Apparatus and method for investigating and / or processing, in particular, biological or medical samples

Abstract

The invention relates in particular to an apparatus for investigating and / or processing biological or medical samples and a method for carrying out the same. In the apparatus (1) and method for investigating and processing biological or medical samples, a discharge unit (3) is used to discharge the sample into a recess of a sample carrier (10). The sample carrier (10) has an aspect ratio of the recess height to the opening diameter or edge length that is greater than 1. The sample is subsequently visually detected through the transparent bottom of the recess by an optical investigation unit (6). The sample carrier (10) is then movable in a horizontally movable receiving part (14) to respective working positions within a working area defined by the respective recess of the sample carrier (10). In the working positions, on the one hand the optical investigation unit (6) and on the other hand, optionally the discharge unit (3), the removal unit (9) or other units are located on mutually opposite sides of the sample carrier (10) on one common axis (17). Reference for the positioning of the sample carrier (10) is performed based on visual detection of the detected sample carrier (10) and / or the respective sample by an optical interrogation unit (6).

Description

[Technical field]

[0001] The present invention relates to an apparatus with a control unit and an implementing method for investigating and / or processing, in particular biological or medical samples, such as cells (including live cells) and / or particles, the apparatus comprising at least one ejection unit for ejecting a sample into at least one sample carrier having a number of recesses with respective openings and a bottom in one common processing chamber, an optical investigation unit, in particular an optical investigation unit having an image sensor, a microscope and / or an objective lens, and a removal unit for removing at least one sample from the sample carrier and discharging or outputting the previously isolated sample. [Background technology]

[0002] Cell culture or even the analysis of individual cells can currently only be achieved with considerable effort or cost due to mechanical and methodological limitations. In particular, the isolation of individual cells from extremely small samples proves to be extremely difficult, since individual cells must be detected under a microscope by laboratory staff and then manipulated by capillaries.

[0003] Particularly in the fields of cell biology and medicine, sample carriers are used for the investigation of biological or medical samples. In most experiments, it is advantageous if the sample can be positioned accurately. Accurate positioning allows for efficient execution of experiments, increased comparability of several experiments and easier evaluation. When filling the sample carrier, accidental positioning of the sample often occurs.

[0004] This kind of sample, especially cell populations, is typically heterogeneous by cell type, cell stage, or even with respect to individual differences, e.g. genetic differences, and possible combinations of these. Studying heterogeneous cell populations necessarily reveals only average properties. This may be the basic ability to produce a certain protein or to study the response to an agent or drug. It may also be studied whether or to what extent the cells have taken up and integrated genes after the introduction of genetic material, for example by transfection or transduction.

[0005] When the properties and / or interactions of individual or small numbers of cells are to be elucidated, the cells have to be treated and investigated individually, for which preferably methods are suitable that allow to spatially separate cells from other cells (compartmentalization).

[0006] Each of the methods provided has its own inherent disadvantages: for example, the individualization of cells on solid media proves to be inconvenient due to the large base surface required, and furthermore, the cells can only be exposed to different conditions or substances in a very limited way.

[0007] US Patent No. 5,399,636 already describes aspirating and retrieving cells based on optical information of a particular cell, where the measurement tip is made of an optically transparent material, the optically transparent material has a recess formed therein, the recess containing a liquid with at least one cell, and the cell is dispensed by an aspirating and dispensing capillary onto a sample carrier at a predetermined position.

[0008] Single cell printers are also known that have a three-axis lab robot, a print head with a print cartridge for a cell suspension, and microscope optics. The functional format is similar to that of an inkjet printer.

[0009] The sample is analyzed inside the print head by means of microscope optics and a high-speed camera. This allows the cells present in the sample to be detected and classified according to optical criteria, such as size and morphology. If the detected contents of the drop to be printed do not meet the requirements, this content is aspirated, so that only the drops with a single cell that meets the criteria are ejected at a predetermined position on the sample carrier, for example a microtiter plate with a large number of partitioned wells (up to 1536 wells). By optical detection using a camera, the identification of single cells can be performed directly during the printing process.

[0010] The treatment of cells by droplet technology, which produces emulsified droplets, as described, for example, in US Pat. No. 5,399,663, in which the droplet phase is produced in a carrier liquid phase, in contrast, certainly requires less base surface and allows the production of larger quantities of "encapsulated" cells, which can also be exposed to different conditions. However, it has been found to be disadvantageous that cells in the carrier liquid phase cannot be optically analyzed optimally, for example due to the difficult fixability of the droplets and their optical properties. In addition, this method is generally only suitable for short-term (less than one day) experiments, since it is difficult to provide fresh nutrients and metabolic products are hardly removed. Furthermore, the oxygen often required cannot be provided optimally. However, the use of a carrier liquid phase, which in practical use is often an oil, can also have a direct effect on the experimental results, for example, the diffusion of pharmaceuticals to the outside, when the carrier liquid phase is not inert. The transferability to an in vivo environment is therefore often rated as insufficient, which limits the use of this technology to experiments of a few hours or even shorter in most cases.

[0011] US Patent No. 5,399,633 discloses a holder with a vacuum bed for a container for biological samples, e.g. a microtiter plate, which is continuously moved with respect to a light beam that illuminates the samples in the wells, while a camera records images of the samples in the wells. To illuminate the sample, the light beam is configured to directly illuminate the entire sample or only a part of the sample. In this case, the light beam is diverted to the other part, so that the entire sample can be imaged using a series of images of different parts of the sample.

[0012] An improvement for long-term experiments and for improved transferability to in vivo environments is the use of biologically inert materials for the sample carriers, in particular glass, silicon and some plastics, e.g. PDMS, COP / COC, PS, PC. Two different approaches are distinguished here:

[0013] In the case of microfluidic approaches, cells are often provided as individualized suspensions in a channel system, which allows spatial sorting of the cells due to the spatial organization of the channel system and a kind of pockets, undercuts or chambers. For example, flow dynamics, electric fields, magnetic fields or acoustic fields may then be used for physical manipulation of the cells, i.e. moving or sorting them. The advantage of this approach is the possibility to test many cells in an inert environment, while requiring little space and reagents.

[0014] What proves to be a disadvantage is that cells can generally only be exposed to consistent conditions, which is often a disadvantage for the investigation of different agents. In addition, the construction, manufacture and use of such microfluidic systems also requires solid expertise, so that such systems prove to be economically feasible only in a few cases.

[0015] For easy handling of samples, microtiter plates are often used. They have a large number (up to 3456) of relatively small recesses or cavities, also called wells, which contain the samples. To simplify their handling, the dimensions of microtiter plates are standardized.

[0016] The use of multi-well or microtiter plates as sample carriers is an established method in practical use. In particular, the versatility of their use, the availability of a wide range of equipment for loading, analyzing or isolating samples from these plates, the manufacturing suitable for mass production of multi-well or microtiter plates at economically attractive costs, and the clarity of their functional form, which does not require much special expertise for use, are advantageous for their use under biologically inert conditions for cell experiments.

[0017] In many cases, the formats provided for practical use, regarding the size of the recesses, for example diameters of 1.1 to 2 mm, prove to be inconvenient, as this is many times larger than the single cells to be investigated, which have a typical size of 10 to 20 μm. Bacteria, for example E. coli, on the contrary, have a typical diameter of 1 to 2 μm and a length of 2 to 6 μm.

[0018] Medicines based on recombinant proteins produced from cells must be traceable back to individual cells according to regulatory authorities ("monoclonality"). Proof that only a single cell is present in one well, in accordance with the stringent requirements for such medicines, requires that large volumes of wells must be scanned over long periods of time in order to finally detect the cell optically and, in particular, to exclude the presence of other cells. This results in an enormous number of microscopic images that must also be evaluated and stored, precisely when the number of individualized cells is large, for example more than 10,000 to 1,000,000 cells. This makes data storage / data processing a considerable cost factor and scan times a significant time factor.

[0019] Furthermore, the required filling volume of the wells is a significant cost factor, especially for large cell populations, since expensive reagents are generally used. It is a disadvantage that in practical use, hundreds or even thousands of microtiter plates have to be handled and incubated quickly for large populations, which results in logistical efforts, causes large space requirements and is prone to errors, since microtiter plates can be mixed up.

[0020] Moreover, the investigation of very large numbers of cells leads to the need to use and coordinate or synchronize various equipment for positioning, examining, isolating and incubating samples, each of which is associated with high procurement costs. In practical use, the collective control of various equipment also proves to be problematic, so that such a strategy presupposes a corresponding enterprise size, and yet compatibility shortcomings arise and the probability of errors increases.

[0021] From patent document 4 an analytical device for isolated cells is known, which comprises a substrate with a number of wells, the wells having a depth of 1 μm to 250 μm, an opening with a diameter of 10 μm to 100 μm and a volume of 1 pL to 10 nL and accommodating a suspension with a cell concentration of 1,000 cells / ml to 500,000 cells / ml. The wells are configured such that at least 25% of the wells contain only a single cell and the isolated cells can be optically investigated.

[0022] Microtiter plates, as are already known, for example from US Pat. No. 5,399,623, US Pat. No. 5,499,637 or US Pat. No. 5,499,627, also count as part of the prior art.

[0023] Furthermore, US Pat. No. 5,999,366 describes a sample carrier which can accommodate a large number of samples to be investigated, in particular having a volume of less than 10 μl. The bottom of the cavity is preferably porous, so that by negative pressure at the underside of the sample carrier, the sample can be sucked out of the cavity via its porous bottom.

[0024] Patent document 9 describes a method for treating wells of a multi-well chip containing cells, where a volume of 30 nL to 50 nL of cell suspension is released into the well. A number of images of the wells in multiple z-planes are detected, and a map is created based on multiple images taken in at least three z-planes to distinguish between empty wells of the multi-well chip and wells containing cells. This image detection may include multiple wells at the same time, whereby the wells are imaged individually one after the other, or in a single microscope image, such that multiple recesses can be imaged simultaneously.

[0025] Patent document 10 originates from the problem that when carrying out a method involving photometric evaluation of aqueous samples using microtiter plates, the results are hindered and erroneous by the formation of a meniscus. Meniscus formation occurs as a result of surface and interfacial tension, which results in the formation of a convex or concave surface, depending on the liquid. To solve this problem, it is proposed to use materials with different physical properties for the container wall and the container bottom, so as to achieve that the sample to be analyzed has a contact angle of about 90° with the container wall, whereby the radiation is substantially not absorbed during the analysis and the light path is not influenced.

[0026] US Patent No. 5,399,633 describes a method for investigating samples contained in sample vessels of a microtiter plate by imaging at least a part of the sample volume through the bottom of the sample vessel with a CCD camera and an evaluation unit, where reflections from the upper or lower surface of the bottom of the sample vessel are detected and a closed-loop control algorithm determines a correction value for focusing the sample inside the sample vessel.

[0027] Patent document 1 describes a device for aspirating cells, which searches for predetermined cells contained within a measurement chip made of a light-transmitting material and having a recess formed therein, based on optical information detected by a sensor.

[0028] Furthermore, US Pat. No. 5,399,633 describes a sample manipulation device in which the actual position is determined by a position measuring unit configured as a microscope.

[0029] According to Patent Document 13, the spatial position of the cell object in the sample carrier is detected by an image capturing unit and an image evaluation unit, and the capillary is first run to a calibration position and then to a position above the cell object.

[0030] During investigations it is generally desirable to keep consumption of sample material and further substances used during the investigation as low as possible, which can essentially be achieved by the miniaturization of microtiter plates.

[0031] However, if the capillary should be immersed in the recess, i.e. has an outer dimension smaller than the inside width of the recess or cavity, the capillary forces will increase undesirably due to the reduced inner diameter of the recess and the capillary used for aspiration as a result of the miniaturization. According to US Pat. No. 5,991, this problem can be avoided in that the capillary is not immersed in the recess, but the cells rise by vortexing and are then aspirated.

[0032] To limit the rise height, the capillary has a ring-shaped hydrophobic coating on the inner wall, in order to precisely meter the liquid and limit the volume that can be drawn up, in particular in a self-regulating manner, according to US Pat. No. 5,399,433. The hydrophobic coating may consist of any suitable hydrophobic material, for example a material based on silicone and / or beeswax.

[0033] Furthermore, the glass capillary has, according to US Pat. No. 5,399,236, an inner diameter in the tip region of less than 10 μm and, at this tip, a polymer coating having properties that repel biomolecules. [Prior art documents] [Patent documents]

[0034] [Patent Document 1] U.S. Patent No. 10101276 [Patent Document 2] European Patent Application Publication No. 2411134 [Patent Document 3] US Patent Application Publication No. 2010 / 0182419 [Patent Document 4] U.S. Patent No. 10032615 [Patent Document 5] DE 19712484 A1 [Patent Document 6] DE 102019003135 A1 [Patent Document 7] German Patent Invention No. 102019109207 [Patent Document 8] European Patent No. 0706646 [Patent Document 9] US Patent Application Publication No. 2019 / 0113457 [Patent Document 10] European Patent Application Publication No. 0648536 [Patent Document 11] German Patent No. 19916749 [Patent Document 12] German Patent No. 102005053669 [Patent Document 13] DE 102007046267 [Patent Document 14] DE 10039195 A1 [Patent Document 15] DE 10334164 A1 Summary of the Invention [Problem to be solved by the invention]

[0035] The problem underlying the present invention is to provide an apparatus and method for preparing, manipulating and investigating multiple, in particular isolated, samples, in particular also single cells, in a sample carrier and selectively removing individual samples from the sample carrier in an integrated and largely automated process, in which the time effort or cost of carrying out the process, the effort or cost of data detection, data processing and control associated with the investigation, as well as the consumption of reagents, chemicals and other consumable materials are substantially reduced compared to the conventional apparatus and methods used. [Means for solving the problem]

[0036] The above object is achieved according to the invention by a device and a method according to the features of claims 1 and 2. Further configurations of the invention can be found in the dependent claims.

[0037] According to the invention, an apparatus and a method are provided, in which, in a processing chamber, at least one sample carrier is movable, in particular by means of at least one receiving part movable in a horizontal plane, to various working positions present in a working area defined by respective recesses of the sample carrier, and in each working position, on the one hand, an optical investigation unit and, on the other hand, a release unit, a removal unit and / or optional further units (tools) are positioned coaxially on opposite sides of the sample carrier, so that the optical axis of the optical investigation unit is present in the working area, at least some of the recesses have a transparent bottom surface at least in some sections, so that optical detection of the sample is carried out through the bottom surface, at least a major proportion of the recesses of the sample carrier have an aspect ratio of the recess height to the opening diameter or edge length greater than 1, and reference setting for the positioning is carried out by a control unit based on the sample carrier and / or the respective sample detected by the optical investigation unit.

[0038] In contrast to the prior art, in which the sample carrier is stationary and the units forming the tool are moved one after the other in a time sequence to the respective working positions in order to carry out the different process steps one after the other, according to the invention the sample carrier is moved, for example by the cross table of the receiving part, and in the working positions set in this way a permanent and uninterrupted observation of the sample is achieved, whereby, if necessary, correction values ​​are determined for the detected residence positions of the sample in the recess or of the sample carrier in the receiving part, and from the correction values ​​a correction of the working position is carried out, if necessary. According to the invention, the mobility of the tool after the completion of the working cycle, in particular for a tool change, is possible and useful.

[0039] Contrary to the preconception among the skilled person that, due to accelerations during the movement of the sample carrier, the samples contained in the respective recesses will also move and therefore can only be detected or investigated with increased effort or cost, it has been found that, taking into account the inventive aspect ratio (depth to width) of the recesses which is greater than 1, due to the small volume in the recesses in connection with the use of so-called well plates as sample carriers, in practical use no undesired movements of the contained samples caused by the positioning of the sample carrier have been found. Moreover, it has been found that with a very high probability the samples do not come off the recesses despite the dynamic forces acting on them.

[0040] As sample carriers preferably well plates are used, which are preferably made of glass or of plastic, preferably transparent plastic (polycarbonate, polystyrene, cyclic olefin polymers, cyclic olefin copolymers, polydimethylsiloxane), or of silicon or metal or a combination thereof, and which have recesses, which preferably have an aspect ratio (depth to width) greater than 1, or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0041] The recess forms a working position according to the invention.

[0042] The recesses may preferably have a circular or rectangular cross section. The diameter of the circular recesses is preferably 10 to 1000 μm or more, preferably 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1000 μm. The depth of the recesses is preferably 20 to 1100 μm or more, preferably 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or 1100 μm.

[0043] The recesses preferably have a center-to-center spacing of 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more of the diameter or edge length. The spacing from the edge of one recess to the adjacent edge of an adjacent recess is preferably 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or more. The density of the recesses is preferably 0.25 per mm 2 ~6,944 per mm 2 , preferably 0.5 per mm 2 , per mm 2 , every 5 mm 2 , every 10 mm 2 , every 20 mm 2 , every 30 mm 2 , every 40 mm 2 , every 50 mm 2 , every 60 mm 2 , every 70 mm 2 , 80 mm 2 , every 90 mm 2 , every 100 mm 2 , every 200 mm 2 , every 300 mm 2 , every 400 mm 2 , every 500 mm 2 , 600 per mm 2 , 700 per mm 2 , 800 per mm 2 , 900 per mm 2 , every 1,000 mm 2 , every 2,000 mm 2 , 3,000 per mm 2 , 4,000 per mm 2 , 5,000 per mm 2 , 6,000 per mm 2 It is.

[0044] In this case, well plates made of glass, in particular produced by the LIDE method (Laser Induced Deep Etching) known per se, have optimal optical and inert properties, so that investigations, in particular investigations by optical methods, are substantially improved. This provides the possibility of error-free detection of the samples through the bottom of the recesses. Well plates made of glass can be produced by laser drilling, wet or dry etching, e.g. plasma etching, ultrasonic drilling, sandblasting, lithographic methods, etc.

[0045] The fabrication method may be combined with a bonding method, such as anodic bonding, fusion bonding, bonding with PDMS or other adhesive methods.

[0046] An example of a well plate is a glass plate made of borosilicate glass having a thickness of 450 μm with through holes having a diameter of 200 μm, which is bonded by fusion bonding to a second glass plate made of borosilicate glass. The second glass plate is used as the bottom of the recess. The second glass plate has a thickness of, for example, 300 μm and is ground and polished to a thickness of 175 μm after bonding. Alternatively, a silicon layer may be deposited on the second glass plate, for example by CVD. The glass plates may then be bonded by anodic bonding.

[0047] Another embodiment of the well plate consists of a silicon plate with an initial thickness of 500 μm, in which through-holes are produced by the Bosch process (deep reactive ion etching), which is bonded by anodic bonding to a glass plate made of borosilicate glass. The through-holes have a diameter of, for example, 300 μm. The glass plate has a thickness of, for example, 175 μm.

[0048] Alternatively, the plates may be joined by adhesive or a PDMS layer.

[0049] Another embodiment of the well plate consists of a glass plate with a thickness of 550 μm with through holes as recesses, which is bonded to a second glass plate with a thickness of 175 μm, both glass plates being adhesively bonded to a plastic body, which has openings that expose the through holes in the first glass plate. The glass plate with the recesses forms the bottom of the plastic body. The diameter of the recesses is, for example, about 100 μm. The plastic body may have several openings instead of one opening. The plastic body may for example be a bottomless microtiter plate, which preferably has 6, 12, 24, 48, 96, 384, 1536, 3456 openings.

[0050] The well plate may have a base surface the size of a microscope object carrier with a width of 25 mm and a length of 75 mm. Such a well plate may consist, for example, of one glass plate with recesses or two or more joined glass plates with recesses. Alternatively, such a well plate may consist of one plastic body to which one glass plate with recesses or a complex of several glass plates are joined as a base.

[0051] Another embodiment of the well plate consists of a plastic body with a circular base surface with a diameter of 35 mm, made of two plastic parts glued together. The upper part has four openings, which expose the second plastic part. The second plastic part has a recess with a diameter of 100 μm and a depth of 250 μm. The plastic parts are produced individually by injection molding.

[0052] Another embodiment of the well plate consists of two glass plates bonded together. The upper glass plate has through holes as recesses with a diameter of 300 μm. The upper glass plate has a thickness of 450 μm. The lower glass plate has a number of channels, which connect the centers of the recesses in pairs. The channels have a width of 10 μm and a triangular cross section with an opening angle of 60°.

[0053] The invention has its origin in miniaturizing the sample carriers used, in particular microtiter plates, and thus overcoming known drawbacks. With the invention, a low consumption of carrier liquid or chemicals required, an overall reduced base surface of the treatment chamber or device, and a recess dimension with a size in the range of the order of microorganisms are thus achieved. Due to the already structurally reduced residence space of the sample, in conjunction with the always possible observation of the sample through the bottom surface of the recess, the unnecessary image detection and data processing of such image data, in particular of areas not important for the investigation, i.e. without samples, can be greatly reduced, and thus the above-mentioned economic and technical disadvantages when investigating and processing single cells can be avoided. The invention thus combines the necessary high quality and biological inertness with important economic aspects for mass screening.

[0054] The device according to the invention allows for a hitherto unattainable precision of release, in particular printing, of cells or reagents in comparison with the prior art, which release can additionally be recorded continuously as a result of visual monitoring possible at any time. Whereas known installations often do not provide for an installation-to-installation interchangeability, the method and device according to the invention provide the prerequisites for a sufficiently fast and automated release of samples into provided recesses, for carrying out optical detection and investigation, and for isolating samples, in particular living cells, with significantly reduced effects.

[0055] The device should thereby be understood as an integrated system for individualizing, investigating and optionally manipulating single cells isolated on a sample carrier, and subsequently isolating the cells from the recesses, in particular through the receiving portion, and ejecting them onto the same or another sample carrier.

[0056] The device is preferably used in cell line development, synthetic biology, investigating immunologically relevant cells (e.g. screening of cells producing antibodies, investigating natural killer cells in interaction with tumor cells), screening the effect of pharmaceuticals on cells (drug screening, drug repurposing), transcriptome analysis, creation of DNA libraries, transfection and transduction experiments, investigating the interaction of different cell types with and without direct physical contact (co-culture) and other biological and medical applications.

[0057] In so doing, the invention opens up the possibility of varying cell types, culture media, reagents and agents in the different wells, in particular by selective release.

[0058] The device according to the invention in particular involves the use of a well plate and one or more processing heads for illumination and / or printing and / or aspiration of the samples, whereby the well plate is manufactured with the utmost precision and with very small tolerances, and its position is thus recognized reliably by the optical sensors of the device. Preferably, one processing head is used which includes all functions.

[0059] Furthermore, the close tolerances achievable with well plates open the prerequisites for considerable miniaturization, which further reduces the possible residence space for the sample as well as the required volume of carrier material.

[0060] The sample typically consists of cells, e.g. bacteria, yeast, animal cells, plant cells, and / or biologically relevant molecules, e.g. proteins, antibodies, DNA, RNA, and / or biomarkers, e.g. fluorescent dyes, antibodies, antigens, suspended in a liquid, which typically consists mainly of water and may additionally contain nutrients, e.g. sugars, amino acids and / or growth factors and / or antibiotics.

[0061] Besides biological or medical samples, samples may also consist of or have particles, e.g. growths, suspended in a liquid medium, most often water and other contents, made of plastic or glass, with a diameter between 1 μm and 50 μm, and optionally with a coating that binds biomolecules, e.g. silanes, antibodies or other protein structures.

[0062] The sample may contain live cells. Mammalian cells, for example CHO cells, HEK cells, immune cells, for example B cells, T cells or NK cells; and also bacteria and yeast. Such cell types may be naturally, genetically engineered, modified or degenerated (cancer cells). Furthermore, they may be in the form of hybrids of different cell types, for example by somatic cell fusion or by cells including antibody producing B cells with cancer cells, for example by hybridoma technology. These cells are suspended in a medium, which may contain as main components water and / or cell culture medium and / or buffer solutions and / or reagent solutions. Preferably, the suspension contains at least 70% live cells. The cell concentration is at least 1,000 cells / mL and up to 2,000,000 cells / mL or more. The percentage of cell clusters is up to 10%, 20%, 30%, 40% or 50%. Does not include cells that are 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times larger in diameter than the diameter of the smallest cell.

[0063] Preferably, different cells can be printed into the wells, e.g., tumor cells can be combined with NK cells in the same well, or when co-cultured, different cell types can be printed into adjacent wells that are connected, e.g., via a channel.

[0064] According to an advantageous development of the invention, the device comprises a processing head, which is attached above the sample carrier on a movement axis independent of the movable receiving part for the sample carrier, in particular the cross table. The processing head can thereby reach the processing position as well as the position of the drop calibration station and the input / output position. The device may also comprise further positions for the processing head, such as a cleaning station, a disinfection station, a temperature adjustment station, a tool change station, a pipette change station, a cannula change station, a coating station, a drying station, an inspection station, a measuring station. In this case, the receiving part for the sample carrier is preferably independently movable by the assigned drive in the direction of two axes in one plane. This plane may also be moved along another axis perpendicular to this plane.

[0065] These shafts are mounted entirely or partly on granite, the properties of which (free of internal stresses, good damping properties, extremely high stability during temperature alternations) allow a constant precession of the shafts.

[0066] The ejection unit may comprise a printing device which ejects the sample into the sample carrier, in particular in the form of droplets. Preferably, the printing process can be triggered by a piezo or solenoid element, where at least one piezo element acts directly on the volume of printing solution containing or forming the sample, or the piezo or solenoid element moves a plunger which acts on the volume of printing solution, thus leading to droplet formation and separation.

[0067] The processing head may have a capillary for aspirating liquid, for example, from a recess in a sample carrier or other vessel.

[0068] After the printing process is completed, the user can select which wells should be further worked on. This can be, for example, samples from such wells containing only exactly one cell, or any range of multiple, e.g., 2-5 cells, or another desired composition, e.g., a combination of a certain number of cell types with a second number of another cell type. This criterion is preferably met by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the wells present in total on the well plate.

[0069] According to another embodiment, interrogation or processing continues only with cells of a certain minimum or maximum size, shape and / or structure, or any range of these values, as detected by the optical interrogation unit.

[0070] Furthermore, the investigation or treatment may be limited to attached or unattached cells, in which case a reaction duration during which the cells must be attached or unattached can be selected, for example at least 12 hours or 24 hours.

[0071] Contrary to what has been the case up to now, advantageously samples from recesses which contain deviant amounts of cells or whose circularity, size or other properties do not correspond to the desired profile can be used in a process step subsequent to the release, thus providing, for example, a comparative sample which can then help to elucidate changes in the reagents which are not caused by cells or which are brought about by unnoticed contaminations.

[0072] When printing according to the dilution principle (Poisson distribution) and the cells are distributed statistically in the wells during each printing cycle or the wells are left empty, it may be advantageous not to refill the empty wells as often as possible and to leave a certain number of wells free, for example 1% or 5% or 10% or more of the wells, or a total of 10 or 100 or 1000 or 10,000 or more wells. In this way, experiments showing changes without cells or contamination can be carried out, for example for comparison purposes. Furthermore, the accuracy of recognizing or not recognizing cells can also be detected, for example by reanalyzing the wells after any defined time and evaluating the deviations. These false negative wells may show cells during a subsequent scan because they have changed position, become larger or divided, increasing their number and making them easier to recognize. Furthermore, this procedure also reveals technical problems, for example vortexes that cause cells to unintentionally end up in empty wells, or convection currents due to heating that cause cells to levitate.

[0073] In one embodiment, this check is used to check the validity of the performed experiment. For this purpose, wells previously identified as empty are analyzed following the experiment. If cells are found in such wells, this suggests that errors have occurred during the performance of the experiment. These errors may have their origin in erroneous initial analysis (cells missed) or handling of the samples (cells transferred from well to well). The threshold for judging the quality of the experiment (e.g. 0.1% of initially empty wells contain cells after the experiment) can be defined by the user.

[0074] The delivery unit, in particular comprising the print head, may have exchangeable cartridges or containers for samples, reagents, cell suspensions, culture media, water or buffer solutions.

[0075] A variation of such a print head may receive liquid from a reservoir by capillary forces or by negative pressure. Furthermore, the print head may be connected to a storage container by a tube system, so that the ejection unit may be supplied with liquid by a pump system. The print head may have exchangeable cartridges, which contain reagents, cell suspensions, culture media, water or buffer solutions.

[0076] In practical use, drop volumes of 20 pL to 70 nL have already proven useful. Drop volumes may, however, be up to 10 μL or even higher. Drop volumes can be determined, for example, by optically observing the drop diameter.

[0077] An optical sensor for detecting the drop volume can further be used for closed-loop control of the parameters of the ejection unit, in particular the piezo-actuated control, such that satellite drops, atomization, drop non-detachment or other undesirable effects are largely avoided. The optical sensor is part of the drop calibration station. As optical methods, drop imaging, Fraunhofer diffraction, laser Doppler or other interferometric methods, Mie-based methods, Lorentz-Mie-based methods can be used. The drop volume can be determined by a capacitive or flow sensor. Alternatively, the optical method can be combined with a weighting method.

[0078] Important influence quantities of the piezo displacement on the printing process of the ejection unit are the absolute displacement, the velocity of the displacement and the time course of the voltage applied to the piezo. The influence quantities can be closed-loop controlled when satellite drops or similar effects are detected and / or when drop break-off is not detected.

[0079] It has proved to be advantageous in this case if the ejection unit, in particular the print head, can be positioned in various vertical positions relative to the sample carrier, without precluding a change in position during sample processing. Prior to the printing process, the print heads that are not used or that are not selected can be raised to increase their distance to the sample carrier. Alternatively, the print heads that are required for the printing process can be advanced to decrease their distance to the sample carrier. Additionally, the selected print heads can be run parallel to the plane of the sample carrier before the printing process. Preferably, all print heads are mounted on a device that allows the distance to the sample carrier to be adjusted.

[0080] Even though the outflow opening of the ejection unit, for example configured as a nozzle, and the optical investigation unit, in particular with an objective lens, are oriented coaxially or perpendicularly to one another, the drops can still be printed with a certain offset. This offset can be small and negligible, or it can have to be corrected. For this, a drop can be ejected onto a surface or into a recess that is located in a common plane with the opening of the recess. By means of the optical investigation unit, in particular a microscope, this offset is optically detected in the x- and y-directions and a correction value for the positioning of the sample carrier is measured. This measurement can also be performed outside the installation. The sample carrier is then run to the corrected position. Alternatively, a print head can be used for the correction in one direction and a cross table for the correction in the orthogonal direction.

[0081] To correct the offset, a drop may be placed in a recess of the sample carrier and a change in the optical properties of this recess, e.g. light transmittance, may be detected. If no optical change occurs, a correction in x and / or y is performed and the process repeated. Typical step sizes in x and / or y are e.g. 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm or more.

[0082] Alternatively, a drop can be dropped at the recess location to correct the offset. If no drop is detected in the recess, a routine can be run in which the offset is first changed by a percentage of the recess diameter or edge length (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 150% of the diameter or edge length), specifically along the x-axis, along the y-axis, and / or diagonally in any order, with different step sizes along the x-axis and y-axis, respectively, and a drop dropped after each change in position. If no drop is detected in the recess after the change in position, the step size of the change in position can be increased (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% increase in step size of the diameter or edge length). Then, again, a check is made to see whether a drop is detected in the recess, and the method can be repeated until a termination criterion is met (e.g. a drop is detected in the recess or a pre-defined number of position changes has been exceeded).

[0083] Additionally, after each change of position, the position of the dropped drop may be compared with the position of the center of the recess or with the position of the edge of the recess. The cutoff criterion of the described routine may in this case be the minimum distance between the drop position and the position of the recess or the maximum distance between the drop position and the position of the edge of the recess. Furthermore, other geometrical features on the sample carrier may be used as a criterion for the correction of the offset.

[0084] Alternatively, a single drop may be placed into the well and the microscope measures the x and y offset above the plane of the bottom of the well and corrects the position of the sample carrier by this amount.

[0085] Alternatively, cross structures or lines may be fabricated on the surface of the substrate or at the bottom of the recess, the size of the cross structures or lines being known. After dropping the drop, the offset between the drop and these structures can be determined. The cross structures may have a spacing of 10-50 μm from each other, for example 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm.

[0086] The above-mentioned structures specifying the offset, in particular the recesses, cross structures and / or lines, may have an edge length of 100 μm to 1 mm or more, preferably 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or more.

[0087] Preferably, the diameter of the droplets emitted by the emission unit is at least 2%, 5%, 10%, 20%, 30%, 40% or 50% smaller than the diameter of the recess or the minimum edge length of the recess.

[0088] Alternatively, for the correction different combinations of movements of the ejection unit and of the receiving part of the sample carrier may be provided.

[0089] The optical investigation unit can be used to perform various microscopy techniques, including bright field microscopy, dark field microscopy, phase contrast microscopy and (epi-)fluorescence microscopy. The optical investigation unit preferably comprises a digital camera sensitive in the wavelength range from 380 nm to 780 nm. The optical investigation unit further comprises an objective lens, which preferably has a magnification of 5x, 10x, 20x, 40x. The optical investigation unit further comprises an illumination unit, which preferably comprises a white LED and / or an LED with emission in the UV and visible spectrum. For fluorescence microscopy, preferably excitation wavelengths of 375 nm, 490 nm and / or 590 nm are used.

[0090] The processing head preferably has illumination as a tool, which can preferably be used for bright field microscopy.

[0091] For optical investigation of the samples in the respective recesses through the bottom surface of the recesses, the receiving part, which for example comprises a cross table, may have an opening or an optically transparent mounting surface for the sample carriers.

[0092] For the optical interrogation or inspection step and scanning of the recess, the distance between the objective lens and the sample carrier can be changed in order to image different planes of the recess, whereby the distance change can be performed in steps of 15 nm to 100 μm. Alternatively, an Electrically Tuneable Liquid Lens (ETL) can be used to change the focal length of the optical interrogation unit and thus image different planes of the recess.

[0093] The microscope may image a rectangular field of view, preferably 0.21 mm x 0.18 mm or 2.8 mm x 2 mm, or a smaller or larger field of view. The field of view may have square faces or other ratios of edge lengths. Within the field of view, preferably one plane of one recess may be completely imaged, depending on the magnification setting. Alternatively, multiple recesses may be imaged, preferably at least 4, 12, 20, 35, 54, 140, 560, 2240 or 10,000 or more recesses.

[0094] The device may comprise an input / output location in which a reservoir is positioned. The reservoir comprises a vessel in which a liquid may be prepared. The reservoir may be a common microtiter plate or may have another format, for example the base surface of a microscope object carrier or a circular format, for example with a diameter of 35 mm. The vessel may contain a sample, for example a reagent or a cell suspension, which may be accommodated by a print head of the device. By means of a capillary the contents may be taken from the recess of the sample carrier and released into the vessel.

[0095] In one variant of the invention, particularly suited to practical use, the sample carrier comprises, at least as the main material component, a plastic, for example COP, COC, PS, PMMA, POM, PC, glass, silicon or a combination of these materials.

[0096] Glass has ideal properties: it is optically transparent, chemically inert, but can be surface-treated by known methods (plasma treatment, silanization, coating), is liquid-tight and does not conduct electricity, which are advantageous for the application of sensors. Glass can, however, be (selectively) coated and thus (selectively) made conductive. A known method is metallization ((partially) selectively, if a mask is used) by deposition using physical vapor deposition (PVD) methods. Chemical metallization, for example by seeding with palladium and subsequent metal deposition without external current or by printing with liquids or pastes containing metals, also comes into consideration. Semiconductors can also be (selectively) deposited, for example by chemical vapor deposition (CVD) methods. Here, silicon and its compounds are used in particular.

[0097] The sample carrier, in particular its surface, may optionally be treated by plasma treatment, preferably with a high percentage of oxygen in the gas, or by wet-chemical methods, for example etching with NaOH, KOH or strong acids, for example HNO3, sulfuric acid, phosphoric acid, in order to improve hydrophilicity and thus wettability. This is achieved at temperatures above 40°C, above 50°C, above 60°C, above 70°C, above 80°C or above 90°C, in particular by contact with chemicals. The duration is then between one minute and several hours. For the manipulation of surface properties, with regard to wettability or for the promotion / opposition of attachment of cells to the surface, various methods are known. Plasma treatment typically makes the surface more hydrophilic, whereas silanization, for example with alkylsilanes or fluoroalkylsilanes, makes the surface hydrophobic. These treatments can be used to make the surface more or less wettable by polar solvents, for example water. Coatings that control cell attachment include, inter alia, poly-L-lysine, poly-D-lysine, poly-ornithine, gelatin, collagen, fibronectin, laminin, vitronectin, osteopontin, etc. Typically, cell surfaces are negatively charged, and therefore negatively charged or hydrophobic coatings make cell attachment difficult or even prevent it.

[0098] The size of the sample carrier can be varied, e.g. 1 cm 2 ~0.1m 2 or more and may be located in the base plane.

[0099] The recess may be configured in a columnar shape, or according to a preferred embodiment, in a cone shape with a wall surface having an inclination angle (taper angle) of 1° to 30° with respect to the normal to the cross-sectional plane.

[0100] Furthermore, the recesses may have a flat base or may have a regular or irregular structuring with pyramidal or spherical substructures.

[0101] Above the liquid or sample containing the cells in the recess, a liquid film or supernatant may be present, which has a height of at least 10 μm to 100 μm, preferably 100 μm to 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or more. The optical investigation through the transparent bottom is then limited to the sample, so that the investigation does not disturb the meniscus that is formed. In particular, the investigation can be completed before reaching the meniscus during a vertical scan of the recess.

[0102] Optionally, all recesses or groups of recesses have such a liquid top. This top can be formed by a glass, silicon, metal or plastic frame. The frame can be glued or joined by fusion bonding (glass to glass) or by anodic bonding (glass to silicon) or clamped by a rubber frame. This allows the meniscus to be formed with a larger distance to the upper edge of the recess and / or to have a larger radius (since the diameter of the frame is always larger than the diameter of the recess) and / or to be convexly shaped, for example when the defining material is hydrophobic (plastic, rubber, coating). This clearly reduces the influence of the meniscus on the imaging of the sample with a microscope.

[0103] The capillary is a vertical hollow body made of glass, plastic or special steel. The capillary is preferably connected to a unit for generating negative pressure. The unit for generating negative pressure can be a pump or a movable plunger, which is advanced by a motor to generate the negative pressure. Alternatively, the negative pressure can be generated via potential energy or via volume expansion due to temperature changes.

[0104] The outer diameter of the capillary in the region of the opening is smaller than the inner diameter of the recess whose contents are to be isolated, so that the capillary can be submerged within the recess.

[0105] Since the sample is immobilized in the recess based on an aspect ratio (depth to width) according to the invention that is greater than 1, there is no risk that capillary movement in the medium in the recess could undesirably displace the cells to be isolated or cells to be isolated later. For this reason, also non-adherent cells can be isolated based on a database generated upon microscopy and subsequent analysis of the image.

[0106] It has already been found that in some cases the location of the cell in the well is so well known that it is not necessary to control or position the capillary by means of an optical interrogation unit: the process of removing the contents from the predetermined well by the capillary can thereby be carried out substantially more quickly.

[0107] The relative positioning of the removal unit, in particular the capillary, is already sufficient when the removal unit is located within the field of view of the optical investigation unit, whereas fine positioning is achieved by the removal unit and the recess being positioned relative to one another by optical control, i.e. the recess itself is used as an inherent reference point.

[0108] Particularly preferably, the position of the removal unit relative to the sample carrier can be navigated manually by means of an operating element, for example by input in the software, or automatically positioned to a starting position above the recess. Further positioning relative to the recess is then also performed manually or fully automatically.

[0109] Another, and likewise particularly advantageous, embodiment of the invention is achieved in that the capillary is completely or partly coated with a hydrophobic coating, for example an alkylsilane or a fluoroalkylsilane.

[0110] It is furthermore advantageous if the capillary has a smaller cross-sectional area in the section facing the opening than in the section facing away from the opening. The change in cross-sectional area along the main extension is then preferably carried out continuously, with a linear or progressive transition. Alternatively, the capillary has a portion with a larger diameter and a portion with a smaller diameter.

[0111] Preferably, the capillary is sized to accommodate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of the volume of the recess.

[0112] Another variant of the invention, which is also particularly suitable for practical use, is achieved by partially filling the capillary with a liquid having a higher density than the carrier liquid from which the contents are to be isolated. For example, when the capillary is immersed, some liquid may be released into the recess. This liquid causes the particles contained in the recess to float above the bottom surface. The contents of the recess can then be more easily aspirated.

[0113] The capillary is immersed in the liquid supernatant at a distance from the bottom surface to such an extent that the sample in the recess is not disturbed by the liquid movement. In the case of a closed capillary, the capillary can remain in this position until the suction action due to the capillary effect is compensated by the back pressure created by the compression of the air, i.e. until an equilibrium occurs (priming of the capillary). In the case of an open capillary, the liquid first rises until gravity and capillary forces are balanced. This rise height is described by the capillary equation. The recess is then positioned, as the case may be, so that the capillary can be immersed in the sample. For aspiration, a valve is opened or a negative pressure is generated for aspiration, for example by the movement of a plunger or other device, so that the capillary is further filled with the contents of the recess. An open capillary must first be closed, for example by a plunger, to generate the negative pressure. This procedure with a primed capillary ensures full control of the suction speed and the forces governing the uptake of, for example, living cells.

[0114] Due to the small scale of the recesses, wicking is significantly affected by capillary forces within the recesses. To reduce this effect, the walls of the recesses may be hydrophobically coated, for example with alkylsilanes or fluoroalkylsilanes.

[0115] The well plate may contain at least one location for "priming" the capillaries, which may be, for example, a well that does not contain cells, or a possibly non-well at the edge area, or any other location that satisfies the condition that the liquid supernatant above the well does not displace the sample uncontrollably from the well.

[0116] The contents of the capillary can be discharged at the input / output location, for example by discharging the liquid into a prepared microtiter plate. It is then particularly advantageous if the capillary accommodates a volume that is substantially larger during the priming step than when drawing up from the recess. The volume during priming is preferably at least 10, 100, 1000 or even more times larger. This ensures that the sample present and contained in the lower region is reliably washed out and does not, for example, come into contact with the capillary wall at the meniscus of the liquid surface, adhere thereto or can only be reliably isolated from the capillary by multiple washings.

[0117] Alternatively, a further supply line may be attached to the upper part of the capillary, which makes it possible to wash the capillary towards the opening and thus ensure the removal of contained particles.

[0118] The capillary may be detachable, for example by a Luer lock or another mechanism, from a unit for generating negative pressure. Such a unit for generating negative pressure may be a pump, plunger, piston or the like, which is movably actuatable to generate negative pressure.

[0119] According to another particularly promising embodiment of the invention, the sample carrier, in particular on its surface or in the region of the bottom of the recess, may be provided or affixed with markings or structures, in particular cross structures or lines, the size and relative positions of which are known, so that the position of the sample carrier can thus be detected optically. Any offsets which may occur when releasing the sample can also be determined by means of these structures.

[0120] It is particularly advantageous if the receiving part and the sample carrier have complementary geometries that only permit mating in a single position and / or orientation. The orientation can be unambiguously indicated, for example, by markings on the sample carrier and / or on the receiving part. The sample carrier can be applied to a stop and locked in position by a clamp or other mechanism.

[0121] The machine housing has an encapsulated treatment chamber that is purged with HEPA and / or ULPA filtered air, and thus sterile air. UV radiation may also be used for sterilization. The encapsulated treatment chamber may be defined by a separate chamber volume or by the volume of the entire device.

[0122] Alternatively, the housing or the separate chamber volume may be sealed against the environment. In both cases, the closed treatment chamber preferably has an opening to lead out the trapped gas volume through a separate HEPA and / or ULPA filter, for example to retain and / or aspirate particles, e.g. microorganisms.

[0123] Furthermore, a sterilizing or purified gas, e.g. air, can be introduced from an external gas supply unit via a line connection or can be generated as part of the device by means of corresponding filters and blowers, e.g. the gas supply unit can be connected to the housing of the device.

[0124] The pressure difference between the encapsulated sterilization chamber and the surroundings may be controlled in a closed loop: when printing, microscopy, isolating cells or during "sleep mode", the positive pressure is up to 1 bar relative to the surroundings, or the positive pressure is limited by the filter used.

[0125] In a particularly advantageous manner, the treatment chamber is separated in a gas-tight manner from the surroundings by at least one closable access opening, in particular a door or flap, and when the access opening is open, the supplied volumetric flow rate of sterile air can preferably be increased in order to prevent the intrusion of non-sterile ambient air.

[0126] When in the closed position, the door or flap is pressure tightly closed by a seal, such as a rubber seal, a Teflon seal, a foam and / or an expandable tube seal.

[0127] To largely avoid vortex motion when the access port is opened and effectively reduce the inflow of non-sterile air from the surroundings into the work area, the door or flap is preferably not pivoted but moved translationally along a flat or curved trajectory, e.g., slid parallel to the housing surface.

[0128] The door or flap may consist of a single movable element which is entirely slidable or of several translationally movable elements which may in particular be moved in the same direction in several parallel planes or may be moved partially or completely in different directions in each case. In practical use, the passage opening can be adapted to the sample carrier, advantageously by all elements or only individual elements being slidable, thus adapting the size of the passage opening as required.

[0129] The door or flap elements can be moved via hydraulic, magnetic or mechanical mechanisms. Linear axes, linear guides, spindle axes are particularly advantageous. The speed at which the door can be moved for opening and closing is preferably limited to 33 mm / s. At higher speeds, a safety mechanism is preferably used to protect the user.

[0130] Furthermore, a sensor, e.g. a magnetic sensor, electrical contact, displacement measuring sensor, light barrier or the like, may be provided to detect whether the door or flap is closed, partially open or fully open, so that a control signal for the volumetric flow rate of the supplied sterile air is triggered depending on the detected state.

[0131] In order to provide suitable conditions for the cells, a further, in particular environmentally controlled, unit may be inserted into the treatment chamber, which defines a sterile area for enclosing a reduced working volume and enclosing the sample carrier. This unit may be equipped with its own supply for a sterile or purified gas volume. The required supplies can be guided through the housing and connected to this unit, for example directly or by means of an adapter piece, only when necessary.

[0132] The environmentally controlled unit may be transparent in at least some sections, thereby allowing optical interrogation through the transparent areas, and possibly also the necessary illumination through the transparent areas.

[0133] The environmentally controlled unit is preferably made of glass, sapphire, COP, COC, PC, PS, POM, PMMA or other transparent materials, and may be removed from the work chamber when it is not needed.

[0134] An alternative connection of the supply may take place through an open door, in which case it is particularly advantageous if the supply is guided through a cover which covers the door opening and provides darkness inside the installation so that microscopy, in particular in the case of fluorescence microscopy, is not hindered by extraneous light which would otherwise result in poorer contrast values.

[0135] One further advantage of such a cover is that the volumetric flow rate of sterile air can be less than when the door is fully open.

[0136] When the door is open, the various processing units are not used and are preferably blocked by the control unit. The installation of the cover allows the use of these units, in particular the optical investigation unit and the receptacle for the sample carrier. For this purpose, the position of the door or flap as well as the presence of the cover are detected, for example by means of magnetic contacts, and corresponding control signals are triggered by the control unit.

[0137] The gas volume can be heated to a predetermined temperature. In practical use, a gas composition of 1%-10% CO2 and / or 5%-30% oxygen and / or 90-98% water vapor has already proven suitable.

[0138] Preferably, the field of view is changed, i.e. scanned to x or y positions, only when the optical investigation is completed over a major part of the well, in particular over the full height or over at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the well height. The image data thus detected are stored together with information on the position (x, y, z) and / or time (absolute or relative to a particular event). The alternative flow, i.e. first scanning all wells in one plane and then defocusing, incurs the disadvantage that cells may move in the wells during the scan along the plane, resulting in erroneous measurements.

[0139] The cross table of the receptacle for the sample carrier has a finite precision, so that tolerances for the sample carrier are unavoidable, which can be recognized as problematic if the edges and / or the center of the recess go outside the tolerance range.

[0140] Furthermore, the sample carrier may have certain positions without recesses or with recesses that are modified (e.g. with a modified diameter) or with recesses that are shifted in the plane of the surface of the sample carrier. These deviations can be used appropriately, for example to recognize erroneous orientations and / or positions or incorrect loading of the sample carrier. For this purpose, the exact position of the field of view is checked or corrected or calibrated by matching the image data with target values ​​of the layout of the sample carrier.

[0141] The images of the optical investigation unit are checked, at least partly automatically by artificial intelligence or machine learning methods, for the presence of cells, cell shape, cell size, intensity or color of the fluorescent signal, cluster formation, cell mobility, cell growth rate, reaction to external stimuli, reaction time and other characteristics and / or combinations thereof, and this information is stored and / or further processed. Based on this information, further steps can be automated, pre-given by the user or manually derived by the user.

[0142] For long term experiments, the optical interrogation unit may interrogate a particular recess multiple times at pre-given intervals and according to a pre-given pattern to detect image data.

[0143] Typically, the cells are distributed in a liquid that partially or completely fills the volume of the well, for example on the side walls, on the bottom or floating freely in the liquid. To detect the cells, imaging in three dimensions is therefore necessary. The optical interrogation unit can therefore be moved in the direction of the depth extension of the well, which allows clear imaging of different planes within the cells.

[0144] From the three-dimensional imaging of the wells, information can be evaluated as to which three-dimensional position the cells or particles are present in. When the capillary receives the sample, this information can be used to receive the cells or particles.

[0145] Imaging of the well contents can be used to automatically identify and catalogue cell types, and this information can be used to automate the selection of specific wells for targeted removal of located cells.

[0146] The wells may be filled with a highly viscous liquid to inhibit cell movement.

[0147] Optionally, the device can access a database to obtain information about the contents of the wells from previous measurements. From this information it can be derived which contents should be received and output from the device at which point in time. Such information can be, for example, the presence of a particular cell type, antibody, protein, or cell growth rate, or other result of a previous evaluation.

[0148] Prior to receiving the sample, the capillary may release a liquid into the well, e.g., a contrast agent, a denser liquid to suspend the cells, and optionally the capillary may repeatedly release and aspirate to suspend the cells or particles or to free them from confinement to the walls or bottom of the well.

[0149] When receiving the sample from the recess, the capillary can be immersed in the recess, whereupon its position within the recess can be detected by the optical interrogation unit, whereby manual steps can be performed by the user, for example correcting the position of the capillary within the recess.

[0150] The capillary tube is optionally attached to the upper end of the printhead.

[0151] Before, during and after the placement of the sample, the contents of the capillary can be checked, for example by the optical interrogation unit or by a separate optical unit, on the basis of which further work steps can be triggered.

[0152] Optionally, the sample carrier may be removed from the equipment during processing within the device and reinserted therein in order to perform another work step, for example centrifugation.

[0153] All of the described work steps can be performed either automated or manually at the user's option.

[0154] The user has the possibility at any time to manually select the wells from which the cells should be removed, or alternatively, this can be performed automated, with a routine being used to select cells with specific properties, pre-specified by the user.

[0155] Optionally, to clean the capillary, a rinsing solution and / or a cleaning solution can be supplied through an opening provided above the tip of the capillary. In this way, the rinsing process is extremely efficient and effective. Alternatively, the rinsing solution and / or the cleaning solution can be accommodated and released again by aspiration. Both rinsing methods can also be combined with each other.

[0156] Optionally, the temperature within the volume of the device is closed-loop controlled within the range of 4°C to 37°C and the relative air humidity may be set to a value above 90%, for example 95%, thereby minimizing evaporation.

[0157] Single-cell experiments can be automated in a single facility and carried out economically with high throughput and a large number of individual experiments (up to 1,000 to 1,000,000). Due to the extremely small volume of the wells, for example compared to microtiter plates, expensive reagents are saved and due to the small dimensions of the wells, even in large numbers, the sample carriers have a significantly smaller base surface. Due to the individualization (compartmentalization), individual cells can be found and compared from large populations of more than 10,000 cells according to pre-given criteria.

[0158] The present invention allows various embodiments, and in order to further clarify the basic principle of the present invention, one embodiment of the present invention is shown in the drawings and will be described below. [Brief description of the drawings]

[0159] [Figure 1] FIG. 2 is a cross-sectional side view of an apparatus according to the present invention in a printing position. [Diagram 2] FIG. 2 is a cross-sectional side view of the apparatus in another printing position. [Diagram 3] FIG. 2 is a cross-sectional side view of the device in an illuminated position. [Figure 4] FIG. 2 is a cross-sectional side view of the device in the removal position. [Diagram 5] FIG. 2 is a cross-sectional side view of the device in a calibration position. [Figure 6] FIG. 2 is a perspective view of an apparatus with a sample carrier. [Figure 7] FIG. 2 is a cross-sectional side view of a sample carrier receiving portion of the device; [Figure 8] FIG. 2 is a side view of the optical interrogation unit of the apparatus. [Figure 9a] FIG. 2 is a side view of the output unit of the device. [Figure 9b] FIG. 2 is a side view of the output unit of the device. [Figure 10a] FIG. 2 is a side view of the ejection unit of the apparatus. [Figure 10b] FIG. 2 is a side view of the ejection unit of the apparatus. [Figure 10c] FIG. 2 is a side view of the ejection unit of the apparatus. [Figure 11] FIG. 2 shows a plan view of a sample carrier of the device having different recesses. [Figure 12] FIG. 2 shows a plan view of a sample carrier of the device having different recesses. [Figure 13a] 4 is a cross-sectional view showing the cross-sectional shape of a recess of a sample carrier. FIG. [Figure 13b] 11A and 11B are cross-sectional views showing different cross-sectional shapes of the recess of the sample carrier. [Figure 13c] It is a cross-sectional view showing another cross-sectional shape of the recess of the sample carrier. [Figure 13d] It is a cross-sectional view showing another cross-sectional shape of the recess of the sample carrier. [Figure 13e] It is a cross-sectional view showing another cross-sectional shape of the recess of the sample carrier. [Figure 13f] It is a cross-sectional view showing another cross-sectional shape of the recess of the sample carrier. [Figure 14a] It is a perspective view of a sample carrier having a plurality of recesses with different aspect ratios respectively. [Figure 14b] It is a perspective view of another sample carrier having a plurality of recesses with different aspect ratios respectively. [Figure 15a] It is a plan view of the recess of the sample carrier shown in Fig. 14a. [Figure 15b] It is a plan view of the recess of the sample carrier shown in Fig. 14a. [Figure 16a] It is a cross-sectional view of one recess of the sample carrier shown in Fig. 14b. [Figure 16b] It is a cross-sectional view of one recess of the sample carrier shown in Fig. 14a. [Figure 16c] It is a cross-sectional view of one recess of the sample carrier shown in Fig. 14a. [Figure 17a] It is a plan view of the recess having a marking on the bottom surface of the sample carrier shown in Fig. 11. [Figure 17b] It is a plan view of the recess having a marking on the bottom surface of the sample carrier shown in Fig. 11. [Figure 18a] It is a cross-sectional view of a sample carrier containing one sample. [Figure 18b] It is a cross-sectional view of another sample carrier containing one sample. [Figure 19] It is a side view of a unit that can be loaded into the processing chamber for housing the sample carrier.

Embodiments for Carrying Out the Invention

[0160] The device 1 according to the invention for investigating or processing a biological or medical sample 2 and the implemented method are explained in more detail below on the basis of figures 1 to 19. In the processing chamber of the device 1, various functional units are arranged, here configured as integral constructional units, comprising a discharge unit 5 with a number of print heads 3 for discharging the sample 2, an illumination unit 16, a removal unit 9 with a capillary tube 8 for removing the sample from the sample carrier 10 and discharging or outputting the previously isolated sample 2, and an optical investigation unit 6 configured as a microscope and having an objective lens 7, spatially separated from these functional units.

[0161] The sample carrier 10 has a number of recesses 11, which are shown in Figs. 11 to 14, each with an opening 12 and a bottom surface 13. The sample carrier 10 is translationally movable in the direction of the arrow 15 in a horizontal plane to various working positions by means of a receiving part 14 configured as a cross table. In each working position, the optical interrogation unit 6 is arranged below the receiving part 14, whereas the currently active units are positioned above the receiving part 14 on the opposite side. In this working position, the optical interrogation unit 6, the currently selected recess 11 and the release unit 5, the removal unit 9 or the illumination unit 16 are located on mutually opposite sides of the sample carrier 10 coaxially on one common axis 17 (corresponding to the optical axis of the optical interrogation unit 6). For this purpose, the bottom surface 13 of the recesses 11 is transparent, so that optical detection of the sample 2 through the bottom surface 13 is performed.

[0162] During processing or investigation of the sample 2, the optical investigation unit 6, at least the objective lens 7, can only be moved in the direction of the arrow 18 towards the sample carrier 10, so that the focus position can be adapted. To investigate the sample 2 in the further recess 11, the movement of the sample carrier 10 is determined by the receiving part 14.

[0163] During operation, the required relative movement of the sample carrier 10 is primarily carried out based on the movement of the sample carrier 10 by the receiving part 14, but in addition, a movement of these units is also provided in order to select the desired units, which in the illustrated variant are moved collectively in translation in the direction of the arrow 19 and thus can also reach, for example, a calibration station 20 for drop calibration for the discharge unit 5, as shown in FIG. 5.

[0164] As shown in Fig. 7, the receiving part 14 has a cutout 21 for the sample carrier 10, so that the sample 2 can be detected unrestrictedly through the bottom surface 13 of the recess 11 by the optical investigation unit 6, which is shown in detail in Fig. 8. Correspondingly, the cross table elements 22 also have corresponding cavities 24 and are each movable on outer guide rails 23.

[0165] For investigating the sample 2, the optical investigation unit 6 has a Z-adjustment for moving the objective lens 7 in the direction of the arrow 18, and also comprises illumination, optics and at least one camera, which are not further shown.

[0166] 9a and 9b and 10a to 10c show, on the one hand, the functional principle of the ejection unit 5 and, on the other hand, the removal unit 9. A capillary 25 made of plastic, glass or stainless steel is used here to eject or receive a liquid of sample 2, for example a cell suspension. A tappet 26 acts in this case on a flexible wall 27 of a chamber 28, which changes the enclosed volume and expels a droplet of sample 2.

[0167] When utilizing the functional principle of the take-out unit 9, the capillary 25 is immersed in the recess 11 of the sample carrier 10, which is filled with sample 2, while the volume of the chamber 28 is reduced against a restoring force by the tappet 26. As soon as the pressure on the tappet is reduced, the chamber 28 expands and sucks in part of the filling volume of the recess.

[0168] 11 to 18b show by way of example several configurations of different sample carriers 10, which are configured as well plates, in particular microwell plates made of glass, in that recesses 11 are produced in the sample carrier 10 by the LIDE method known per se. As can be seen in the plan views of Figures 11 and 12, the recesses 11 may have a circular or polygonal, for example rectangular, cross-sectional shape and may have identical or different cross-sectional areas and arrangements in the sample carrier 10.

[0169] At the same time, different configurations of the bottom surface 13 are also possible, which can be seen, again by way of example, in the cross-sectional views of different recesses 11 with, for example, a cylindrical or pyramidal basic shape and respectively different opening angles α, as shown in Figures 13a to 13f. The residence position of the sample 2 in the recess 11 can be predetermined with a statistical probability, for example by generating a topography or structuring with regular ridges in the bottom surface 13, as can be seen in Figures 13a, 13c, 14a, 15a, 15b, 16b and 16c. In this case, it is already known that the sample 2 does not leave the position it has once occupied in the structuring, as shown, for example, in Figures 18a and 18b, even if the sample carrier 10 is moved. This reduces the time required to carry out the investigation and manipulation.

[0170] By means of the optical interrogation unit 6, these structures in the bottom surface 13 can be identified, so that a reference for the positioning of the sample carrier 10 can be performed on the basis of the structures detected by the optical interrogation unit 6 and / or the respective sample 2. In particular, this makes it possible for the first time to orient the sample carrier 10 on the basis of the detected residence location of the sample 2, such that not only the recess 11 but also in particular the sample 2 itself is located on the optical axis 17, as shown in Figures 18a and 18b. Additionally, if for example the bottom surface 13 is flat according to the recess 11 shown in Figures 13d, 13e, 13f, 14b and 16a, a marking 34 can also be made into the bottom surface 13, for example in the glass material, or on the outer surface opposite the recess 11, which marking 34 can be optically detected and thus alternatively or additionally can be used as a positioning reference.

[0171] In so doing, the recess 11 preferably has an aspect ratio of the recess height H to the opening diameter D or edge length L that is greater than one.

[0172] To further optimize the observation of the sample volume of the sample 2, but also its manipulation, the sample carrier 10 may have a layer or coating 36 in the area of ​​the recess allocated to the opening 12, which allows a targeted setting of the characteristics of the meniscus 35 formed, which appears as a convex or concave surface depending on the wetting of the liquid due to surface and interfacial tension. Setting a flat liquid surface is likewise realizable, and the use of an inert, adjusted liquid film covering the sample 2 as a layer or coating 36 has already proven useful.

[0173] Finally, Fig. 19 shows a closable unit 29 which can be inserted into the treatment chamber of the device 1, which defines a sterile area confining the sample carrier 10. The unit 29 has inlet and outlet openings 30 and 31 for gas, a bottom 32 which is transparent in at least some sections, and a cover 33 which is also transparent in at least some sections, so that interrogation by the optical interrogation unit 6 can be carried out through the bottom and lid sides without having to open the unit 29. [Explanation of symbols]

[0174] 1 device 2. Sample 3 Emission Unit 4 Print Head 5 Emission Units 6 Research Unit 7 Objective Lens 8 Capillary 9 Ejection unit 10 Sample Carriers 11 Recess 12 aperture 13 Bottom 14 Receiving part 15 Arrow direction 16 Lighting Unit 17 axis 18 Arrow Direction 19 Arrow Direction 20 Calibration Station 21 Notch 22 Cross Table Elements 23 Guide rail 24 Vacant Space 25 Capillary 26 Tappet 27 Wall 28 Chamber 29 units 30 Air intake opening 31 Exhaust opening 32 bottom 33 Cover 34 Marking 35 Meniscus 36 Coating α Opening angle D diameter H Height L Edge length

Claims

1. A device (1) for investigating and / or processing a sample (2), in particular a biological or medical sample, such as cells and / or particles, comprising: In one common processing chamber, at least one ejection unit (3) for ejecting said sample (2) into at least one sample carrier (10) having a number of recesses (11) each having an opening (12) and a bottom surface (13); an optical investigation unit (6), in particular having an image sensor, a microscope and / or an objective lens (7); a removal unit (9) for removing at least one sample (2) from said sample carrier (10) and for discharging or outputting the previously isolated sample (2); Equipped with In the apparatus (1), Within said treatment chamber, at least one sample carrier (10) is movable by means of at least one receiving part (14) to various working positions within a working area defined by respective recesses (11) of said sample carrier (10); and in each working position, on the one hand, the optical interrogation unit (6) and, on the other hand, the release unit (3), the removal unit (9) and / or optional further units are positioned coaxially in the working area along an optical axis (17) of the optical interrogation unit (6) on mutually opposite sides of the sample carrier (10), at least some of the recesses (11) have a transparent bottom surface (13) at least in some sections, so that optical detection of the sample (2) is performed through the bottom surface (13), A device (1) for investigating and / or processing, in particular biological or medical samples (2), such as cells and / or particles, characterized in that:

2. A method for investigating and / or processing a sample (2), in particular a biological or medical sample, such as cells and / or particles, comprising: outputting said sample (2) by means of an ejection unit (5) into at least one recess (11) of a sample carrier (10) defined by a transparent bottom surface (13); and The sample (2) is optically detected through the bottom surface (13) by an optical interrogation unit (6); and a removal unit (9) for removing the material from said recess (11) and providing it for further use as required; In the method, moving said sample carrier (10) in relation to selected recesses (11) into various working positions, in particular by means of a motor-movable receiving part (14); and in each working position, on the one hand, the optical investigation unit (6) and, on the other hand, the ejection unit (5), the removal unit (9) and / or optional further units are coaxially positioned in a working area along an optical axis (17) of the optical investigation unit (6) on mutually opposite sides of the sample carrier (10), 1. A method for investigating and / or processing a sample (2), in particular a biological or medical sample, such as cells and / or particles, comprising:

3. 3. An apparatus (1) or method according to claim 1 or 2, characterized in that the reference for the positioning is performed by a control unit on the basis of the sample carrier (10) and / or the respective sample (2) detected by the optical investigation unit (6).

4. 4. The device (1) or method according to claim 1, characterized in that at least a major proportion of the recess (11) of the sample carrier (10) preferably has an aspect ratio of the height (H) of the recess (11) to the cross-sectional area, diameter (D) and / or edge length (L) of the opening (12) that is greater than 1.

5. 5. Apparatus (1) or method according to at least one of the claims 1 to 4, characterized in that the receiving part (14) has a cross table, in particular with at least one cavity (24).

6. 6. Apparatus (1) or method according to at least one of the preceding claims, characterized in that the ejection unit (5) comprises at least one printhead (4).

7. 7. Apparatus (1) or method according to at least one of claims 1 to 6, characterized in that the sample carrier (10) is configured as a well plate made of glass.

8. 8. Apparatus (1) or method according to at least one of the claims 1 to 7, characterized in that the recesses (11) in the sample carrier (10) are created by a LIDE method (Laser Induced Deep Etching).

9. 9. The device (1) or method according to at least one of claims 1 to 8, characterized in that the optical investigation unit (6) detects the minimum size, maximum size, shape, in particular the roundness and / or structure of the sample (2).

10. 10. The device (1) or method according to claim 1, characterized in that the emission unit (5) and / or the optical investigation unit (6), in particular the objective lens (7) of the optical investigation unit (6), can be positioned in various vertical positions relative to the sample carrier (10).

11. 11. The device (1) or method according to claim 1, characterized in that at least some of the recesses (11) provided in the sample carrier (10) are configured in at least some sections as cylindrical and / or pyramidal and / or with an opening angle (α) greater than zero.

12. 12. A device (1) or method according to at least one of claims 1 to 11, characterized in that in a first step, the sample (2) contained in a carrier liquid is released into the recess (11) and then another liquid is placed on top of the carrier liquid as a cover or liquid film.

13. 13. The device (1) or method according to at least one of claims 1 to 12, characterized in that the removal unit (9) comprises a capillary (8) having a first cross-sectional area on the side of the opening and a second cross-sectional area on the side opposite the opening, the second cross-sectional area being larger than the first cross-sectional area.

14. 14. Device (1) or method according to at least one of the claims 1 to 13, characterized in that the capillary (8) and / or the recess (11) have, at least in some sections, a hydrophobic coating (36).

15. 15. The device (1) or method according to claim 1, characterized in that the sample carrier (10) has a marking (34) or structure, in particular a cross structure or a line, in particular in the region of the bottom surface (13) of the recess (11).

16. 16. The device (1) or method according to at least one of the claims 1 to 15, characterized in that the receiving part (14) and the sample carrier (10) are connected in a form-fitting manner only at predetermined target positions.

17. 17. Apparatus (1) or method according to at least one of the preceding claims, characterized in that the treatment chamber is sterile and / or sterilizable or is sterilized.

18. 18. An apparatus (1) or a method according to at least one of claims 1 to 17, characterized in that the apparatus (1) comprises a closable unit (29) which can be inserted into the treatment chamber, in particular a replaceable unit, which defines in particular a sterile area confining the sample carrier (10).

19. 19. The device (1) or method according to claim 1 , characterized in that during the investigation by the optical investigation unit (6), the field of view is directed successively at different cross-sectional planes within the recess (11).

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