EP3229965A2

EP3229965A2 - Temperature-control element for a multiwell plate and method and device for freezing and/or thawing biological samples

Info

Publication number: EP3229965A2
Application number: EP15798324.8A
Authority: EP
Inventors: Günter R. Fuhr; Heiko Zimmermann
Original assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Priority date: 2014-12-10
Filing date: 2015-11-06
Publication date: 2017-10-18
Also published as: KR20170093173A; US10987673B2; CN107257710B; DE102014018308A1; KR102103237B1; WO2016091344A2; JP6715841B2; US20170333905A1; CN107257710A; JP2018510321A; WO2016091344A3

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.

Description

DESCRIPTION

TEMPERATURE BODY FOR A MULTIWELL PLATE AND METHOD AND DEVICE FOR FREEZING AND / OR DEFROSTING

OF BIOLOGICAL SAMPLES

The invention relates to a tempering body for a multiwell plate. The invention further relates to an apparatus and a method for freezing biological samples, in particular for cryopreservation, and / or for thawing biological samples, in particular a cryopreserved sample.

The proliferation of institutional and commercial cryobiobanks, especially for the storage of living cell material, with sample numbers of tens of thousands to several millions, requires an automation of the processes. On the one hand, this is necessary to achieve cost-effective location conditions and, on the other hand, the consistent implementation of SOP conditions (SOP: Standard Operation Procedures), complete documentation and the exclusion of subjective influences by laboratory personnel, as increasingly demanded in the biomedical field.

Automated sampling standards have gained acceptance in the pharmaceutical, medical and large-scale biotechnology sectors, which were mainly developed during the development of high-throughput screening (so-called SBS standard) in laboratory practice multi-well substrates of columnar and cellular arranged, forming a round honeycomb structure, very small reaction spaces, also called wells, cavities or wells, used, in each of the smallest proportions of a sample, z. As cell material, a blood sample, etc., are introduced. These are multi-well substrates, with 6, 8, 16, 24 to 96 and more single wells in flat plastic versions. These formats are now standardized worldwide and are used in pipetting machines, cell culture machines as well as the device platform of analysis technology and diagnostics.

Such multi-well substrates are also referred to as multi-well plates or microtiter plates. The exact dimensions (length ^χ width ^χ height) are 127.76 mm ^χ 85.48 mm * 14.35 mm according to the ANSI standard recommended by the Society for Biomolecular Screening (SBS). Standards for microtiter plates have been published by the ANSI on the recommendation of the Society of Biomolecular Screening (SBS), in particular concerning the dimensions and positions of the wells in 96, 384 and 1536 well microtiter plates. These are the standards ANSI / SBS 1 to 4 - 2004 and the standard SBS-6 - 2009.

To the chain of processing, characterization and handling of samples in medicine, pharmaceutical development

and biotechnology more and more include cryobiobanks, in which the samples, especially living cells and here

Stem cells are deposited by animals and humans and fed back into use if needed. This usually takes place via cryopreservation, defined freezing and thawing protocols as well as a storage temperature below minus 140 ° C, however, in individual tubs, straws, individual plastic containers, etc., so that the suspensions contained in the corrugated substrates must be removed and transferred. As numerous experiments have shown, the quality of a biological sample, in particular when cells grow adhering to surfaces, decreases with each transfer, since they have to be detached by enzyme treatment or mechanical treatment and are therefore subject to considerable stress. It is also important that all samples of a corrugated plate, z. B. at 96 wells, treated in the same or predeterminable manner and thus also frozen, stored and thawed.

In addition, both controlled cooling and heating systems are known from practice, such as the "Cryo Freezers" from Planar Plc with programmable temperature programs or simple cryoboxes, such as the cryobox of the type sold under the name "Mr. Frosty"

ThermoFisher Scientific Inc .. In particular, in important medical cell types, such as immune cells, stem cells, especially the Induced Pluripotent Stem cells, recently controlled freeze and thaw protocols have proven to be extremely important for the quality of the sample and its vitality. Very good results are achieved with very quick cooling and warming. All this and in particular rapid cooling and heating below 1 ° / sec are not yet available for multiwell substrates, which is why the bioprobes have to be transferred to other vessels. But even in the conventional plastic tubes, due to the thickness of the plastic wall and the arrangement of the volumes, such exact, but above all, rapid temperature profiles can not be achieved.

It is thus an object of the invention to provide an improved apparatus for freezing biological samples, in particular for cryopreservation, and / or for thawing biological samples, in particular a cryopreserved sample, to avoid the disadvantages of conventional techniques and in particular allows a cryopreservation with a rapid cooling and / or heating and an increased vitality rate. A further object is to provide such a device which is process efficient in automated high throughput methods, e.g. As high-throughput screening method, can be integrated. Furthermore, it is an object of the invention to provide an improved method for freezing biological samples, in particular for cryopreservation, and / or for thawing biological

Samples, in particular cryopreserved samples to provide, are overcome with the vitality-affecting disadvantages of conventional techniques and in particular a simplified post-processing of the thawed bi- ologischen sample allows.

These objects are achieved by devices and methods having the features of the independent claims. Advantageous embodiments and applications of the invention will become apparent from the dependent claims and are explained in more detail in the following description with partial reference to the figures.

According to a first aspect of the invention, a temperature body is provided for a multi-well plate, for

Freezing and / or thawing of samples, in particular. biological samples located in the wells of the multiwell plate. In this case, the multiwell plate has, in a manner known per se, a plurality of cavities arranged in rows and columns.

The tempering body according to the invention comprises a body which can be flowed through by a tempering fluid, preferably of a thermally conductive material, preferably high heat. mekapazität, and a plurality of arranged on an upper surface of the body in rows and columns protruding tempering fingers, which are thermally conductively connected to the base body, wherein a grid spacing of the tempering corresponds to a grid spacing of the cavities of the multiwell plate. The distance between adjacent tempering fingers thus corresponds to the distance between adjacent cavities.

Thus, the invention comprises the general technical teaching to provide a tempering, which is adapted to the matrix-like, regular arrangement of the cavities of the multiwell plate and for this purpose has a corresponding maxtrixartige arrangement of tempering fingers in the pitch of the arrangement of the cavities. Here, the cooling fingers can be brought with their end faces with the bottom plates of the cavities in connection, wherein preferably each tempering finger of a cavity is assigned. In this case, the tempering fingers can be made rod-shaped or suppository-type, and the end faces of the tempering fingers are designed such that they form planar supports for the bottoms of the cavities of the multiwell plate.

A particular advantage of the invention is therefore that samples for freezing and thawing no longer in Einzeltubes, Straws or special plastic containers, etc. must be transferred, but frozen by means of the tempering according to the invention directly into and with the multiwell plate and subsequently thawed again , This can increase the vitality rate in the cryopreservation of biological samples. Another advantage is that a tempering body designed in this way allows a rapid freezing and / or thawing, since the tempering fingers can be positioned very close to the sample and high cooling and Heating rates can be generated directly in the bioprobe on the bottom wall of the cavities.

Furthermore, the time-consuming transfer into separate freezing containers is eliminated; instead, commercially available multi-well plates can be used throughout the process ^chain , so that the processing speed and efficiency, in particular for high-throughput processes, can be increased.

By the term "sample" is meant any article which undergoes cryopreservation in the cavity. The sample material typically includes biological material such as cells, tissues, cell components or biological macromolecules and optionally a nutrient solution, reagents, cryoprotectants or other substances.

According to a variant of the invention, an electrically controllable heating and / or cooling element may be integrated in at least some of the tempering fingers, preferably in all. Examples of such heating elements can also be microwave or high-frequency elements, which can set a defined heat input with appropriate control against the fluid cooling of the body. This variant offers the advantage that individual tempering fingers or subgroups of tempering fingers, for example individual rows and / or columns, can be tempered differently. As a result, the heat or cold input can be selectively varied via the arrangement of the cavities and adapted to the samples stored in the individual cavities.

According to another variant, there is the possibility that for monitoring the heat or cold entry into the cavities in the front region of at least one of the tempering fingers a temperature sensor, such as. As a thermoelectric sensor is integrated. For example, the temperature sensor may be designed as a flat design of a platinum resistance temperature sensor, such as a PT 100 or PT 1000 sensor.

In order to achieve the fastest possible freezing or thawing of the samples, it is advantageous if the end faces of the tempering fingers are highly polished, preferably with roughnesses below 20 pm, and / or have a coating of high thermal conductivity, preferably with a thermal conductivity comparable to that of copper or silver, preferably a coating of graphite or diamond.

According to the dimensions of commercially available multi-well plates, the tempering fingers can be arranged within an area with a length of 127.8 mm and a width of 85.5 mm. Further, the number of tempering fingers may correspond to the number of wells of the multi-well plate and preferably have one of the following values: 6, 8, 12, 16, 24, 48, 96, 384 or 1536. Further, there is a possibility that one of them may be different Number of Temperierfingern is arranged on the body, in particular a multiple of the aforementioned variants, for example, to temper a plurality of multi-well plates simultaneously with a tempering.

The tempering fingers are preferably made of a material of high heat capacity and high thermal conductivity, preferably of a metallic material. In particular, the heat capacity of the tempering fingers is higher than that of a commercially available multiwell plate. The main body of the tempering body comprises at least one line through which a cooling fluid can flow, with a supply connection and an outflow connection for connecting the at least one line to a cooling circuit and / or a heating circuit. The line course of the at least one line is preferably meander-shaped or spiral in order to achieve a desired temperature profile evenly distributed over the base body. An advantageous variant provides in this case that the flow of a tempering fluid through the at least one line is controllable so that predetermined individual tempering fingers and / or at least one predetermined subgroup of Temperierfinger different in temperature compared to the rest of the tempering fingers are tempered. This can be realized, for example, by means of a plurality of fluid lines or line sections which can be connected and disconnected for different regions of the heat sink, in order to temper sub-groups of tempering fingers differently. The end faces of the tempering fingers can be flat or slightly curved. This embodiment is preferably suitable for the tempering of multi-well plates which have cavities with a flat bottom or a slightly curved round bottom.

A further variant of this embodiment is characterized by an inclination of the end faces of the tempering fingers, which increases from the middle to two opposite edge regions of the temperature body, with respect to a planar upper side of the base body. The increasing inclination may be formed by an increasing inclination of the tempering fingers arranged on the planar upper side of the main body or by an increasing bevel of the end faces of the tempering fingers. According to this embodiment, uses that multiwell plates mostly made of plastic, z. B. made of polystyrene or polyvinyl chloride, are manufactured and bend slightly under pressure. This bending can be exploited by the increasingly inclined outwardly embodiment of the end faces of the tem- perierfinger advantageously to determine safe ^¬ that all cavities come into planar contact with the temperature conditioning.

A further alternative embodiment provides that an outer wall of the bottoms of the cavities and an end face of the tempering fingers for forming a local positive connection each have a shape-corresponding non-planar surface shape complementary to one another. In other words, the end face of the tempering finger according to the key-lock principle is designed as a counter contour to the contour of the underside of the cavities. For example, a surface shape of the end faces of the tempering fingers and a surface shape of the outer walls of the bottoms of the cavities to form a local form fit can be designed as interlocking toothing. This alternative embodiment has the advantage that even with cavities with small diameters, a large surface contact between the cavity and corresponding cooling finger and thus a comparatively large cooling surface and faster temperature control are made possible.

The invention further relates to an arrangement of a tempering, as disclosed herein, and a multi-well plate whose pitch of their arranged in rows and columns cavities corresponds to the pitch of the tempering fingers.

According to a further aspect of the invention, a temperature control device for freezing samples, in particular for cryopreservation, and / or for thawing samples, in particular particular of a cryopreserved sample. The samples may in particular be biological samples. The tempering device comprises a tempering as disclosed in this document. The temperature control device comprises WEI direct a positioning means for positioning the Tem ^¬ perierkörpers a multiwell plate in a predetermined position relative to each other, wherein the pitch of the ^¬ hen in Rei and columns arranged cavities of the multiwell plate correspond to the grid spacing of the Temperierfinger and wherein in the predetermined position, the multi-well plate above the tempering and the cavities are each positioned in alignment with the longitudinal axis of one of the tempering fingers. The tempering device further comprises a device for contacting the tempering fingers of the tempering with the bottoms of the cavities of a positioned in the predetermined position multiwell plate.

One possibility of realization in this case provides that the device for contacting brings about a pressing body which can be pressed from above onto a multiwell plate positioned above the tempering body, as a result of which the bottoms of the cavities of the multiwell plate are to bring the contact pressure in contact with the end faces of the tempering fingers. The pressing body preferably comprises a contact surface with the multiwell plate of at least the same

Length and width as the matrix-like arrangement of the cavities of the multiwell plate.

In a further advantageous embodiment variant of the temperature control device, the device for bringing into contact comprises a plurality of electrically controllable actuators, which are designed to indirectly on the top of a positioned above the temperature control multiwell plate, z. B. on the aforementioned Anpresskörper, or directly to attack in order to change a relative distance between the multi-well plate and the tempering when driving the actuators to bring the temperature control fingers and the bottoms of the cavities in contact and / or out of contact. The electrically controllable actuators may be embodied as micromechanical actuators or as piezoelectric actuators.

It is also advantageous to carry out the temperature control device that individually the plurality of electrically controlled actuators of the tempering and / or can be controlled in ^'part groups to individual cavities and / or subsets of cavities, z. B. individual rows or columns, regardless of the other cavities in contact and / or to bring out of contact with the tempering. This embodiment in turn makes the flexibility of plastic multi-well plates to advantage, by controlling only those actuators, which are arranged in a selected area above the multiwell plate, only those cavities in this area in contact or out of contact with the tempering can be brought.

Unlike in cryomicroscopes, in which the cooling and heating rate is controlled by the temperature of a cooling medium, this is done according to the temperature control via a preferably very rapid change in the contact preheated heat sink to multiwell plate. In this case, the tempering fingers can all be pressed or returned to the underside of the cavities of the multiwell plate at the same time or in groups, in some cases only a single tempering finger, so that a temperature bridge is established between the bottom of the multiwell plate and the tempering fingers and dissolved, so that the heat can be led out of or into the sample.

Another advantage of the embodiment in which the electrically controllable actuators as high-precision micro ^¬ mechanical actuators or piezoelectric actuators are carried out, is that such actuators by a control unit of the device for in-contacting can be controlled so that a sequential in-contact matching, an out-contacting and re-in-contact accommodating multiwell plate and tempering frame within a time can be carried out in the range of 1 ms (milli ^¬ seconds) to 1 s (second ) and this is feasible with a distance ^¬ accuracy <Ιμπι. Deflections of the multiwell plate in the direction of Temperierkörpers in the range of 1 are pect According to another As- means of electrically controllable actuators μηι to 1 mm produced, characterized Kgs ^¬ NEN virtually any temperature programs and Temperaturgradi ^¬ ducks gradients can be realized.

As already explained above, the tempering device for handling a multi-well plate, the grid spacing of which is arranged in rows and columns cavities corresponds to the grid spacing of the tempering of a tempering of the temperature control executed. Furthermore, the temperature control device may comprise such a multiwell plate.

The multiwell plate may be a commercially available multiwell plate. Furthermore, the multiwell plate may differ from commercial multiwell plates and be adapted for use for cryopreservation and for use with the tempering body and / or the tempering device. In this case, in the context of the invention in particular the possibility that in the bottoms of the cavities each one electrically controllable heating and / or cooling element, preferably a Peltier element, is integrated and / or that in at least one of the bottoms of the cavities, a temperature sensor is integrated. Furthermore, the bottoms of the cavities can be made thin and made of a heat-conducting material or be provided with a structure on the underside, resulting in a larger surface contact with the tempering.

The modified according to the above variants multiwell plate should also be disclosed as an independent subject and claimable.

The tempering device may further comprise, in a manner known per se, a temperature-controlled cooling chamber or housing which can be filled and / or filled with a dry gas and, when cooled, has a vertical temperature stratification in the temperature-control chamber with a lower cold layer and an upper warm layer. and at least one provided on a housing wall of the Temperierkaramer lock for introducing and / or carrying out a multiwell plate. Advantageously, two such locks are provided: a first lock, via which a multiwell plate can be introduced or carried out in the chamber in the warm state, and a second lock, via which a multiwell plate is introduced into the chamber in the cold state or can be executed.

It has already been stated above that the tempering device comprises a tempering body according to the invention. In particular, the tempering device may be arranged in the lower cold layer of the temperature control chamber, connected to a cooling circuit first temperature control body for cryopreserving biological samples and / or arranged in the upper warm layer, attached to a heat cycle. include closed second tempering for thawing cryokonser- vierter biological samples.

According to another aspect of this variation, a mounted in the lower cold layer of the temperature-control chamber, we ^¬ nigstens containing a thawed sample multiwell plate may be positioned above the second temperature conditioning means of the positioning device. Furthermore, a multi-well plate which is introduced into the temperature-control chamber via the at least one lock and contains at least one sample to be frozen can be positioned above the first temperature-control body by means of the positioning device. For this purpose, the positioning device may have a suitably designed guide mechanism in order to move the multiwell plate within the temperature chamber. According to this aspect, a multi-well plate positioned above the first and / or second temperature control body can be lowered or raised in a controlled manner by means of the device for bringing it into contact and / or out of contact with the thermal element to become.

Alternatively, the positioning device can also be designed instead to move the or the tempering body toward the multiwell plate.

In another aspect, the tempering chamber may be cooled with liquid gases such as LN2, N2 gas or a Sterling engine. For example, in a trough at the bottom of the tempering chamber open or introduced into a spongy material liquid nitrogen can be stored, resulting in the vertical temperature stratification.

Furthermore, it is advantageous to arrange an ice trap in the tempering chamber in order to prevent ice formation over the ice trap. force if humid air should penetrate into the tempering chambers from the outside by introducing or carrying out a multiwell plate. According to a further aspect of the vertical Temperaturver ^¬ running is set within the temperature chamber so that the warm layer having a temperature which corresponds essentially to a predetermined starting temperature of a freezing process or a specified target temperature of a thawing process corresponds, while the cold layer has a temperature which Substantially corresponds to a predetermined target temperature of the freezing process or a predetermined starting temperature of the thawing process. A further aspect of the invention relates to a method for freezing biological samples, in particular for cryopreserving, and / or thawing biological samples, in particular cryopreserved samples, using a tempering body as disclosed in this document, and / or a tempering device, as in disclosed in this document.

According to a preferred embodiment, the method comprises applying a substance to a sample stored in a cavity of the multiwell plate.

According to an advantageous variant, the applied substance is a solution which, when it solidifies, terminates a surface of the cavity contents with respect to the outer space, preferably closes in a gastight manner, so that no lid or the like is required as a closure. The substance may be, for example, a natural or synthetic oil, a liquid or a gel which is immiscible with an aqueous solution, or solid CO 2. According to a particularly advantageous variant, the substance is applied to the already frozen sample, wherein the substance after and / or thawing of the sample causes a predetermined reaction or interaction with the sample. Preferably, a substance is used, from the state of which it can be deduced whether, after freezing the sample, an intermediate thawing has taken place.

According to a further advantageous variant, the substance has a higher density than the nutrient solution surrounding the sample, so that the sequence of the sample and the substance turns over after thawing, so that, for example, floating cells can be easily removed in a nutrient solution.

According to a further advantageous variant, a substance is applied, wherein information about the sample can be derived from the state of the introduced substance and / or the substance causes a predetermined reaction or interaction with the sample during the thawing of the sample.

For example, it is possible to add liquids which give rise to a specific pattern during freezing or have a temperature sensor function from which it is possible to detect whether thawing has occurred in the meantime or by which the structure, color, mixture, etc. have been changed. These can also be recrystallization processes which are not visible macroscopically but can easily be detected and quantified by means of scattered light, fluorescence, Raman measurements or the like. For example, the substance may be a diluent and wash solution or antifreeze, act as a differentiation factor with respect to the sample, or be a substance containing antioxidants, anti-apoptotic substances, and / or live-dead dyes. It is emphasized that the abovementioned method aspects relating to the application of the substance to a sample stored in the cavity are also possible independently of the use of the temperature control device and / or of the temperature control element and are therefore claimed independently of the use of the temperature control device and / or the temperature control element can .

In order to avoid repetition, features disclosed purely in terms of apparatus should also be considered and be able to be claimed as part of the manufacturing process.

In summary, with the present invention, multi-well plates can be used directly for cryopreservation, so that every transfer into new containers is omitted. By

Adapting the tempering to the format of the multi-well plate used in each case can be used in principle multiwell plates of any number of cavities directly for cryopreservation. As a result, known cryotechnologies can be efficiently integrated into the existing high-throughput process chains so that the automation chain of the multi-well plate-based device platforms is also closed in the cryogenic area. Further details and advantages of the invention will be described below with reference to the accompanying drawings. Show it:

Figure 1 is a perspective view of a multi-well plate and a tempering according to an embodiment of the invention; an arrangement of a multi-well plate and a tempering, from which a section is enlarged and reproduced in section; a cross section of a multi-well plate and a tempering according to another embodiment of the invention; schematically a tempering and a tempering according to an embodiment of the invention;

FIG. 5 schematically shows the application of a substance according to an embodiment of the method;

FIGS. 6A and 6B show a unit consisting of a tempering finger and a filled cavity;

FIGS. 7A and 7B show a cross-section of a multi-well plate on a tempering body according to another embodiment; and

FIGS. 8A and 8B show a cross section of a multiwell plate and a tempering element according to a further embodiment.

Identical parts are provided in the figures with the same reference numerals.

Figure 1 shows a multiwell plate 1 and a first Ausfüh tion form of the tempering 4 according to the invention in a perspective view. In this case, in the center, a commercially available plastic multiwell plate 1 in the standardized 96-well format is shown schematically in an oblique view. shows. According to the standard, the cavities (wells) 2 are arranged side by side in matrix form in eight rows of twelve cavities each and represent depressions for receiving the test pieces) on such a multiwell plate 1. According to the standard ANSI / SBS 4-2004, the grid spacing of adjacent cavities is for a 9 mm 96-well multiwell plate.

Such multi-well plates 1 can be covered with a plastic lid 3, which can also be omitted in the machine for filling, emptying and other manipulations. On the underside, the cavities 2 are planarly closed with a thin plastic disc or foil, which generally allows microscope images of adherent cells in their optical quality.

Below the multiwell plate 1, an exemplary tempering 4 for the multiwell plate 1 is shown in Figure 1. The tempering body 4 comprises a cuboid basic body 6, through which a tempering fluid can flow, and a plurality of projecting cylindrical tempering fingers 5 arranged in rows and columns in rows and columns in rows and columns exactly in the pattern of the 96-well multiwell plate. The tempering body 4 is made of one Material made of high heat capacity or good heat conduction. Usually these are metals, such as silver or alloys.

Corresponding to the multi-well plate 1, therefore, likewise 96 tempering fingers 5 are arranged in a matrix-like manner in eight rows of twelve tempering fingers 5 each. The grid spacing of the tempering fingers 5 corresponds to a grid spacing of the cavities 2 of the multiwell plate 1, ie, the distance between adjacent tempering fingers thus corresponds to the spacing of adjacent cavities and is therefore also 9 mm in the present case. The tempering fingers 5 are in each case of essentially the same shape and are regularly divided in two into the contact area with the multi-surface Well plate 1 spanning and substantially perpendicular to each other surface directions each arranged substantially equidistantly. The tempering fingers 5 can be provided in one piece with the main body 6. The Temperierfinger 5 are in very good, usually thermi ^¬ rule contact with the arranged below tempering

The base body 6 can be flowed through by at least two openings 7a, 7b with a temperature control gas or a temperature control liquid. For this purpose, a meandering or even spiral course of a fluid guide connecting the two openings is realized in the tempering body 6 so that a uniform or desired temperature profile is achieved, via which the tempering fingers 5 each assume the temperature prevailing at their location.

The temperature control fingers 5 have the highest possible heat capacity, which is far greater than that of the bottom regions of the multi-well plates, so that they dominate and determine the temperature of the cavity region with the bioprobe when in contact with them. D. h., The cooling and heating are essentially limited only by the thermal conductivity of the bottom regions of the multiwell plate 1 and the bioprobe.

For cooling and / or heating of biosamples stored in a multiwell plate of a different format, for example in a multiwell plate with 8, 12, 16, 24, 48, 96, 384 or 1536 cavities, a corresponding This format adapted tempering are used, which then according to 8, 12, 16, 24, 48, 96, 384 or 1536 Temperierfinger 5 whose pitch is adapted to the pitch of the multiwell plate. The principle of cooling a 96-well multiwell plate 1 from room temperature to a target temperature of z. B. minus 150 ° C is explained below using the example of a similar cooling all 96 cavities 2. By different tempering of the rows or columns of tempering fingers 5 or into the

Tempering fingers 5 heating elements located (not shown) and different temperatures at the individual Temperierfingern 5 can be realized.

For the freezing of a 96-well multiwell plate 1, this is first brought to a temperature between 1 ° C and 15 ° C, in which the antifreeze medium is added from above via pipettes. In the meantime, the tempering body 4 has been brought to the target temperature via the passage of nitrogen gas at a temperature of -150 ° C. to -195 ° C., so that all temperature control fingers 5 also assume this temperature. The 96-well multi-well plate 1 is then pressed onto the tempering body 4 by means of a press-on body 8 pressing surface-on from above in such a way that the end faces 5a of the tempering fingers 5 are in direct material contact with the individual bottoms of the 96 Cavities 2 of the multiwell plate 1 come. Instead of the pressing body 8, it is also possible to use a piezo-controlled device for bringing the tempering fingers 5 into contact with the bottoms of the cavities 2 for pressure (shown in FIG. 3), which permits contact of the multiwell substrate with the tempering fingers 5 to open by vertical movement and close again. For this only small gaps in the micrometer range are required. By repeated repetition alone on this way a temperature profile of the entire plate can be driven. Additionally or alternatively, the temperature of the gas stream can be changed by the base body 6, which can drive slow T-profiles, as they are also common in the cryopreservation of cells (for example in the range of a few fractions ° C per minute, some ° C per Minute). In the case of heating, the procedure is contrary: Very quickly, the multiwell plate 1 is brought into contact with a temperature control body 4 brought to a high temperature. This can be with a warm or

hot gas or else a corresponding liquid to be flowed through, whose or whose temperature in the simplest case of the target temperature, for. B. 10 ° C, at which then the antifreeze is washed out, or directly to 37 ° C. In this case, the multi-well plate 1 is also pressed rapidly to the tempering 4.

For extremely rapid warming, which is desirable in stem cells and in particular IPS, the tempering 4 is brought to 40 ° C to 300 ° C and only as long brought into thermal contact with the multiwell plate 1, until the target temperature is reached. Even during heating, the temperature profiles can be controlled by opening and closing the thermal contact between the tempering fingers 5 and the cavities 2.

FIG. 2 shows, in section, in the lower part, a tempering body 24, which in turn has a base body 6, through which a tempering fluid can flow, and a plurality of projecting tempering fingers 25 arranged in rows and columns on the upper side 6a of the base body 6. The grid spacing of the tempering fingers 25 again corresponds to the grid spacing of the cavities 2 of the multiwell plate 1, which is enlarged in the middle in a detail in FIG. 2 and reproduced in section. In addition, the multiwell plate 1 is inclined Top view shown with the marked area used as a cut.

In the cavities 2 is located in each case above a gas space 23 and the biosample 20 with adherent cells 21 on the Obersei ^¬ te the bottom plate 11 of the cavities 2. In the embodiment, the multiwell plate 1 is still covered with a cover 3.

In order to achieve a good contact pressure and thus thermal contact between the tempering 24 and the multiwell plate 1, the tempering fingers 25 are according to this embodiment not vertically standing on the surface 6a of the base 6, but increasingly inclined to the edges of the multiwell plate 1 , This is exaggerated in the figure by the dashed line 5c and the two longitudinal axes 5b of tempering fingers 25 arranged in the outer region, which are tilted outward in comparison with the longitudinal axis 5d of a centrally arranged tempering finger 25. Due to the surface pressure from above or below the multiwell plate 1 is bent a little lenticular, which ensures that all cavities 2 get with their bottom bottom 11 in the same good planar contact with the tempering fingers 25. The surface of the temperature control fingers, in particular the end face 25a, as exemplified by an example cylinder in Figure 2, with a good thermal conductivity

Layer 9 can be covered, whereby very rapid cooling and heating are possible. In addition, heating or cooling elements 10 may be integrated in the tempering fingers 25, via which individual elements are controlled in temperature.

For this purpose are located near or on the end face of the tempering 5 temperature sensors 12, z. B. in flat design 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 high thermal conductivity or the heating or cooling elements 10 shown schematically only for one or two Temperierfinger 25, but can be vorgese ^¬ hen at all Temperierfingern 25.

FIG. 3 shows, in analogy to FIG. 2, a cross section of a multiwell plate 1, which is in contact with the temperature control body 24. The peculiarity of this embodiment is that piezoelectric actuators 30 are fixedly arranged on the cover of the multiwell plate 1, so that over the expansion or shrinkage of the piezoelectric actuators 30 (shown by the arrows), the contact of the cavities 2 to the tempering fingers 25 is made or interrupted. These are deflections in the range of 1 to a few 100 μπκ

FIG. 4 shows by way of example a tempering device 40 for the automated and direct cryopreservation of biological samples stored in multi-well plates 1. The device 40 includes fully a temperature-controlled chamber 48, no ^moisture-¬ ness is in the ^"so that no humidity may condense as ice. The chamber 48 comprises further regions which at least the output temperature of the multiwell plate and the have desired target temperature.

At the bottom of the temperature chamber 48 is for this purpose a trough 43 in which open or in a sponge-like material, for. As steel wool, porous aluminum, etc., liquid nitrogen (LN2) is located. This is covered with a perforated plate 44, which should prevent that parts can fall into the nitrogen lake at a temperature of -196 ° C. By the evaporation of LN2 a tro ^¬ ckene nitrogen atmosphere, the structured in horizontal Schich ^¬ th that an almost linear T-gradient with a lower cold layer 43 at about -196 ° C and an upper warm layer 43b is formed in the interior about 10 ° C or warmer develops.

Furthermore, 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 multi-well plate 1 is warmly introduced or carried out in the temperature-control chamber 48. Via the lock 47b, a multiwell plate 1 can be cold introduced or performed in the temperature control chamber 48. If, due to the introduction or removal of a multi-well plate 1, moist air enters the tempering chamber from the outside

48 should penetrate, the ice is over an ice trap

49 forced. This is a cooled body in the warm area 43b. To bring about the interventions no moisture, again a hood (not shown) on top of the

Temperature chamber 48 and via the locks 47a, 47b are placed, through which the gaseous dry nitrogen escapes. The overall system 40 is not sealed pressure-tight, but has a siphon-like outlet tube at the top (not shown here).

In the tempering chamber 48 is fixed a first cooled tempering 41 for the cooling of introduced biosamples or the multiwell plate 1 and a second heated tempering 42 for heating the biosamples or multiwell plates. Both do not have to be identical. So z. For example, the area of the end faces in the heating temperature control unit 42 of the shrunken multiwell substructure geometry can be adapted at -150 ° C., ie the area the end faces of the tempering 42 for heating are made slightly smaller than the end faces of the tempering 41 for cooling. The device 40 further comprises a positioning device (not shown), by means of which the multiwell plates 1 to be tempered within the chamber 49 can be moved in accordance with the travel paths illustrated by the arrows 45a-c or by the arrows 46a-c, and by means of which the multi-well plates can be positioned in particular exactly aligned above the tempering bodies 41 and 42. The arrows 45a-c show here the temporal and spatial sequence when heating a cryogenic multiwell plate 1. The arrows 46a-c illustrate the course of a cooling of a multiwell plate 1. The paths indicated by the arrows are by means of mechanical elements of the Go through positioning, the drives are preferably outside the temperature control chamber 48 and a conventional guide mechanism, such. As rods, coils, etc. (not shown), the multi-well plates - l - method.

Thus, for example, a multiwell plate 1 containing bioprobes to be frozen is introduced via the lock 47a into the temperature control chamber 48 (arrow 46a) and moved by means of the positioning device into the cold layer 43a and there above the first, standing on the perforated plate 44 Temperature control 41 positioned (arrow 46b). The positioning takes place in such a way that the cavities of the multi-well 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 by means for contacting (not shown), such as. B. described above in connection with Figure 3, controlled or regulated lowered to be brought into contact with the tempering 41,

After reaching the target temperature, the multiwell plate 1 can either be stored for storage in the lower cold layer 43a or for further processing via the second

Lock 47a are led out of the temperature control chamber 48 (arrow 46c). FIG. 5 schematically illustrates the application of a substance to the biological samples stored in the cavities 2, and shows a series of cavities 2 with a liquid filling 20 and cells 21 on the cavity bottoms 11 the temperature control fingers 5. Before, during cooling or heating and then in the frozen or thawed state further substances 51 with a pipetting 50, z. B. with a pipetting robot, added to the cavities 2. These may be, for example, antifreeze agents, particle suspensions, gels and the like which solidify, which are helpful in freezing, but may also be closure material which, when solidified, seals the surface of the actual cavity contents with respect to the exterior so that there is no cover or other is needed as a closure, which simplifies the automation processes. But it can also be added liquids that give a certain pattern during freezing o- have a temperature sensor function, where you can tell whether there has been a thawing in the meantime, or by the structure, color, mixture, etc. has been changed , These can also be recrystallization processes which are not visible macroscopically but can easily be detected and quantified by means of scattered light, fluorescence, Raman measurements or the like. Of particular importance is the application of substances in solid or liquid form in the cavities 2, when the contents 20 is already frozen. This can be differentiation factors for stem cells that become effective immediately after thawing, protective materials, or genetic material that only binds after thawing with the underlying solution. These can also be diluent media that reduce the concentration of antifreeze after thawing.

Figures 6A shows an arrangement of a tempering finger 5 and a cavity 2. In the cavity 2 is a filling, which consists of three materials. At the bottom of the bottom, the culture medium 60, in which the bioprobes (here shown as cells on the bottom plate) are located, is a medium 61 which has been applied after freezing of the medium 60, so that it is not mixed with it , The whole is covered with another medium 62 which provides a gas tight seal to the outside atmosphere. The medium 62 can be a natural or synthetic oil, a liquid-immiscible liquid, a gel, or solid CO 2. The advantage of such arrangements is that they can be optimally adapted to the process of thawing or freezing. The

The type and arrangement of the media determines the reaction during thawing. Thus, staggered liquefaction takes place at different temperatures. Depending on the composition of the filling media can shift so that a new order arises, as shown in Figure 6B.

FIG. 6B shows two different states of an arrangement of a tempering finger 5 and a cavity 2. In the first state, designated "I", the arrangement is in the cold state. In the second state, designated "II", the arrangement in the warm state in which the nutrient solution 60 has thawed is shown, for example, when frozen, a silicone oil 63 is applied to the solid culture medium 60, which has a higher density than the nutrient solution 60, then this layer will turn over in sequence after the two phases have become liquid as the cells 64 easily detach from the surface during thawing and go into suspension, after thawing, they float in the upward-rising, final-layered nutrient solution 60, which can be easily removed from a vending machine without having to remove a cover Alternative variants can be developed for freezing, for example by using glycerol solutions which remain liquid up to temperatures of -40 ° C and lower or which do not assume a solid state at all A particular advantage of this arrangement and method is the possibility of checking the maintained frozen storage of samples and the combination of materials in solid and liquid state, which are not possible at normal temperature. FIGS. 7A and 7B show two classic corrugated sheets 70, 71 with larger cavities 72, with a diameter of approximately 2 to 3 cm. The corrugated sheets 70, 71 are made as an injection-molded part in a modified form for freezing in the entirety of the multiwell plate. The bottom plate 75 is made of a heat-conducting thin material, for. B. of a polymer, a metal, a metal coating or diamond, so that the heat well above the cooling or

Warming room 74, which is located in a stable cooling or heating body 73, can be initiated or initiated. The corrugated sheets 70, 71 are pressed from above ^onto the tempering unit 73 and can be bent over the cavities 76 in the corrugated sheet by creating a slight negative pressure. This variant of the total cooling of the Multiwellsub- strate is a simplified form that combined with the Temperierkör ^¬ pervarianten corresponding to FIGS 1 to 6 who can ^¬. Such a possibility of the combination is the introduction of the tempering 4, 24 in the space 74. This is traversed by a cooling or heating liquid or the tempering gas, whereby the corresponding temperature profiles are transmitted via the multiwell substrate bottom 75 in the cavities 72. In all variants, in turn, temperature sensors can be integrated, for. Example in the form of flat Pt-100 or Pt-1000 sensors, which are arranged on the bottom plate 75 o- or in each cavity 72. Alternatively, a temperature sensor which supplies the control values for the control of the temperature profiles can also be arranged in a reference cavity with a similar or identical filling. FIGS. 8A and 8B show a cross-section of a multi-well plate and a tempering body according to a further embodiment. Here, Figure 8A shows a cross section of an arrangement of a multi-well plate 1 and a tempering 4, both of which are not yet in contact with each other. FIG. 8B shows a single cavity 2 with the tempering cylinder 25 below. Characterized by the dashed lines, a greatly enlarged section of the cavity bottom area with the cells 21 can also be seen in FIG. 8B. The cooling contact can now be substantially increased for faster temperature-gradient processes by the topography of the located on the bottom plate 11 of the cavities 2 layer 82 is performed with a counterpart on the tempering according to the key-lock principle. In other words, the end faces 25a of the elements Perierzylinder 25 and the lower bottom side 11 of the cavities 2 for forming a, local form fit in each case a mutually complementary form-corresponding non-planar surface shape 82, 83. The topography shown here is just one example.

Although the invention has been described with reference to particular embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for without departing from the scope of the invention. In addition, many modifications can be made without leaving the associated area. Accordingly, the invention should not be limited to the disclosed embodiments, but should include all embodiments which fall within the scope of the appended claims. In particular, the invention also claims protection of the subject matter and the features of the subclaims independently of the claims referred to.

Claims

Requirements 1. Temperature control body (4; 24) for a multi-well plate (1), which has a plurality of rows and columns

Cavities (2), for freezing and / or thawing of biological samples comprising

a body (6) through which a temperature control fluid can flow; and

a plurality of on a top of the base body (6) arranged in rows and columns projecting tempering fingers (5; 25), wherein a grid spacing of the tempering fingers (5; 25) corresponds to a grid spacing of the cavities (2) of the multiwell plate (1).

2. tempering (24) according to claim 1, characterized

in that an electrically controllable heating and / or cooling element (10) in at least some of

Temperature control finger (25) is integrated.

3. tempering (24) according to claim 1 or 2, characterized in that in the end region (25 a) of at least one of the tempering fingers (25), a temperature sensor (9) is integrated.

4. tempering according to one of the preceding claims, characterized in that the end faces (5a, 25a) of the tempering fingers (5; 25)

(a) form flat supports for the bottoms (11) of the cavities (2) of the multiwell plate (1); and or

(b) have a coating of graphite or diamond.

5. tempering body (4; 24) according to one of the preceding claims, characterized in that (a) that the tempering fingers (5; 25) are disposed within an area 127.8 mm long and 85.5 mm wide; and or

(b) that the number of tempering fingers (5; 25) corresponds to the number of cavities (2) of the multiwell plate (1) and

preferably has one of the following values: 6, 8, 12, 16, 24, 48, 96, 384 or 1536.

6. tempering body (24) according to any one of claims 1-3 or 5, characterized

(A) that an outer wall of the bottoms (11) of the cavities (2) and an end face (25a) of the tempering fingers (5) for

Forming a local form fit in each case a mutually complementary form-corresponding non-plane

Surface shape (82, 83) have; and or

(B) that a surface shape (83) of the end faces (25a) of the tempering fingers (25) and the outer walls (82) of the floors

(11) the cavities (2) to form a local

Positive fit are designed as interlocking teeth.

7. temperature control body (24) according to one of the preceding

Claims, characterized by an inclination of the end faces (25a) increasing from the middle to two opposite edge regions of the temperature body (24)

Temperature control finger (25) relative to a planar top (6a) of the base body (6).

8. temperature control body (24) according to claim 7, characterized

characterized in that the increasing inclination through a

increasing inclination of the tempering fingers (25) arranged on the planar upper side (6a) of the basic body (6) or by an increasing bevel of the end faces of the

Temperierfinger is formed.

9. tempering body (4; 24) according to one of the preceding claims, characterized in that in the base body (6) at least one of a tempering fluid can be flowed through

Line is integrated, with an inflow port (7a) and an outflow port (7a) for connecting the at least one line to a cooling circuit and / or a

Heating circuit, wherein the flow of the tempering fluid is controlled by the at least one line, such that

predetermined individual tempering fingers (5; 25) and / or

at least one predetermined subgroup of tempering fingers (5; 25) different in comparison to the rest

Temperature control fingers (5, 25) are temperature controlled.

10. Arrangement off

(A) a tempering according to any one of claims 1 to 9; and

(B) an ultiwell plate, the grid spacing of their arranged in rows and columns cavities corresponds to the grid spacing of the tempering fingers of the tempering.

-

11. tempering device (40) for freezing biological samples, in particular for cryopreservation, and / or for

Thawing of biological samples, especially one

cryopreserved sample comprising

a tempering body (4; 24; 41, 42) according to one of the

Claims 1 to 9,

a positioning device for positioning a

Multiwell plate (1), the grid spacing of their arranged in rows and columns cavities the pitch of the

Temperierfinger (5; 25) corresponds, and the tempering (4; 24; 41, 42) in a predetermined position relative

to each other, wherein in the predetermined position, the multiwell plate above the tempering (4; 24, 41, 42) and the cavities (2) in each case to the longitudinal axis of the tempering fingers (5; 25) are aligned; and a means for contacting the

Temperature control finger (5) of the tempering (4, 24, 41, 42) with the bottoms (11) of the cavities (2) of a positioned in the predetermined position multiwell plate (1).

12. tempering device (40) according to claim 11, characterized in that the means for bringing into contact comprises a pressing body (8) from the top of a

positioned above the tempering body (41, 42)

Multiwell plate (1) is pressable to the bottoms (11) of the

Cavities (2) of the multiwell plate (1) in contact with the end faces (5a) of the tempering fingers (5; 25) to bring.

13. tempering device (40) according to claim 11 or 12, characterized in that the means for contacting a plurality of electrically controllable actuators comprises, which are carried out at the top of a temperature control device (4, 24, 41, 42 ), in order to change a relative distance between the multiwell plate (1) and the tempering body (4, 24, 41, 42) when the actuators are actuated in order to move the tempering fingers (5, 25) ) and the bottoms (11) of the cavities (2) in contact and / or out of contact.

14. Temperature control device (40) according to claim 13, characterized in that

(A) that the electrically controllable actuators as micromechanical actuators or as piezoelectric

Actuators (30) are executed; and or

(b) that the majority of the electrically controllable

Actuators of the temperature control (4, 24, 41, 42) individually and / or in sub-groups are controlled to individual cavities (2) and / or sub-groups of cavities (2)

regardless of the other cavities in contact and / or except To bring contact with the tempering body (4; 24; 41, 42); and or

(C) that by means of the electrically controllable actuators, a deflection of the multiwell plate (1) in the direction of

Temperierkörpers (4; 24; 41, 42) in the range of 1 μιη to 1 mm can be generated;

and or

(d) that the electrically controllable actuators are controllable by a control unit of the device for bringing into contact such that a successive contacting, a disengaging and a re-contacting of multiwell Plate (1) and tempering (4; 24; 41, 42) is feasible within a time ranging from 1 ms (millisecond) to 1 s (second).

15. Temperature control device according to one of claims 11 to 14, comprising a multiwell plate (1), the grid spacing of their arranged in rows and columns cavities corresponds to the grid spacing of the tempering fingers (5; 25).

, ^, -

16. tempering according to claim 15, characterized

in

(A) that in the bottoms of the cavities (2) in each case an electrically controllable heating and / or cooling element, preferably a Peltier element is integrated; and or

(B) that in at least one of the bottoms of the cavities (2), a temperature sensor is integrated; and or

(C) that the bottoms of the cavities (2) are designed thermally conductive.

17. tempering device (40) according to any one of claims 11 to 16, characterized by

a temperature-controlled from below cooling chamber (48), which is filled with a dry gas and / or filled and in the cooled state, a vertical temperature stratification in the Tempering chamber (48) having a lower cold layer (43a) and an upper heat layer (43b);

at least one lock (47a, 47b) provided on a housing wall of the temperature-control chamber (48) for introducing and / or executing a multiwell plate (1);

a first tempering body (41) arranged in the lower cold layer (43a) and connected to a cooling circuit for the cryopreservation of biological samples and / or one arranged in the upper warm layer (43b)

Heat cycle connected second tempering (42) for thawing cryopreserved biological samples.

18. tempering device according to claim 17, characterized

in

(a) that a multi-well plate (1) containing a sample to be thawed by means of the positioning over the second

Temperature control (42) is positionable; and

(b) that a sample to be frozen which is introduced into the tempering chamber (48) via the at least one lock (47a)

-containing multiwell plate (1) by means of

Positioning device over the first tempering (41) can be positioned; and

(c) that a multiwell plate (1) positioned above the first and / or second tempering body (41, 42) is replaced by means of the

Device for bringing in controlled or lowered lowered and / or raised to be in contact

and / or out of contact with the tempering (41, 42) to be brought.

19. Device according to one of claims 17 or 18, characterized

(a) that the tempering chamber (48) is cooled with liquid nitrogen (LN2), nitrogen (N2) gas or a Sterling engine;

and or (B) that in the tempering chamber (48) an ice trap (49) is arranged; and or

(C) that the heat layer (43b) has a temperature substantially a predetermined starting temperature of

Freezing process or a predetermined target temperature of a thawing process corresponds, while the cold layer (43a) has a temperature which substantially corresponds to a predetermined target temperature of the freezing process or a predetermined starting temperature of the thawing process.

20. A method for freezing biological samples, in particular for cryopreservation, and / or for thawing biological samples, in particular cryopreserved samples, under

Use of a tempering body according to one of claims 1 to 9 and / or a tempering device according to one of

Claims 11 to 19.

21. The method according to claim 20, characterized by the

Step:

Applying a substance (51, 61, 62, 63) to a sample (20) stored in a cavity (2) of the multiwell plate (1).

22. The method according to claim 21, characterized

(a) that the applied substance is a solution (62) which when solidified forms a surface of the cavity contents

opposite the outside closes, preferably gas-tight closes; and or

(b) that the substance is a natural or synthetic oil, a liquid or a gel which is mixed with an aqueous

Solution are immiscible, or solid carbon dioxide

(C02) is; and or

(c) the substance (63) has a higher density than the nutrient solution (60) surrounding the sample.

23. The method according to claim 21 or 22, characterized

(a) that the substance is thawed when thawing the sample

causes predetermined reaction or interaction with the sample; and or

(b) the substance is a diluent and wash solution or antifreeze, acts as a differentiation factor with respect to the sample, or is a substance which

Contains antioxidants, anti-apoptotic substances and / or live-dead dyes.

24. The method according to any one of claims 21 to 23, characterized

(a) that the substance (63) is applied to the already frozen sample (60, 64) and the substance after and / or during

Thawing the sample (60, 64) causes a predetermined reaction or interaction with the sample (60, 64); and or

(b) that the substance is a substance (63), from the state of which it can be deduced whether, after the freezing of the sample, an intermediate thawing has occurred.

* * * * *