10 V to 50 V. Furthermore, the at least one electrical signal is supplied continuously or discontinuously, preferably in a pulsed manner.

[0268] In step 303, the variable electric field may be used to move at least one biological cell to a predetermined location within the container, which is a manipulation of the at least one biological cell. For example, the cell may be located at the bottom of the container.

[0269] The electric field may exert a force on the cells in the container in the range of 1 pN to 1000 pN at a distance of 10 μm to 5 mm. At least one cell may be polarized by the electric field.

[0270] In another optional step 304, the at least one cell may migrate to at least a minimum of the electric field.

[0271] In an optional step 305, the suspension may include at least two cells. The electric field may form the at least two cells in the suspension into at least one cell aggregate.

[0272] According to an optional step 306 of method 300, the strength of the electric field may be reduced to cause the at least one cell aggregate to settle to the bottom of the container.

[0273] In a further optional step 307, the electrical signal is switched off as soon as at least one cell aggregate has settled.

[0274] Additionally, method 300 may include an optional step 308. In step 308, the electric field exerts an aggregation force on at least one of the precipitated cell aggregates, which prevents cells of the cell aggregates from migrating sideways, and thus prevents flattening of the cell aggregates.

[0275] Further, in optional step 309, the electrical signal may be applied either constantly or intermittently with short overall application times of the electric field.

[0276] In an optional step 315 of method 300, cryopreservation of the suspension can be performed by directly contacting a coolant with at least one electrode. Preferably, the cells are disposed at the bottom of the container. Because the electrodes typically comprise a metallic material and the separator layer is very thin, cooling the electrodes can efficiently cool the suspension and the cells therein.

[0277] Method 300 may further comprise optional step 316. In step 316, the frozen suspension of step 315 or another frozen suspension may be thawed. Thawing may be initiated by heating at least one electrode by providing at least one electrical signal at a frequency in the range of 1 kHz to 10 MHz to the at least one electrode. As explained by analogy in step 315, the metallic material of the electrodes and the thin separator layer may efficiently transfer heat to the suspension and cells.

[0278] At least one cell may be observed microscopically within the vessel and the behavior and growth of the cell may be recorded.

[0279] Additionally, at least one cell is measured for electrical and / or dielectric polarization induced by the electric field.

[0280] The steps of method 300 described above may be performed in any order that is practical and reasonable.

[0281] FIG. 31 is a flow chart of a method 310 for fabricating a separator layer of the device described above.

[0282] In a first step 311 of the method 310, at least one liquid polymer having a first dielectric constant is provided. The liquid polymer may be a cyanoresin selected from the group consisting of cyanoethyl pullulan (CRS), cyanoethyl poly(vinyl alcohol) (CRV), and cyanoresin type M (CRM). Further, the first dielectric constant may be greater than 10.

[0283] In a second step 312, a plurality of particles (preferably crystallites) having a second dielectric constant are mixed with at least one liquid polymer to obtain a separator layer mixture. For example, the particles may be CaTiO3, SrTiO3, BaTiO3, Ba 1-x Sr x It may comprise an alkaline earth metal titanate selected from the group consisting of TiO3 and / or combinations thereof.

[0284] The material mixture of the separator layer may contain 10% to 60% by volume of these particles, more preferably 30% to 50% by volume, and most preferably 40% or less by volume.

[0285] The particles may have a uniform size or may have at least two different sizes.

[0286] In optional step 314 of method 310, the separator material mixture is applied onto at least one electrode in a sealing layer, the application method being preferably sputtering, spin coating, screen printing and / or sol-gel processing.

[0287] Moreover, the method 310 further comprises step 313. In step 313, the separator material mixture is cured to obtain a separator layer.

[0288] During at least a portion of the curing process, an electric field may be applied to the separator material mixture to induce alignment, aggregation, and chain formation of particles in the liquid polymer, which can produce well-ordered columnar aligned particle domains and / or particle agglomerates within the separator layer.

[0289] Additionally, the separator material mixture may be cured to form the separator layer, and during curing, an electric field is applied perpendicular to the separator material mixture.

[0290] FIG. 32 is a flow chart of a method 320 for manufacturing the device described above.

[0291] In a first step 321, a vessel for culturing biological cells is provided. Furthermore, a number of vessels may be provided, for example as wells of a microwell plate.

[0292] In a second step 322, a layer of the separator layer may be formed. This layer may be formed by the method 310 described above.

[0293] In a third step 323, at least one electrode for forming and manipulating biological cells is provided. A separator layer separates the electrode from the interior space of the container.

[0294] Some embodiments of the above-mentioned invention refer to method type claims, while other embodiments refer to device type claims. Unless otherwise stated, a person skilled in the art will realize that in addition to any combination of features belonging to one type, the present application also discloses any combination of features related to different types. However, all features can be combined to produce synergistic effects that are more than the simple sum of the features.

[0295] The drawings and the foregoing description are merely illustrative and non-limiting examples of the present invention. The disclosed embodiments are not limiting. Those skilled in the art of practicing the claimed invention can understand and implement other variations to the disclosed embodiments by studying the drawings, the disclosure, and the dependent claims.

[0296] Reference signs in the claims should not be construed as limiting the scope of the invention to the illustrative elements used in the drawings.

Claims

1. A device for manipulating biological cells, the device (10) comprising: At least one container (22) for culturing biological cells, said container (22) having an interior space (11), At least one electrode (14) for manipulating biological cells; the device (10) comprises at least one separator layer (20), the at least one separator layer (20) being arranged between the at least one electrode (14) and the internal space (11) such that the at least one electrode (14) is arranged outside the internal space (11), device.

2. characterised in that at least one bottom element (21) of said at least one container (22) comprises said separator layer (20), and said at least one electrode (14) is arranged outside said interior space (11) in said bottom element (21), The device of claim 1 .

3. characterised in that the at least one bottom element (21) has, at least at the at least one electrode (14), a thickness of less than or equal to 200 μm, preferably less than or equal to 100 μm, and even more preferably less than or equal to 25 μm; The device of claim 2.

4. characterised in that the at least one electrode (14) is arranged between the bottom element (21) and an electrically insulating layer (144), A device according to claim 2 or 3.

5. the at least one electrode (14) is disposed in at least a portion of the at least one container (22) and is covered with a separator layer (20) at least inside the container (22). The device of claim 1 .

6. The separator layer (20) comprises a material having a relative dielectric constant in the range of 10 to 10,000, preferably 10 to 1,000, more preferably 20 to 500, for a voltage-current waveform having a frequency in the range of 1 kHz to 10 MHz, and preferably has ferromagnetic properties. A device according to any one of claims 1 to 5.

7. said separator layer (20) is characterized in that it comprises particles (122, 123) having a crystalline structure, preferably microcrystalline, particles (122, 123) having a uniform size in the range of 100 nm to 1000 nm, preferably 300 nm, or particles (122, 123) having different sizes in the range of 100 nm to 1000 nm, preferably 300 nm and 700 nm, A device according to any one of claims 1 to 6.

8. the particles (122, 123) are arranged in a columnar shape in the separator layer (20) between two opposing side surfaces of the separator layer (20), The device of claim 7.

9. The separator layer (20) is made of an alkaline earth metal titanate, preferably CaTiO ３ , SrTiO ３ , BaTiO ３ , Ba １－ｘ Sr ｘ TiO ３ and / or a combination thereof, preferably in an amount of 10% to 60% by volume, more preferably 30% to 50% by volume, and most preferably 40% by volume or less, A device according to any one of claims 1 to 8.

10. The separator layer (20) is characterized in that it contains at least one polymer (121), preferably a cyano resin, more preferably CRS, CRV and / or CRM, and even more preferably a polymer having a relative dielectric constant of 10 or more. A device according to any one of claims 1 to 9.

11. The separator layer (20) is characterized in that it has a total relative dielectric constant in the range of 10 to 200, preferably 16 to 120, and more preferably 20 to 120, with respect to a voltage-current waveform having a frequency in the range of 1 kHz to 10 MHz. A device according to any one of claims 1 to 10.

12. The separator layer (20) is characterized in that it has at least one curved area (192) in the internal space (11). A device according to any one of claims 1 to 11.

13. The at least one curved area (192) includes at least one convex portion and / or at least one concave portion. The device of claim 12.

14. the separator layer (20) comprises at least two regions (224, 225), the at least two regions (224, 225) having different dielectric constants, the at least two regions (224, 225) preferably comprising different materials and / or different material mixture ratios, A device according to any one of claims 1 to 13.

15. the at least one electrode (14) comprises a base metal, in particular aluminum or nickel; an alloy of a base metal; or at least one plotter-writable conductive ink or paste; A device according to any one of claims 1 to 14.

16. said device (10) comprising at least two electrodes (14), or at least four electrodes (14) in a quadrupole configuration, or at least eight electrodes (14) in an octupole configuration, A device according to any one of claims 1 to 15.

17. The electrodes (14) are electrically connected to different phases of a multi-phase voltage source or are electrically connected in pairs to one phase of a multi-phase voltage source.

17. A device according to claim 15 or 16.

18. The at least one electrode has at least one section, the section being cross-shaped or Y-shaped. A device according to any one of the preceding claims.

19. At least one electrode (14) is characterized in that it has an end piece having a circular, triangular, square or T-shaped cross section. A device according to any one of the preceding claims.

20. said at least one electrode (14) being linear or zigzag and / or having a plurality of, preferably triangular, projections extending along the base element (21); A device according to any one of claims 1 to 19.

21. the device (10) comprises a plurality of reservoirs (22) and a plurality of electrodes (14), at least one of the plurality of electrodes (14) being disposed on each reservoir (22), the device (10) being preferably formed as a microwell plate, A device according to any one of claims 1 to 20.

22. a first group of the plurality of electrodes (14) electrically connected to a first phase of a multi-phase voltage source via a first wire (71) and a second group of the plurality of electrodes (14) electrically connected to a second phase of the multi-phase voltage source via a second wire (72), at least one electrode (14) of each group being disposed on each container (22). A device according to any one of claims 1 to 21.

23. the device (10) further comprises at least one energy storage device (111) and at least one electronic circuit (112) for generating a voltage having a frequency in the range of at least 1 kHz to 10 kHz, the at least one electronic circuit (112) electrically connecting the at least one electrode (14) to the at least one energy storage device (111); A device according to any one of claims 1 to 22.

24. The at least one electrode (14) is disposed on a first module (106); the at least one electronic circuit (112) is disposed on a second module (105) that is detachable from the first module (106), and the at least one electrode (14) is electrically connected to the at least one electronic circuit (112) via a detachable electrical contact disposed between the first module (106) and the second module (105).

24. The device of claim 23.

25. the separator layer (20) separates the conductive or insulating island element (162) from the internal space (11), the separator layer (20) separating the conductive or insulating island element (162) from the internal space (11), A device according to any one of claims 1 to 24.

26. The at least one conductive or insulating island element (162) has a length in the range of 1 μm to 200 μm.

26. The device of claim 25.

27. The internal space (11) comprises at least one bead, tube (163) and / or wire. A device according to any one of claims 1 to 26.

28. At least a portion of the device (10) has a functional material surface, A device according to any one of claims 1 to 27.

29. The biological cells are stem cells, preferably induced pluripotent stem cells. A device according to any one of claims 1 to 28.

30. A method for manipulating biological cells by means of a device according to any one of claims 1 to 29, comprising the steps of: The method (300) comprises: - introducing into said at least one container a suspension comprising at least one biological cell (301), - applying an electrical signal to said at least one electrode to generate a variable electric field (302); - moving said at least one biological cell to a predetermined location within said container by said variable electric field, thereby manipulating said at least one biological cell (303); A method for providing the above.

31. The electrical signal has a voltage peak-to-peak value in the range of 4V to 100V, preferably 10V to 100V, more preferably 10V to 50V. The method of claim 30.

32. The at least one electrical signal is supplied continuously or discontinuously, preferably in pulses.

32. The method of claim 30 or 31.

33. the device comprises at least two electrodes, and the electrical signal is configured such that the at least two electrodes generate an electric field in the vessel having a stable temporal pattern of minima and maxima.

33. The method according to any one of claims 30 to 32.

34. The electric field exerts a force in the range of 1 pN to 1000 pN on the cells in the container at a distance of 10 μm to 5 mm.

34. The method of claim 33.

35. The at least one cell moves at least at a local minimum of the electric field (304).

35. The method of claim 30 or 34.

36. The at least one cell is polarized by the electric field. The method according to any one of claims 30 to 35.

37. The suspension comprises at least two cells, and the cells in the suspension form at least one cell aggregate by the electric field (305).

37. The method of claim 36.

38. The at least one cell aggregate is precipitated to the bottom of the container by reducing the strength of the electric field (306).

38. The method of claim 37.

39. and as soon as the at least one cell aggregate has settled, the electrical signal is turned off (307).

39. The method of claim 38.

40. The electric field exerts an aggregation force on the at least one precipitated cell aggregate (308).

39. The method of claim 38.

41. The electrical signal is applied either constantly or intermittently by applying the electric field for short periods (309).

41. The method of claim 40.

42. After the step of introducing the suspension (301), the suspension is conditioned (310) with at least one group of cells, The method according to any one of claims 30 to 41.

43. After preparing the suspension (310), the at least one group of cells is separated and aggregated (311) in the container by the electric field.

43. The method of claim 42.

44. and introducing (312) at least one further cell into the suspension, the at least one further cell being moved to the minimum of the electric field and held there for a predetermined period of time.

44. The method of claim 43.

45. The at least one cell is observed microscopically in the container and the behavior and proliferation of the cell is recorded. The method according to any one of claims 30 to 44.

46. The method further comprises, prior to the step of introducing the suspension (301), The method further comprises the step of generating a positive or negative dielectrophoretic motion for the at least one biological cell by selecting the suspension according to a preferred electrical conductivity and by selecting a frequency of a variable electrical signal (313). The method according to any one of claims 30 to 45.

47. the at least one cell is lifted (314) and moved in the vessel by negative dielectrophoresis; 47. The method of claim 46.

48. After the step of applying the electrical signal to the at least one electrode (302), the method further comprises: - storing said suspension frozen while simultaneously cooling said at least one electrode by direct contact with a cooling agent (315); and / or - heating said at least one electrode by supplying at least one electrical signal with a frequency ranging from 1 kHz to 10 kHz to said at least one electrode when the frozen suspension thaws (316); The method further comprises at least one of the following steps:

48. The method according to any one of claims 30 to 47.

49. At least one cell is electrically and / or dielectrically measured by the polarization induced by the electric field, 49. The method according to any one of claims 30 to 48.

50. The biological cells are stem cells, preferably stem cells, preferably induced pluripotent stem cells.

50. The method according to any one of claims 30 to 49.

51. A method for producing a separator layer of the device according to any one of claims 1 to 29, comprising the steps of: The method (310) comprises: - a supplying step (311) of supplying at least one liquid polymer having a first dielectric constant; - a mixing step (312) of a plurality of particles, preferably crystallites, having a second dielectric constant with said at least one liquid polymer to obtain a separator layer mixture; - A curing step (313) of curing said separator material mixture to obtain a separator layer. A method for providing the above.

52. wherein during at least a portion of the curing step, an electric field is applied to the separator material mixture to align, aggregate and form chains of the particles within the liquid polymer, forming well-ordered columnar particle domains and / or particle agglomerates within the separator layer.

52. The method of claim 51.

53. In the mixing step (312) of mixing the plurality of particles with the at least one liquid polymer, the particles have a uniform size and preferably account for 40% or less by volume of the liquid polymer.

53. The method of claim 51 or 52.

54. In the step of mixing (312) the plurality of particles with the at least one liquid polymer, the particles have at least two different sizes.

54. The method according to any one of claims 51 to 53.

55. the separator material mixture is cured to form a separator layer, and the electric field is applied perpendicular to the separator material mixture during curing.

55. The method according to any one of claims 51 to 54.

56. characterised in that prior to curing (313) the separator material mixture is applied (314) onto the at least one electrode in a sealing layer, preferably by sputtering, spin coating, screen printing and / or a sol-gel process, 56. The method according to any one of claims 51 to 55.

57. A method for manufacturing a device according to any one of claims 1 to 29, comprising the steps of: Providing (321) said vessel for culturing biological cells; Providing (322) a layer of the separator layer; Providing (323) the at least one electrode for forming and manipulating the biological cells; Equipped with the electrodes are separated from the interior space of the container by the separator layer; method.

58. The separator layer is produced according to the method of any one of claims 51 to 56.

58. The method of claim 57.

59. Use of a device according to any one of claims 1 to 29 for manipulating living cells.

60. 1. Use of a variable electric field capacitively coupled from outside a vessel containing a suspension containing at least one biological cell to manipulate said biological cell in said suspension.

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