TEMPERATURE-CONTROL ELEMENT FOR A MULTIWELL PLATE AND METHOD AND DEVICE FOR FREEZING AND/OR THAWING BIOLOGICAL SAMPLES

Abstract

The invention relates to a temperature-control element (4) for a multiwell plate (1), which comprises a plurality of cavities (2) arranged in rows and columns for freezing and/or thawing biological samples. The temperature-control element (4) comprises a base body (6) which is made of a thermally conductive material and is flown through by a temperature-control fluid; and a plurality of protruding temperature-control fingers (5) arranged in rows and columns on an upper side of the base body (6), which are connected in a thermally conductive manner to the base body (6), wherein a grid spacing of the temperature control fingers (5) corresponds to a grid spacing of the cavities (2) of the multiwell plate (1). The invention further relates to a device and method for freezing biological samples, in particular for cryopreservation, and/or thawing biological samples, in particular a cryopreserved sample.

Claims

1-24. (canceled)

25. A temperature-control body for a multiwell plate, which comprises a plurality of cavities, arranged in rows and columns, for at least one of freezing and thawing of biological samples, comprising a base body through which a temperature-control fluid can flow; and a plurality of protruding temperature-control fingers arranged in rows and columns on an upper side of the base body, wherein a grid spacing of the temperature-control fingers corresponds to a grid spacing of the cavities of the multiwell plate.

26. The temperature-control body according to claim 25, wherein at least one of an electrically controllable heating and an electrically controllable cooling element is integrated into at least some of the temperature-control fingers.

27. The temperature-control body according to claim 25, wherein a temperature sensor is integrated in a face area of at least one of the temperature-control fingers.

28. The temperature-control body according to claim 25, wherein end faces of the temperature-control fingers have at least one of the features (a) the end faces form flat support surfaces for bases of the cavities of the multiwell plate; and (b) the end faces comprise a coating of graphite or diamond.

29. The temperature-control body according to claim 25, wherein the temperature-control fingers have at least one of the features (a) the temperature-control fingers can be arranged within an area with a length of 127.8 mm and width of 85.5 mm; and (b) the number of temperature-control fingers corresponds to the number of cavities of the multiwell plate.

30. The temperature-control body according to claim 29, wherein the number of temperature-control fingers has one of the following values: 6, 8, 12, 16, 24, 48, 96, 384 or 1536.

31. The temperature-control body according to claim 25, having at least one of the features (a) an outer wall of bases of the cavities and an end face of the temperature-control fingers in order to form a local form fit each comprise a non-planar surface form complementary in shape to each other; and (b) a surface form of end faces of the temperature-control fingers and outer walls of the bases of the cavities are designed as interlocking toothing in order to form a local form fit.

32. The temperature-control body according to claim 25, comprising an inclination of end faces of the temperature-control body in relation to a planar surface of the base body which increases from a middle to two opposite marginal areas of the temperature-control body.

33. The temperature-control body according to claim 32, wherein the inclination is formed by an increasing oblique position of the temperature-control fingers arranged on the upper side of the base body or an increasing bevel of the end faces of the temperature-control fingers.

34. The temperature-control body according to claim 25, wherein integrated in the base body is at least one line, through which a temperature-control fluid can flow, with an inflow connection and an outflow connection for connecting at least one line with at least one of a cooling circuit and a heating circuit wherein the flow of the temperature-control fluid through the at least one line is controllable in such a way that predetermined, at least one of individual temperature-control fingers and at least one predetermined partial group of temperature-control fingers can be temperature controlled differently in relation to the remaining temperature-control fingers.

35. An arrangement of (a) a temperature-control body according to claim 25; and (b) a multiwell plate, the grid spacing of whose cavities arranged in rows and columns corresponds to the grid spacing of the temperature-control fingers of the temperature-control body.

36. A temperature-control apparatus for at least one of freezing and thawing of biological samples, comprising a temperature-control body according to claim 25, a positioning device for positioning a multiwell plate, the grid spacing of whose cavities arranged in rows and columns corresponds to the grid spacing of the temperature-control fingers, and the temperature-control body in a predetermined position relative to each other, wherein in the predetermined position the multiwell plate is positioned above the temperature-control body and the cavities are each positioned flush to the longitudinal axis of one of the temperature-control fingers; and a device for bringing the temperature-control fingers of the temperature-control body into contact with bases of the cavities of a multiwell plate positioned in the predetermined position.

37. The temperature-control apparatus according to claim 36, which is configured for at least one of a cryopreservation of biological samples and thawing of cryopreserved biological sample.

38. The temperature-control apparatus according to claim 36, wherein the device for bringing into contact comprises a pressing body which can be pressed from above onto a multiwell plate positioned above the temperature-control body in order to bring the bases of the cavities of the multiwell plate into contact with end faces of the temperature-control fingers.

39. The temperature-control apparatus according to claim 36, wherein the device for bringing into contact comprises a plurality of electrically controllable actuators which are designed to act directly or indirectly on an upper side of a multiwell plate positioned above the temperature-control body, in order, on controlling of the actuators to change a relative distance between the multiwell plate and the temperature-control body so as to move the temperature-control fingers and the bases of the cavities into contact or out of contact.

40. A temperature-control apparatus according to claim 39, wherein the electrically controllable actuators have at least one of the features (a) the electrically controllable actuators are designed as micromechanical actuators or as piezo-electrical actuators; (b) the plurality of the electrically controllable actuators can be controlled by the temperature-control apparatus at least one of individually and in groups in order to bring at least one of individual cavities and partial groups of cavities into or out of contact with the temperature-control body, irrespective of the other cavities; (c) by use of the electrically controllable actuators a displacement of the multiwell plate in the direction of the temperature-control element in the range of 1 μm to 1 mm can be produced; and (d) the electrically controllable actuators can be controlled by a control unit of the device for bringing into contact in such a way that consecutive bringing into contact, taking out of contact and bringing back into contact of the multiwell plate and temperature-control body can be carried out within a time in a range of 1 ms (millisecond) to 1 s (second).

41. The temperature-control apparatus according to claim 36, comprising a multiwell plate, the grid spacing of whose cavities arranged in rows and columns corresponds to the grid spacing of the temperature-control fingers.

42. The temperature-control apparatus according to claim 41, having at least one of the features (a) integrated into the bases of each of the cavities is at least one of an electrically controllable heating and an electrically controllable cooling element; (b) in a least one of the bases of the cavities a temperature sensor is integrated; and (c) the bases of the cavities are designed to be thermally conductive.

43. The temperature-control apparatus according to claim 36, comprising a temperature-control chamber, coolable from below, which is fillable or is filled with a dry gas and in a cooled state has a vertical temperature layering in the temperature-control chamber with a lower cold layer and an upper warm layer; at least one lock provided on a housing wall of the temperature-control chamber for at least one of introducing and removing a multiwell plate; and at least one of a first temperature-control body, arranged in the lower cold layer and connected to a cooling circuit, for the cryopreservation of biological samples, and a second temperature-control body, arranged in the upper warm layer and connected to a heating circuit, for thawing out cryopreserved biological samples.

44. The temperature-control apparatus according to claim 43, comprising (a) a multiwell plate containing samples to be thawed that can be positioned by use of the positioning device above the second temperature-control body; and (b) a multiwell plate containing samples to be frozen and introduced into the temperature-control chamber via the at least one lock that can be positioned by use of the positioning device above the first temperature-control body; and (c) a multiwell plate positioned above the at least one of the first and second temperature-control body that can be at least one of lowered and raised in a controlled or regulated manner by use of the device for bringing into contact in order to be brought into contact or taken out of contact with the temperature-control body.

45. The temperature-control apparatus according to claim 41, having at least one of the features (a) the temperature-control chamber is cooled with liquid nitrogen (LN2), nitrogen (N2) gas or a Sterling motor; (b) an ice trap is arranged in the temperature-control chamber; and (c) the warm layer has a temperature gradient which essentially corresponds with a predetermined starting temperature of a freezing process or a predetermined target temperature of a thawing process, whereas the cold layer has a temperature which essentially corresponds with a predetermined target temperature of the freezing process or a predetermined starting temperature of the thawing process.

46. A method of at least one of freezing and thawing of biological samples, said method comprising at least one of freezing and thawing the biological samples with a temperature-control body according to claim 25.

47. The method according to claim 46, including at least one of cryopreservation of biological samples and thawing cryopreserved biological samples.

48. The method according to claim 46, further comprising applying a substance to a sample stored in cavity of the multiwell plate.

49. The method according to claim 48, having at least one of the features (a) the substance is a solution which on hardening closes off a surface of the cavity contents from the outside; (b) the substance is a natural or synthetic oil, a liquid or a gel which cannot be mixed with an aqueous solution, or is solid carbon dioxide (CO.sub.2); and (c) the substance has a greater density than a nutrient solution surrounding the sample.

50. The method according to claim 49, wherein the applied substance is a solution which on hardening closes off the surface of the cavity contents from the outside in a gas-tight manner.

51. The method according to claim 48, having at least one of the features (a) on thawing of the sample the substance brings about a predetermined reaction or interaction with the sample; and (b) the substance is a dilution or washing solution or a cryoprotection agent, acts on the sample as a differentiation factor in relation to the sample, or is a substance which contains antioxidants, anti-apoptosis substances or live/dead staining agents.

52. The method according to claim 49, having at least one of the features (a) the substance is applied to the already frozen sample and at least one of after and during thawing of the sample the substance brings about a predetermined reaction or interaction with the sample; and (b) the substance is a substance whose condition reveals whether the sample has been frozen or thawed.

Description

[0051] Further details and advantages of the invention will be described below with reference to the attached drawings. In these show:

[0052] FIG. 1 a perspective view of a multiwell plate and a temperature-control body according to an embodiment of the invention;

[0053] FIG. 2 an arrangement of a multiwell plate and a temperature-control body from which a section is enlarged and shown in cross-section;

[0054] FIG. 3 a cross-sectional view of a multiwell plate and a temperature-control body according to another embodiment of the invention;

[0055] FIG. 4 schematically a temperature-control apparatus and temperature-control method according to an embodiment of the invention;

[0056] FIG. 5 schematically the application of a substance in accordance with an embodiment of the method;

[0057] FIGS. 6A and 6B a unit comprising a temperature-control finger and filled cavity;

[0058] FIGS. 7A and 7B a cross-sectional view of a multiwell plate on a temperature-control body according to another embodiment; and

[0059] FIGS. 8A and 8B a cross-sectional view of a multiwell plate according to another embodiment.

[0060] Equal components are given the same reference numbers in the figures.

[0061] FIG. 1 shows a perspective view of a multiwell plate 1 and a first embodiment of the temperature-control body 4 according to the invention. In the middle a schematic oblique view shows a commercially available plastic multiwell plate 1 in the standardized 96 well format. According to the standard the cavities (wells) 2 are arranged next to each other in a matrix-like manner in eight rows of twelve cavities each and constitute recesses for accommodating the sample(s) on such a multiwell plate 1. In accordance with the standard ANSI/SBS 4-2004 the grid spacing of adjacent cavities in a 96 well format multiwell plate is 9 mm.

[0062] Such multiwell plates 1 can be covered with a plastic cover, which can also be left out in the case of machines for filling, emptying or other manipulations. On the underside the cavities 2 are closed off in a planar manner with a thin plastic sheet or film which in terms of its optical quality generally allows microscope images of adhered cells.

[0063] Shown underneath the multiwell plate 1 in FIG. 1 is an example temperature-control body 4 for the multiwell plate 1. The temperature-control body 4 comprises a cuboid base body 6 through which a temperature-control fluid can flow and a plurality of protruding cylinder-shaped temperature-control fingers 5 arranged in rows and columns on an upper side of the base body 6 exactly matching the pattern of the 96-well multiwell plate 1. The temperature-control body 4 is made of a material with a high thermal capacity and good thermal conductivity. As a rule, metals such as silver or alloys are used.

[0064] Corresponding to the multiwell plate 1, 96 temperature-control fingers 5 are thus also arranged in eight rows of twelve temperature-control fingers 5 each in a matrix-like manner. The grid spacing of the temperature-control fingers 5 thus corresponds to a grid spacing of the cavities 2 of the multiwell plate 1, i.e. the distance between adjacent temperature-control fingers correspond to the spacing between adjacent cavities and in this case is thus also 9 mm. The temperature-control fingers 5 are each essentially identically formed and regularly arranged essentially equidistantly in area directions at right angles to each other spanning the contact surface with the multiwell plate 1. The temperature-control finger 5 can be provided in one piece with the base body 6. The temperature-control fingers 5 are in very good, generally thermal contact with the temperature-control bodies 6 arranged underneath.

[0065] Via at least 2 openings 7a, 7b a temperature-control gas or a temperature-control liquid can flow through the base body 6.

[0066] For this in the temperature-control body 6 a meandering or spiral course of a fluid guide connecting the two openings is provided so that an even or desired temperature profile is achieved, via which the temperature-control fingers each assume the temperatures prevailing at their location.

[0067] The temperature-control fingers 5 have as high a thermal capacity as possible which is much greater than that of the base areas of the multiwell plates so that during bringing into contact they dominate and determine the temperature of the cavity area with the biological sample, i.e. cooling and heating are essentially now only limited by the thermal conductivity of the base areas of the multiwell plate 1 and the biological sample.

[0068] For cooling and/or heating biological samples which are stored in a multiwell plate with a different format, for example in a multiwell plate with 8, 12, 16, 24, 48, 96, 384 or 1536 cavities a temperature-control body appropriately adapted to this format can be used, which then has 8, 12, 16, 24, 48, 96, 384 or 1536 temperature-control fingers 5, the grid spacing of which is matched to the grid spacing of the multiwell plate.

[0069] The principle of cooling a 96-well multiwell plate 1 from room temperature to a target temperature of, for example, −150° C. will be explained below by way of the example of identical cooling of all 96 cavities 2. Through different temperature controlling of the rows or columns of the temperature-control fingers or via heating elements (not shown) in the temperature-control fingers 5 different temperatures can also be brought about on the individual temperature-control fingers 5.

[0070] For freezing of a 96-well multiwell plate 1, it is initially brought to a temperature of between 1° C. and 15° C. at which the cryoprotection medium is added from above via pipettes. In the meantime the temperature-control body 4 has been brought to the target temperature by way of passing though nitrogen gas at a temperature of −150° C. to −195° C. so that all the temperature-control fingers 5 also assume this temperature. By means of a mechanism described below in the context of FIG. 4 the 96-well multiwell plate 1 is pressed now from above onto the temperature-control body 4 by a flat pressing body 8 so that the end faces 5a of the temperature-control fingers 5 come into direct material contact with the individual bases of the 96 cavities 2 of the multiwell plate 1. Instead of the pressing body 8 a piezo-controlled device can also be used for bringing the temperature-control fingers 5 into contact with the bases of the cavities 2 (shown in FIG. 3), which allows the contact of the multiwell substrate with the temperature-control fingers 5 to open and close again through perpendicular movement. For this only small gaps in the micrometer range are required. Through multiple repetition, solely in this way a temperature profile of the entire plate can be run.

[0071] Additionally or alternatively the temperature of the gas flow through the base body 6 can be altered, through which slower T profiles can be run as is also usual in the cryopreservation of cells (for example in the region of several fractions ° C. per minute, a few ° C. per minute). In the case of heating the procedure is reversed: The multiwell plate 1 is very quickly brought into contact with a temperature-control body 4 heated to a high temperature. A warm or a hot gas or also a corresponding liquid can flow through this, the temperature of which in the simplest case corresponds to the target temperature of, for example 10° C. at which the cryprotection medium can be washed out, or directly to 37° C. Here the multiwell plate 1 is also pressed rapidly to the temperature-control body 4.

[0072] For extremely rapid heating as required in the case of stem cells and in particular IPS, the temperature control body 4 is brought to 40° C. to 300° C. and is only brought into thermal contact with the multiwell plate 1 until the target temperature is reached. Via opening and closing the thermal contact between the temperature-control fingers 5 and the cavities 2 the courses of the temperature during heating can also be controlled.

[0073] In the lower section FIG. 2 shows a cross-section of a temperature-control body 24 which has a base body 6 through which a temperature-control fluid can flow and a plurality of protruding temperature-control fingers 25 arranged in rows and columns on an upper side of the base body 6. The grid spacing of the temperature-control fingers 25 again corresponds to the grid spacing of the cavities 2 of the multiwell plate 1, which in the middle of FIG. 2 is partially shown enlarged and in cross-section. Shown above it is the multiwell plate 1 in an oblique view with the marked area used as the cross-section shown.

[0074] In each of the cavities 2 there is a gas space 23 at the top and the biological sample 20 with adhered cells 21 on the upper side of the base plate 11 of the cavities 2. In the embodiment the multiwell plate 1 is still covered with a cover 3.

[0075] In order to achieve good pressing and thereby thermal contact between the temperature-control body 24 and the multiwell plate 1, in this variant of embodiment the temperature-control fingers 25 are not perpendicularly upright on the surface 6a of the base body 6 but are increasingly inclined towards the edges of the multiwell plate 1. This is shown in the figure in an exaggerated manner by the dashed line 5c and the two longitudinal axes 5b of temperature-control fingers 25 arranged in the outer area, which in comparison with the longitudinal axis 5d of a centrally arranged temperature-control finger 25 are tilted outward. Through the flat pressure from above or below the multiwell plate 1 is bent slightly in a lens-shape manner, which ensures that with their base sides 11 all the cavities 2 come into equally good planar contact with the temperature-control fingers 25. The upper surface of the temperature-control fingers, in particular the end face 25a can, as illustrated by an example cylinder in FIG. 2, be covered with a well thermally-conducting layer 9 through which very rapid cooling and heating become possible. Heating/cooling elements 10 can additionally be integrated into the temperature-control fingers 25 via which the temperature of individual elements can be controlled. To this end, near or on the end face of the temperature-control fingers 25 temperature sensor 12 are provided, for example with a flat configuration of a platinum resistance temperature sensor, such as a PT 100 or PT 1000 sensor. To simplify the illustration the aforementioned temperature sensors 12, the layer 9 of higher thermal conductivity or the heating/cooling elements 10 are only shown schematically for one or two temperature-control fingers 25, but can be provided on all temperature-control fingers 25.

[0076] In analogy to FIG. 2, FIG. 3 shows a cross-section of a multiwell plate 1 which is in contact with the temperature-control body 24. The special feature of this embodiment is that piezo-electric actuators 30 are firmly arranged on the cover of the multiwell plate 1 so that by way of expansion or shrinkage of the piezo-electric actuators 30 (shown by the arrows) contact between the cavities 2 and the temperature-control fingers 25 can be established or interrupted. This involves movements in the range from 1 to several 100 μm.

[0077] FIG. 4 shows as an example of a temperature-control apparatus 40 for the automated and direct cryopreservation of biological samples stored in the multiwell plates 1. The apparatus 40 comprises a temperature-controlled chamber 48 in which there is no moisture so that no relative humidity of the air can precipitate as ice. The chamber 48 also has areas which are at least at the initial temperature of the multiwell plate as well as at the required target temperature.

[0078] At the base of the temperature-control chamber 48 there is a trough 43 containing liquid nitrogen (LN2) openly or in a sponge-like materials, e.g. steel wool, porous aluminum etc. This is covered with a perforated metal sheet 44 which is intended to prevent parts falling into the nitrogen pool with a temperature of −196° C.

[0079] By the evaporation of the LN2a dry nitrogen atmosphere is produced in the interior which is structured in horizontal layers in such a way that an almost linear T-gradient with a lower cold layer 43a at around −196° C. and an upper warm layer 43b at around 10° C. or warmer is formed.

[0080] In addition two locks 47a and 47b are shown which are arranged on the housing wall of the temperature-control chamber 48. Via the lock 47a a multiwell plate 1 is introduced into the temperature-control chamber 48 or removed when warm. Via the lock 47b a multiwell plate 1 can be introduced into the temperature-control chamber 48 or removed when cold.

[0081] If humid air penetrates into the temperature-control chamber 48 through introducing or removing a multiwell plate 1, ice formation is forcible brought about by means of an ice trap 49. This is a cooled body in the warm area 43b. In order not to bring in humidity via the procedures, a hood (not shown) can again be placed on top of the temperature-control chamber 48 and over the locks 47a, 47b via which the gaseous dry nitrogen escapes. The entire system 40 is not closed in a pressure-right matter but has a syphon-like outlet pipe (not shown here) at the top.

[0082] In the temperature-control chamber 28 there is a fixed first cooled temperature-control body 41 for cooling introduced biological samples or the multiwell plate 1 and a second heated temperature-control body 42 for heating the biological samples or multiwell plates. Both do not have to be designed identically. Thus, for example, the surface of the end faces of the heating temperature unit 42 can be adapted to the shrunken multiwell substrate geometry at −150° C., i.e. the surface of the end faces of the temperature-control body 42 for heating can be slightly smaller than the end faces of the temperature-control body 41 for cooling.

[0083] The device 40 also comprises a positioning device (not shown) by means of which the multiwell plates 1 to be temperature-controlled can be moved within the chamber 49 in accordance with the displacement paths illustrated by the arrows 45a-c or by the arrows 46a-c and by means of which the multiwell plates can, in particular, be positioned in a precisely aligned manner above the temperature-control bodies 41 and 42. The arrows 45a-c show the temporal and spatial sequence when heating a cryogenic multiwell plate 1. The arrows 46a-c illustrate the sequence when cooling a multiwell plate 1. The paths indicated by the arrows are traversed by mechanical elements of the positioning device, the drives of which are preferably located outside the temperature-control chamber 48, and the multiwell plates 1 are moved by means of a conventional guide system, for examples rods, coils etc. (not shown).

[0084] For example, a multiwell plate 1 containing biological samples to be frozen is introduced into the temperature-control chamber 48 via the lock 47a (arrow 46a) and by means of the positioning device is moved into the cold layer 46a and there positioned above the first temperature-control body 41 standing on the perforated plate 44 (arrow 46b).

[0085] Positioning takes place in such a way that the cavities of the multiwell plate 1 are each positioned in alignment with the longitudinal axis of one of the temperature-control fingers of the temperature-control body 41. Subsequently the thus positioned multiwell plate is lowered in a controlled or regulated manner by means of a device for bringing into contact (not shown), such as described above in connection with FIG. 3, in order to be brought into contact with the temperature-control body 41.

[0086] After reaching the target temperature, the multiwell plate 1 can either be placed for storage in the lower cold layer 43a or removed for further processing from the temperature-control chamber 48 via the second lock 47a (arrow 46c).

[0087] FIG. 5 schematically illustrates the application of a substance on the biological samples stored in the cavities 2 and for this shows a row of cavities 2 with a fluid filling 20 and cells 21 on the cavity bases 11. In the row of cavities 2 the base plates 11 are in thermal contact with the temperature-control fingers 5. Before, during cooling or heating as well as afterwards in the frozen or thawed state, other substances 51 are added to the cavities 2 with a pipetting device 50, e.g. with a pipetting robot. For example, these can be cryoprotection agents, particle suspensions, solidifying gels and similar, which are useful during freezing, but can also be closing material which on solidification closes off the surface of the actual cavity contents from the outside so that no cover or other object is required as a closure, which simplifies the automation processes. However, fluids can also be added which on freezing produce a particular pattern or have a temperature sensor function by means of which it can be seen whether thawing has taken place in the meantime and the structure, color, mixture etc. has been changed. This can involve re-crystallization processes which are not macroscopically visible, but can be easily recognized and quantified via scattered light measurements, fluorescence measurements, Raman measurements or similar.

[0088] Of particular importance is the application of substances in solid or liquid form into the cavities 2 if their content 20 is already frozen. These could be differentiation factors for stem cells, which become active immediately after thawing, protective materials or genetic material which only combine with the solution below it after thawing out. They can also be dilution media which after thawing out reduce the concentration of the anti-freeze agents.

[0089] FIG. 6A shows an arrangement of a temperature-control finger 5 and a cavity 2. In the cavity 2 there is a filling consisting of three materials. At the bottom on the base is the culture medium 60 in which the biological samples (here shown as cells on the base plate) are located, above this is a medium 61 which is applied after freezing of the medium 60 so that it does not mix with it. This is all covered with a further medium 62 which produces a gas-tight closure with regard to the outside atmosphere. The medium 62 can be a natural or synthetic oil, a fluid that cannot be mixed with aqueous solutions, a gel or also solid CO2. The advantage of such arrangements is that they can be optimally adapted to the thawing process or deep-freezing. The nature and arrangement of the media determine the reaction when thawing out. Staggered liquefaction therefore takes place at different temperatures. Depending on the composition of the filling, media can be displaced in such a way that a new sequence comes about, as shown in FIG. 6B.

[0090] FIG. 6B shows two different states of an arrangement of a temperature-control finger 5 and a cavity 2. In the first state, designated “I” the arrangement is shown in the cold state in which nutrient medium 60 is frozen, whereas in the second state, designated “II” the arrangement is shown in the warm state in which the nutrient medium 60 is thawed out. For example, if in the frozen state a silicone oil 63 is applied to the solid nutrient medium, which has a higher density than the nutrient solution 60, the sequence of this layering will change around after both phases become liquid on increasing the temperature. As during thawing cells 64 easily detach from the surface and pass into suspension, after thawing they float in the nutrient solution 60 which rises and ultimately becomes the upper layer, which in automatic systems can be very easily removed and without having to take off a cover.

[0091] Alternative variants can be developed for freezing, in that, for example, glycerin solutions are used which remain liquid at temperatures down to −40° C. or do not take on a solid state at all. A particular advantage of this arrangement and method is the possibility of monitoring the observed cryogenic storage of samples and combination of materials in a solid and liquid state which is not possible at normal temperature.

[0092] FIGS. 7A and 7B show two classic well plates 70, 71 with larger cavities 72 with a diameter of approximately 2 to 3 cm. The well plates 70, 71 are designed as an injection-molded component in a form modified for freezing the multiwell plate in its entirety. The base plate 75 comprises a thin material that conducts heat well, e.g. a polymer, a metal, a metal coating or also diamond, so that the heat can be easily removed and introduced via the cooling and warming space 74 located in a stable cooling or warming body 73. The well plates 70, 71 are pressed from above onto the temperature-control unit 72 and through the generation of a slight negative pressure over the hollow spaces 76 can be bent in the well plate. This variant of overall cooling of the multiwell substrates is a simplified form which can be combined with the temperature-control body variants according to FIGS. 1 to 6. The introduction of the temperature-control body 4, 24 into the space 74 is one such combination possibility. A cooling or heating fluid or the temperature-control gas flows through this space, wherein the corresponding temperature courses are transferred via the multiwell substrate base 75 into the cavities 72. In all variants temperature sensors can in be integrated, e.g. in the form of flat Pt-100 or Pt-1000 sensors which are arranged on the base plate 75 or in each cavity 72. Alternatively, a temperature sensor can be arranged in a reference cavity with a similar or identical filling which provides the regulating values for control the temperature courses.

[0093] FIGS. 8A and 8B show a cross-section through a multiwell plate and a temperature-control body according to a further embodiment. Here, FIG. 8A shows a cross-section of an arrangement of a multiwell plate 1 and a temperature-control body 4, both of which are not in contact with each other yet. Shown in FIG. 8B is an individual cavity 2 with the temperature-control cylinder 25 below it. Characterized by the dashed lines in FIG. 8B a greatly enlarged excerpt of the cavity base area with the cells 21 can be seen. The cooling contact can now be considerably increased for more rapid temperature gradient processes in that the topography of the layer 82 located on the base plate 11 of the cavities 2 is designed with a counter-piece on the temperature-control body on the basis of the key and lock principle. In other words, the end faces 25a of the temperature-control cylinder 25 and the lower base side 11 of the cavities 2 each have a non-planar surface form 82, 83 corresponding in shape to each other in order to bring about a local form fit. The shown tip topography is only one example possibility.

[0094] Although the invention has been described with reference to certain examples of embodiment, for a person skilled in the art it is evident that different modifications can be carried out and equivalents used as substitutes without leaving the scope of the invention. Additionally, many modifications can be carried out without departing from the relevant area. Consequently the invention is not to be restricted to the disclosed examples of embodiment, but should cover all examples of embodiment which come under the scope of the attached patent claims. In particular, the invention also claims protection for the subject matter and the features of the sub-claims irrespective of the claims to which reference has been made.