Coolable carrier and device and method for producing frozen sample spheres

Abstract

Coolable carrier (1), comprising at least one structured surface (2) with a plurality of receiving structures (A), each of which serves to receive and position a liquid sample (5), wherein - each receiving structure (A) has a concave recess (3) in the shape of a spherical segment, - a ratio of a diameter of the recess (3) to a diameter of the receiving structure (A) is at least 0.7, the spherical segment is flatter than a hemisphere.

Description

[0001] The invention relates to a coolable carrier according to the preamble of the main claim, its use and a device and a method for producing frozen sample spheres.

[0002] In the following, the term "cryo-beads" refers in particular to spherical frozen solutions (sample spheres) containing various active ingredients, especially therapeutic agents, excipients, enzymes, proteins, genetic material, especially sections of DNA and / or RNA, primers or oligonucleotides, salts, organic substances, complexes and / or other substances or molecules, vesicles, chromosomes, cell organelles, and complete cells, also referred to herein as reagent mixtures. After production, the cryo-beads can advantageously be preserved even at ambient temperatures above their respective freezing temperature by removing the water contained in the cryo-beads.

[0003] The advantage of such dried cryo-beads lies in the transportability of originally sensitive materials without the mandatory maintenance of cold chains and thus their usability even in processes and / or laboratories in countries with incomplete or missing cooling infrastructure.

[0004] To achieve the best possible ratio of mechanical stability and volume to surface area for such cryo-beads, they are manufactured as spheres whenever possible. In this form, both the cryo-beads and their dried forms (lyo-beads) are most stable against mechanical stress and, thanks to their compact shape, can be easily transported and used without material loss due to abrasion and in a space-saving manner.

[0005] There are essentially three known methods for the production of cryo-beads. The first method involves pressing ice particles, such as dry ice pellets, into spheres or pellets using stamps or stamp pairs and dies. However, this method is technologically difficult to master for use in pharmaceutical and diagnostic applications.

[0006] A second manufacturing method involves freezing a reagent mixture in or on liquid nitrogen or other cryogenic liquids, such as silicone oils. If the number of droplets of the reagent mixture is sufficiently small, the liquid droplets initially float on the evaporating nitrogen due to the "inverse Leidenfrost effect" and then solidify into spherical shapes. As the liquid droplets cool, the temperature difference to the still boiling nitrogen decreases, causing the droplets to sink. If the liquid droplet is too large or too heavy, the evaporating nitrogen cannot hold the droplet on the surface. As it sinks, the liquid then solidifies into a droplet shape. This is particularly true for non-boiling cryogenic liquids such as silicone oil.

[0007] As the nitrogen boils, small volumes, so-called "satellite droplets," can detach from a still-liquid droplet, leading to an undesirable and uncontrollable decrease in the droplet's volume. As the nitrogen boils, the droplets suspended on the nitrogen vapor move back and forth, and upon collision with other droplets, they merge into an undesirably large droplet. This results in a mixture of unequal-sized beads, i.e., an inhomogeneous size distribution of the beads. Furthermore, before they completely freeze, the droplets come into direct contact with the nitrogen and any impure substances it may contain, or agents that may contaminate the product.

[0008] In a third approach, drops of the solution to be processed into cryo-beads are applied to an (ultra)cold plate, whereupon they freeze. To assist the droplets in spherical formation (i.e., the formation of a spherical shape), the plate can exhibit repulsive, particularly hydrophobic, properties, at least in the relevant areas of its surface. This method usually produces hemispheres, spherical segments, or flattened, elliptical lenses, rather than true spherical shapes. The cryo-beads produced on a plate must then be mechanically removed from the plate. This can easily damage the individual cryo-beads, which then no longer exhibit the desired shape or contain the desired amount of reagent mixture.

[0009] Examples of the production and optional drying of the cryo-beads can be found in the documents US 4 848 094 A, US 2014 / 0294872 A1, US 2016 / 0252300 A1, WO 2009 / 092703 A1, WO 2010 / 125087 A1 and WO 2013 / 066769 A1.

[0010] The invention is based on the object of proposing an improved method for producing frozen sample spheres. The object of the invention is to provide device- and method-related possibilities for achieving this object.

[0011] This object is achieved with a carrier according to the subject matter of the main claim and its use, as well as with a device and a method according to the subject matter of the subordinate claims. Advantageous further developments can be found in the dependent claims.

[0012] The coolable carrier comprises at least one surface structured with a plurality of receiving structures, each of which serves to receive and position a predetermined amount, for example an aliquot, of a liquid sample, hereinafter referred to simply as a “sample.” In addition to aqueous solutions of various viscosities, the term “liquid sample” also includes gels, particularly if these can assume a spherical shape (sphere) under suitable conditions due to their molecular interactions. A characteristic feature here is that each receiving structure has a depression, in particular a concave one. The receiving structures are designed in such a way that they exhibit a certain holding capacity relative to the sample in question (reagent mixture) and prevent it from undesirably flowing or rolling away.For example, the receiving structure optionally has a slightly to moderately repelling, i.e., particularly hydrophobic, coating and / or surface structure over the area where it comes into direct contact with the sample, to support the formation of a sphere and achieve a large contact angle between the sample and the surface of the support. The dimensions of the recess also determine the positioning, particularly of the still-liquid sample, and provide mechanical spatial resistance to prevent the sample from rolling away.

[0013] Particularly suitable materials for the carrier are those that allow effective cooling of the receiving structures, for example metals and metallic alloys, composites, especially with metallic layers, i.e. materials with the highest possible thermal conductivity.

[0014] In further embodiments of the carrier according to the invention, the area of ​​the surface surrounding a depression is also provided with a (hydrophobic) coating and / or surface design in order to achieve the largest possible contact angle between the reagent mixture and the surface.

[0015] In a further embodiment, a peripheral edge is provided around each of the recesses present in the structured surface. This edge extends beyond the structured surface.

[0016] The depression itself advantageously has the shape of a spherical segment to support the formation of a sphere. The spherical segment is characterized by the radius of the associated sphere and the height of the spherical segment. If the height of the spherical segment is equal to the radius of the sphere, the spherical segment corresponds to a hemisphere. In preferred embodiments, the spherical segment is flatter than a hemisphere, i.e., the ratio of the height of the spherical segment to the radius of the sphere is less than 1 to simplify removal of the cryo-beads. In particularly preferred embodiments, the ratio of the height of the spherical segment to the radius of the sphere is less than 0.7. Even more preferably, the ratio of the height of the spherical segment to the radius of the sphere is less than 0.4.

[0017] In addition to or as an alternative to a correspondingly repellent coating and / or design of the surface of the recess, the receiving structure can be free from the structured surface, wherein these each protrude, advantageously at least 1 nm, in particular at least 1 µm, above a surface of the structured surface immediately surrounding it.

[0018] For this purpose, a depression or trench can be created around some or all of the receiving structures using an abrasive process such as milling, spark erosion (sinking, drilling, wire erosion, etc.). As a result of their exposure created in this way, the receiving structures comprise a support structure at their base, for example in the form of a column. The column can, for example, have a round, oval, or n-sided cross-section. As a result of their exposure, the receiving structures also protrude above the floor of the immediately surrounding depression or the floor of the immediately surrounding trench.

[0019] Advantageously, the trench around a single receiving structure is at least so large that the clear width of the trench wall surrounding the receiving structure corresponds at least to the diameter of the sample sphere to be formed.

[0020] If the receiving structures present on the structured surface are arranged so close to one another that their respective trenches touch or penetrate one another completely or partially, only the end faces and / or remnants of the trench walls remain from the original surface of the structured surface.

[0021] The trench walls can also be completely eliminated in the case of complete penetration, so that only the receiving structures and, optionally, a support border arranged around all receiving structures protrude at least 1 nm above the bottom of the trenches. In other words, in this design, the receiving structures are machined out of the original surface using an abrasive process, without leaving secondary structures such as trench wall remnants between or next to the receiving structures. The optional border can preferably serve as a support and guide for a scraper (see below), with which the produced frozen sample spheres can be automatically removed from the support.

[0022] Similarly, in other embodiments, some or all of the receiving structures can extend beyond the at least one surface, for example by producing the receiving structures on the surface using an additive process such as a 3D printing process (e.g., SLS, SLA, FDM, etc.) or sputtering. In these embodiments, too, the receiving structures comprise a support structure at their base, which is designed in particular in the form of a column. The column can, for example, have a round, oval, or n-shaped cross-section. The receiving structures, in turn, extend at least 1 nm beyond the structured surface.

[0023] In order to minimize the risk of two adjacent sample spheres merging, the average distance between two receiving structures (the distance between their imaginary center lines or longitudinal axes) is advantageously at least one time and particularly preferably at least 1.1 times the diameter of the sample spheres to be formed.

[0024] In each of the aforementioned embodiments, the formation of a sample sphere can be supported by a ratio of a diameter of the recess of the receiving structure to a diameter of the receiving structure being at least 0.7, advantageously at least 0.8, and preferably at least 0.9. This means that the receiving structure or the edge (see above) projecting beyond the immediately surrounding surface has, from its outer side to the recess, only a very sharply defined area that extends as little as possible in a direction parallel to the structured surface. Such a design supports the formation of a sample sphere because contact with a material of higher density—and thus potentially higher wettability—is reduced. In this way, even highly wetting reagent mixtures with a small contact angle can be securely held and frozen in the recess of the receiving structure.In addition, a coating with a hydrophobic effect, for example, can be formed on the front side of the edge or the wall of the recess.

[0025] The receiving structures can, for example, have diameters selected from a range of 0.1 mm to 20 mm. This allows, for example, liquid sample volumes from less than 1 µl to 4000 µl to be applied and processed. Each carrier advantageously, but not exclusively, has receiving structures of one type and one size to allow for efficient production of frozen sample spheres.

[0026] It is also possible to equip carriers with receiving structures of different types and / or dimensions. Such carriers can be used, for example, to find, test, and, if necessary, optimize suitable receiving structures for a specific reagent mixture (sample).

[0027] The invention serves, not least, to enable the efficient production of a large number of frozen sample spheres. For this purpose, the receiving structures are advantageously arranged in a regular pattern, in particular in rows and / or columns, on the structured surface of the carrier. A regular arrangement allows, on the one hand, advantageous utilization of the available area of ​​the structured surface and, on the other hand, predictable positioning of the recesses or the frozen sample spheres, preferably for automated handling (see below).

[0028] For effective loading of the carrier as well as for simple and, if possible, automated detachment of the frozen sample spheres ("harvesting"), the carrier according to the invention is designed, for example, in the form of a plate. In order to connect several plate-shaped carriers to one another, a base body of the carrier can be designed as a rod with an n-sided cross-section, wherein at least one of the n side surfaces is a structured surface. The at least one plate-shaped carrier can be mounted interchangeably on the base body. Such a base body with several structured surfaces thereon can advantageously be rotatable in a controlled manner about its longitudinal axis, so that a specific surface can be delivered to a designated working position at a time. For example, one of the surfaces can be loaded with liquid sample at its current position, while another surface is harvested at its current position.

[0029] Continuing the concept of a rotatable arrangement of surfaces, the coolable support according to the invention can be configured with a curved, structured surface. A curved, structured surface can be a cylindrical section. To support the unidirectional movement of a support with a curved surface with a view to automation, the curved surface can be the outer surface of a cylinder.

[0030] The object of the invention is achieved, in addition to the carrier according to the invention, by a device for producing frozen sample spheres (cryo beads). The device comprises at least one carrier according to the invention and a cooling device for cooling the carrier. The cooling device can, for example, be an electrical Peltier element arranged on the carrier. Alternatively, a refrigeration machine can transport a cooling liquid to the carrier, and the carrier can have cooling channels through which the coolant circulates. Cooling using liquid nitrogen or dry ice is also possible; to enlarge the surface area, the side of the carrier facing away from the structured surface (underside) can, for example, have cooling fins for this purpose. A scraper is provided in order to be able to separate frozen sample spheres produced during use of the device from the receiving structures.The structured surface and the wiper are arranged to be movable relative to each other, whereby the wiper can be movable along the stationary surface, the structured surface against the stationary wiper or both against each other.

[0031] To remove the frozen sample spheres from the wells without damaging them, the scraper can be designed with an inclined contact surface. The inclination is advantageously directed at an obtuse angle to the direction of relative movement between the structured surface and the scraper, so that the scraper lifts the frozen sample spheres from the respective wells in a wedge-like manner, as described further below.

[0032] In a further embodiment of the device, a device for the automated application of the liquid sample into the wells can be provided, which can be designed, for example, as a pipetting head, a dispenser, or a multi-channel pipetting system, such as a 12-channel pipetting system. If, for example, a pipetting head in a 16x24 format is used, 384 wells can be filled with liquid sample simultaneously. Other grids or formats such as 1x8, 8x12, 32x48, etc. are also conceivable. Such parallelization of the process reduces the so-called cycle time for each liquid sample to be applied to a few milliseconds, in contrast to prior art methods, for example compared to dripping into liquid nitrogen.

[0033] The device as well as a drive of the carrier and / or the scraper can be controlled and moved or actuated in a coordinated manner by means of a control system.

[0034] The device can preferably be operated in a controlled gas atmosphere. For this purpose, the device can have a supply with an outlet opening for a gas and optionally a housing. The supply can be rigidly directed towards the carrier or arranged to be movable. A movable supply advantageously enables the outlet opening to be adjusted over a respective pipetting position. The housing can enclose at least the carrier and optionally the wiper, the pipetting head and / or the cooling system. The gas can in particular be a protective gas or a gas mixture (including atmospheric air) with the lowest possible water content.

[0035] The carrier according to the invention or the device according to the invention can be used in a method for producing frozen sample spheres.

[0036] Such a method comprises the steps of providing a carrier according to the invention or a device according to the invention. This is followed by applying a liquid sample to at least one of the receiving structures. The liquid sample can be applied, for example, by pipetting or dispensing. A total volume V can be applied, for example, by simultaneously dispensing a number of individual volumes corresponding to the number of receiving structures, for example using an appropriately dimensioned pipetting head, or by rapidly sequentially dispensing a corresponding number of droplets with droplet volumes TV in the pL, nL, or µL range at frequencies f from a few Hz to several hundred kHz. For dispensing, the combination of total volume V and repetition frequency f of the droplet volume dispenses TV is selected such that the total duration t of the total volume dispensing V is in the range t = 0.0.1 s to t = 60 s, preferably in the range t = 0.5 s to t = 15 seconds. Other methods of liquid transfer, such as ultrasound-based transfers or others, are also conceivable.

[0037] The liquid sample introduced into the respective wells is cooled significantly, creating a frozen sample sphere in each case. The temperature can be controlled using, for example, liquid nitrogen, dry ice, electrical Peltier elements, coolant provided by a refrigeration machine, etc. For temperature control, the carrier itself is cooled or by direct contact of the carrier with, for example, an actively cooled base plate. The carrier, particularly in a design as a cylinder or n-cornered column, can in other designs be temperature-controlled by the action of a coolant (e.g. liquid nitrogen, tempered (silicone) oil, etc.) via a refrigeration machine. The coolant can flow through the carrier and does not come into direct contact with the liquid sample or the frozen sample spheres.

[0038] To harvest the frozen sample spheres, they are optionally removed from the respective receiving structure, for example, by means of a scraper. In further embodiments, the carrier can be oriented after the sample spheres have frozen so that they fall out of the recesses due to the force of gravity and can be collected. For this purpose, the recesses can be provided, for example, with a coating and / or surface structure on which the frozen sample spheres cannot find sufficient grip, for example, in an inclined or overhead position.

[0039] The collected sample spheres must continue to be cooled to such an extent that they do not experience an uncontrolled increase in temperature or thaw again or even thaw.

[0040] The frozen sample spheres produced in this way can be stored in a chilled state and used later. However, to ensure their storage, transport, and use at temperatures above their respective freezing point, the sample spheres are preferably dried, particularly by lyophilization.

[0041] Advantages of the invention include, firstly, that the liquid sample and the frozen sample spheres do not come into direct contact with the coolant, thus eliminating contamination. Frozen sample spheres can be produced with minimal manufacturing-related variance in their dimensions and volume, which is crucial for the reproducibility of applications in which the manufactured sample spheres, possibly in a dried state, are used. Accordingly, the scrap rate is low. Efficient production, coupled with a comparatively simple design of the manufacturing device, allows for the production of high-quality frozen sample spheres using small and cost-effective systems.

[0042] Using the method according to the invention, sample spheres can be produced, for example, from a diameter range of 0.5 to 20 mm and with volumes of less than 1 up to 4000 µl. In experiments, several thousand sample spheres with a volume of 10 µL each were produced as mock beads, as were several thousand RT-qPCR beads with volumes of 5 µL, 10 µL, and 25 µL, as well as several hundred LAMP beads with a volume of 10 µL each. These were then freeze-dried and functionally tested. The performance parameters after freeze-drying did not differ from the standard method. The number of cycles can reach 72,000 per hour; the production of 125,000 sample spheres, for example, requires only 1.75 hours.

[0043] In their freeze-dried state, the sample spheres produced according to the invention can be used in particular as precursors, for example as an additive for a detection reaction or as an inactive precursor of a ready-to-use reaction mixture or a ready-to-use buffer solution, etc. These are used, for example, for RT-qPCR (and other PCR variants), isothermal amplification, immunoassays, enzymes and proteins, conjugated antibodies, collagens, pharmaceutical active ingredients or therapeutics or medications, etc.

[0044] The invention is explained in more detail below with reference to illustrations and exemplary embodiments. They show: Fig. 1 a schematic representation of a first embodiment of a carrier according to the invention; Fig. 2 a schematic representation of a second embodiment of a carrier according to the invention; Fig. 3 a schematic representation of a third embodiment of a carrier according to the invention; Fig. 4 a schematic representation of a fourth embodiment of a carrier according to the invention; Fig. 5 a schematic representation of a fifth embodiment of a carrier according to the invention; Fig. 6 a schematic representation of a sixth embodiment of a carrier according to the invention; Fig. 7 a schematic representation of a seventh embodiment of a carrier according to the invention; Fig. 8 is a schematic representation of an eighth embodiment of a carrier according to the invention; Fig. 9 a schematic representation of a ninth embodiment of a carrier according to the invention; Fig. 10 is a schematic representation of a tenth embodiment of a carrier according to the invention; Fig. 11 is a schematic representation of an embodiment of a device according to the invention for producing frozen sample spheres; Fig. 12 a schematic representation of a further embodiment of a device according to the invention for producing frozen sample spheres and Fig. 13 a schematic sequence of an embodiment of the method according to the invention.

[0045] The illustrations are schematic and not to scale. The same reference symbols indicate the same technical elements in the different illustrations.

[0046] A carrier 1 according to the invention has at least one structured surface 2, in particular a side surface, in which at least one depression 3 serving as a receiving structure A is formed. Fig. 1 shows, by way of example, three rows each with three recesses 3. The recesses 3 are, in particular, concavely curved into the material of the carrier 1 and each represent, for example, a spherical segment.

[0047] In a second embodiment, the recess 3 is surrounded by an edge 4 ( Fig. 2). Depression 3 and edge 4 together form the receiving structure A. The edge 4 is kept narrow in the radial direction on its end face facing away from the structured surface 2 in order to allow a liquid sample 5 introduced into the depression 3 (see Fig. 3) to offer a small potential wetting surface. In Fig. 2, only one receiving structure A is shown as an example. However, a carrier 1 can have a plurality of such receiving structures A.

[0048] To minimize the potential wetting surface and to support the formation of the liquid sample 5 into a sphere, each of the depressions can be surrounded by a groove 6, which is created, for example, by local material removal from the structured surface 2. The receiving structure A created in this way is exposed and protrudes beyond the immediately surrounding surface 7 (bottom of the respective groove 6). As a result of the exposure, the receiving structure A comprises a basally arranged support structure T. Fig. 3 shows two different shapes of receiving structures A. On the right in the image, the receiving structure A, which remains as a column, has a comparatively small diameter and a very shallow depression 3. Such a design is suitable, for example, for small amounts of a liquid sample 5 and / or for reagent mixtures with a strong tendency to spherize.

[0049] A liquid sample 5 is applied to one of the receiving structures A shown on the left in the image. Its quantity and composition allow the formation of a spherical shape (sphere) due to molecular interactions. This process is further supported by the fact that the depression is shaped as a distinct spherical segment.

[0050] In the Fig. In the fourth exemplary embodiment shown in Figure 4, the positions of the individual receiving structures A are so close to one another and the respective grooves 6 are dimensioned so large that the latter partially penetrate one another. In the example, a diameter D1 of the recess 3 is in a ratio of slightly more than 0.7 to a diameter D2 of the receiving structure A. The annular end face of the receiving structure A facing away from the remaining surface 7 can optionally be provided with a coating and / or surface structuring that repels the liquid sample 5.

[0051] An even more pronounced mutual penetration of the trenches 6 can be seen in a fifth embodiment in the enlarged detailed view ( Fig. 5). Of the original surface 2, only a few columns with pillow-shaped cross-sections remain. The carrier 1 is designed as a plate in SBS format and has 768 support structures A for the production of frozen sample spheres 5 ( Fig. 3) or 10 (Cryo-Beads, see Fig. 11) with 10 µl each. The recording structures A are arranged in rows and columns, with every second one offset from each other by the distance of a recording structure A and a portion of the diameter of the surrounding groove 6 in the direction of the columns in order to effectively utilize the available space.

[0052] The Fig. Figure 6 shows a sixth embodiment of the carrier 1 according to the invention with receiving structures A also arranged in rows and columns and offset from one another, the trenches 6 of which partially penetrate one another. The number of receiving structures A in the example is 192.

[0053] The same basic embodiment is followed by a seventh embodiment ( Fig. 7) with 384 recording structures A.

[0054] In contrast, the trenches 6 of the 384 receiving structures A do not penetrate each other according to an eighth embodiment ( Fig. 8). These are again arranged in rows and columns, but without being offset from each other.

[0055] Likewise, the receiving structures A or the depressions 3 on the structured surface 2 of a carrier 1 shown as a section in the form of a plate according to a ninth embodiment according to Fig. 9. The carrier 1 has a number of differently designed receiving structures A, wherein the receiving structures A of each two adjacent columns are identical. For the sake of clarity, the receiving structures A are assigned to one of three sections S1 to S3 in the direction of the rows. The receiving structures A of the first section S1 shown furthest to the right are formed as concave depressions 3 in the structured surface 2.

[0056] To the left follow receiving structures A of the second section S2, each surrounded by a trench 6, whereby the trenches do not penetrate each other. The relationships between the diameters of the receiving structures A and the trenches 6, which remain constant in diameter, change every two columns, whereby the receiving structures A each have very shallow depressions 3.

[0057] In the third section S3, the receiving structures A are also surrounded by grooves 6. The ratios between the diameters of the receiving structures A and the constant-diameter grooves 6 increase toward the left, i.e., the diameters of the receiving structures A become smaller every two columns. The recesses 3 present on the end faces facing the observer are concave, in particular in the form of spherical segments.

[0058] A carrier 1 formed according to the ninth embodiment can advantageously be used to determine a suitable receiving structure A for a specific reagent mixture and / or for desired volumes of the frozen sample spheres 10 to be produced. This is advantageously done by taking into account the interaction with the respective production conditions, such as a respective temperature of the carrier 1 and the liquid sample 5 and a time period for the formation of a sample sphere and its complete conversion into the frozen state.

[0059] The above-described carriers 1 in the form of plates can be used in manual or automated production processes for frozen sample spheres 10. To promote a continuous loading of the carrier 1 with the liquid sample 5 and the harvesting of frozen sample spheres 10, according to a tenth embodiment, the carrier 1 is formed with a cylindrical or n-cornered base body 11. In the Fig. 10 shows a carrier 1 according to a tenth embodiment with a cylindrical base body 11, the outwardly facing surface of which is formed by the structured surface 2. The receiving structures A are arranged in rows, with adjacent rows being offset from one another in the embodiment shown in order to achieve better utilization of the available surface area.

[0060] The cylindrical carrier 1 can be present in a device 8 (see also Fig. 11). The loading of the receiving structures A with the liquid sample 5 can be carried out by means of a device 13 designed for this purpose, which can be designed, for example, as a dispensing or pipetting head 17.

[0061] An embodiment of a device 8 for producing frozen sample spheres 10 is shown in simplified form in Fig. 11. The carrier 1 is brought to and maintained at the desired temperature by means of a cooling device 9. The liquid sample 5 is introduced into the respective well 3 of a receiving structure A (see above) by means of a device 13, implemented as a pipetting head 17, for applying the liquid sample 5, and freezes there. The frozen sample spheres 10 thus produced are removed from the wells 3 by means of a scraper 14. This can be done purely mechanically by means of a compressive force transmitted to the frozen sample sphere 10 by the scraper 14. If the wells 3 are designed as spherical segments, in particular as (almost) hemispherical segments, an inclined scraper 14 is advantageous. Fig. 11 shows a wiper 14 whose contact surface 15 is inclined against relative movement with respect to the carrier 1. An existing controller 12 is connected to the pipetting head 17, a drive 16 for the wiper 14, and the cooling device 9 in a manner suitable for transmitting data and controls them with control commands.

[0062] In the example, the scraper 14 is guided from left to right against the frozen sample spheres 10. The contact surface 15 of the scraper 14 is inclined to the left, so that it lifts the frozen sample spheres 10 from the corresponding recesses 3 like a wedge without damaging the frozen sample spheres 10. A relative movement between the carrier 1 and the scraper 14 is generated by the drive 16.

[0063] In addition, the device 8 of the Fig. 11 has a feed 19 through which a protective gas can be supplied to the carrier 1. To optimally maintain a controlled gas atmosphere, the device 8 is also provided with a housing 18.

[0064] In the embodiment of the Fig. 12 is a device 8 with a coolable roller-shaped carrier 1 (cf. Fig. 10). The cylindrical carrier 1 rotates one position further after each release of liquid sample droplets 5 onto the receiving structures A arranged in rows on the curved structured surface 2. After a rotation of ≥ 90° to < 360°, preferably between ≥ 180° to < 360° and ideally at 270°, the already applied and now frozen sample spheres 10 reach the preferably stationary scraper 14. As the carrier 1 rotates further, the sample spheres 10 are pressed against the scraper 14 and are thereby released or harvested from the recesses 3. In this exemplary embodiment, the contact surface 15 of the scraper 14 is also inclined.

[0065] The harvested frozen sample spheres 10 can be placed in a collection container for further processing or storage. To enable storage, transport, and use of the frozen sample spheres 10 without further cooling, they are advantageously subsequently freeze-dried (lyophilized).

[0066] An embodiment of the method according to the invention is described in Fig. 13 is shown in simplified form. After at least one carrier 1 according to the invention has been provided, it is cooled to a process temperature at which the reagent mixture to be used freezes. The process temperature is typically in a range between 10 K and 350 K, advantageously 75 K to 277.15 K. A temperature of the carrier 1 in a range of ± 50 K around the melting temperature T m or the eutectic temperature T eut the reagent mixture or the collapse temperature T C .

[0067] On the carrier 1, which has the desired process temperature, the corresponding volumes of liquid sample 5 are introduced into the respective wells 3 of the receiving structures A. There, the respective volumes of the liquid sample 5 form a sphere due to molecular interactions of the ingredients, which is subsequently frozen.

[0068] The frozen sample spheres 10 thus produced are harvested and subsequently optionally dried. Reference symbol 1 carrier 2 structured surface 3 Deepening 3a Frontal surface 3b Support structure 4 edge 5 liquid sample 6 trench 7 Trench bottom, remaining surface 8 Device 9 Cooling device 10 frozen sample spheres 11 Basic body 12 Control 13 Furnishings 14 scrapers 15 Contact surface 16 Drive 17 Pipette head 18 Enclosure 19 (Protective gas) supply A recording structure D1 Diameter of the recess 3 D2 Diameter of the receiving structure 3 T support structure

Claims

[1] Coolable carrier (1), comprising at least one structured surface (2) with a plurality of receiving structures (A), each of which serves to receive and position a liquid sample (5), wherein - each receiving structure (A) has a concave recess (3) in the shape of a spherical segment, - a ratio of a diameter of the recess (3) to a diameter of the receiving structure (A) is at least 0.7, the spherical segment is flatter than a hemisphere. [2] Coolable carrier (1) according to claim 1, characterized by that at least some of the receiving structures (A) comprise a basally arranged support structure (T), whereby these receiving structures (A) each protrude beyond a surface (7) of the structured surface (2) immediately surrounding them. [3] Coolable carrier (1) according to claim 1 or 2, characterized by that a trench (6) is formed in the structured surface (2) around each of the receiving structures (A). [4] Coolable carrier (1) according to claim 2 or 3, characterized by that the recess (3) is formed on an end face (3a) of each receiving structure (A). [5] Coolable carrier (1) according to one of the preceding claims, characterized by that the receiving structures (A) have diameters selected from a range of 0.1 mm to 20 mm. [6] Coolable carrier (1) according to claim 1, characterized by that the ratio of the diameter of the recess (3) to the diameter of the receiving structure (A) is at least 0.8 and preferably at least 0.

9. [7] Coolable carrier (1) according to one of the preceding claims, characterized by that the receiving structures (A) are formed row by row and / or column on the structured surface (2). [8] Coolable carrier (1) according to one of the preceding claims, characterized by that the carrier (1) is designed in the form of a plate. [9] Coolable carrier (1) according to one of the preceding claims, characterized by that a base body (11) of the carrier (1) is designed as a rod with an n-cornered cross-section and at least one of the n side surfaces is a structured surface (2). [10] Coolable carrier according to one of claims 1 to 8, characterized by that the carrier (1) is formed with a curved structured surface (2). [11] Device (8) for producing frozen sample spheres (10) comprising a carrier (1) according to one of the preceding claims, a cooling device (9) for cooling the carrier (1) and a scraper (14) with an inclined contact surface, which serves to scrape off frozen sample spheres (10) present on the receiving structures (A), wherein the carrier (1) and the scraper (14) are movable relative to one another. [12] Device (8) according to claim 11, characterized bythat the device (8) has, for the purpose of operation in a controlled gas atmosphere, a supply with an outlet opening for a gas and optionally a housing enclosing at least the carrier (1). [13] Use of a carrier (1) according to one of claims 1 to 10 in a method for producing frozen sample spheres (10). [14] Method for producing sample spheres (10), comprising the steps - Providing a carrier (1) according to one of claims 1 to 10; - applying a liquid sample (5) to at least one of the cooled receiving structures (A); - freezing the liquid sample (5) so that a frozen sample sphere (10) is created; and - optionally stripping the frozen sample sphere (10) from the respective receiving structure (A). [15] Method according to claim 14, characterized by that the sample spheres (10) are dried, in particular lyophilized.

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