Method and device for dispensing individualized biological cells

Abstract

The invention relates to a method and a device for dispensing individualized biological cells (20). The method has the steps of providing a cell suspension and providing a dispensing device (10) comprising a support (12) in which a channel (14) for conducting the cell suspension is formed, wherein the channel (14) extends in a longitudinal direction (L) and has a circumferential channel wall (16) with at least one lateral opening (18) and a cell trap (22) in the vicinity of the opening (18). The method additionally has the steps of generating a flow of the cell suspension in the channel (14), trapping an individual cell (20) in the cell trap (22), the location of the trapped cell (20) being located at the same height as the lateral opening (18) with respect to the longitudinal direction (L), and generating a pressure surge in the surroundings of the lateral opening (18), said pressure surge resulting in a force, which acts in the direction of the opening (18), on the trapped cell (20).

Description

[0001] Method and device for dispensing isolated biological cells

[0002] Description

[0003] The invention relates to a method and a device for dispensing individual biological cells. Dispensing is part of a printing process by which the biological cells are applied individually, one after the other, or in parallel, to a substrate in order to produce organic tissue, organ parts, or entire organs.

[0004] In particular, the demand for organs for transplantation, especially skin, kidneys, liver, and hearts, is currently very high and cannot be met with the available donor organs. Long waiting times are often the result, which in many cases lead to the death of waiting patients. Therefore, it is desirable to produce organs in the laboratory quickly and close to the patient. Organs grown or manufactured in vitro could also be immunologically personalized, which would significantly reduce the rejection rate and the use of immunosuppressants. The illegal organ trade could also be curbed or even completely eliminated with affordable, in vitro-produced transplants.

[0005] Initial approaches to 3D bioprinting already exist. For example, an extrusion process is known, which is primarily used in regenerative medicine research. Here, a cell suspension is dispensed as a continuous bioink jet. A large number of cells of the same type are deposited simultaneously on a substrate. The hope is that cells will self-organize to form functional units. This approach is widely pursued in the field of 3D bioprinting. However, it is unsuitable for the precise "cell-by-cell" construction of complex organ structures.

[0006] In a well-known single-cell printing technology, dispensing systems continuously eject drops of a cell suspension. The contents of the microdroplets are monitored by photodetection. Each empty droplet or one containing multiple cells is discarded after ejection before it reaches the target surface of the substrate. Only microdroplets containing single cells are deposited on the substrate. The principle was published, for example, in the scientific article Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter (2013): Single-cell printer: automated, on demand, and label free; Journal of laboratory automation 18 (6). DOI: 10.1 177 / 2211068213497204. This process, as well as further developments of it, as described in the article Riba, J. Schoendube, S. Zimmermann, P. Koltay and R.Zengerle, "Single-cell dispensing and 'real-time' cell classification using convolutional neural networks for higher efficiency in single-cell cloning," published in 2020 (doi.org / 10.1038 / s41598-020-57900-3), results in an insufficient and unpredictable print rate that only represents a usable statistical value as an average over a long period of time. The value is on the order of one cell per minute. Such systems are therefore more suitable for non-time-critical single-cell analyses, cloning, or cell therapy.

[0007] A third approach, which pipettes individual cells from a reservoir directly onto the substrate, is known from the article Jeffries, Gavin DM; Xu, Shijun; Lobovkina, Tatsiana; Kirejev, Vladimir; Tusseau, Florian; Gyllensten, Christoffer et al. (2020): 3D micro-organism printing of mammalian cells to generate biological tissues, Scientific reports 10 (1), DOI: 10.1038 / s41598-020-74191 -w). Here, too, each dispensed drop that does not contain an isolated cell is photodetected and aspirated again, resulting in the same limitations as in the case of the previously mentioned method.

[0008] The article by Schober, Lena; Büttner, Evy; Laske, Christopher; Traube, Andrea; Brode, Tobias; Traube, Andreas Florian; Bauern-hansl, Thomas (2015): Cell dispensing in low-volume range with the immediate drop-on-demand technology (l-DOT); Journal of laboratory automation 20 (2); DOI: 10.1177 / 2211068214562450, describes a method in which empty drops are dispensed into a cavity under optical monitoring until a cell is finally located underneath. The next cavity is then filled. The volume of accumulated liquid at the same deposition location is therefore variable, depends on chance, and far larger than the actual dispensing requires. The individual drop volumes are therefore significantly too large to place cells sufficiently close to one another, which prevents the necessary intercellular communication and connection. The process is therefore not well suited for 3D printing.

[0009] Another process is known as Laser Induced Forward Transfer (“LIFT”), see Schiele, Nathan R.; Corr, David T.; Huang, Yong; Raof, Nurazhani Abdul; Xie, Yubing; Chrisey, Douglas B. (2010): Laser-based direct-write techniques for cell printing; Biofabrication 2 (3). DOI: 10.1088 / 1758-5082 / 2 / 3 / 032001. This technique is based on the laser-induced ejection of individual cells from a cell-bearing gel layer that is open at the bottom. To detect cells in the carrier layer, which can be randomly isolated or suspended in a uniform grid, an optical method is used and the laser is positioned accordingly. This leads to a reduction in print rate. Furthermore, unwanted satellite droplets are created during drop ejection, resulting in unintentional splashes in the print image. Finally, there is no continuous renewal of the cell carriers in use, which makes the process unsuitable for practical use.

[0010] Yet another method is known from document CN 105238676 A, in which a curved channel is arranged in a microfluidic chip through which a cell suspension is passed. A cell is caught in a hook-shaped microbarrier, after which the channel is flushed with a cell-free buffer solution to ensure that no further cells remain in the channel. The inlet and outlet of the channel are then closed, the flow stops, and compressed air is introduced into the channel via a separate inlet, causing the cell to be flushed backwards out of the microbarrier and out of the channel through a lateral outlet line. The process takes 8 seconds. This makes it far too slow for the production of complex organs. In addition, the achievable droplet size of 10 nL is far too large to deposit cells close enough to one another for them to bond.

[0011] In addition, methods of so-called thermal drop-on-demand inkjet bioprinting, in which a gas bubble is thermally generated, are known, for example from UDDIN, J. et al.: Thermal Inkjet Printing: Prospects and Applications in the Development of Medicine; Technologies (2022) 10 (5) 108, printed pages 1-29; or from KUMAR, P. et al.: Inkjet printing of mammalian cells - Theory and applications; Bioprinting (2021) 23. e00157, printed pages 1-18.

[0012] Recreating the complexity of an organ and its microstructures precisely yet sufficiently quickly is a challenge that existing cell printing technologies cannot yet address for a number of reasons. In summary, none of the currently known processes combines the following properties, which are essential for 3D organ printing according to current knowledge:

[0013] 1) "Single-cell printing": the isolation of identical or different cells and their subsequent dispensing in a predefined grid onto a suitable substrate.

[0014] 2) Small dispensed microdroplets.

[0015] 3) "High print resolution" with a maximum of 10pm between adjacent printed cells.

[0016] 4) “Contamination-free” microdroplets, cells, and the final printed product.

[0017] 5) Print rates > 1000 Hz. The print rate of > 1000 Hz is based on previous experience with 3D-printed, homogeneous skin and the realization that sterility can only be guaranteed if the tissue is printed within 24 hours. This was achieved in preliminary clinical studies for additively manufactured, homogeneous skin without complex microstructures, such as sweat glands and hair follicles, because single-cell printing technology is not required for these. However, if one wants to produce large, complex organs such as a fully grown liver with a cell count of > 1 billion at a print rate of 1,000 cells / second using single-cell printing, the process would take more than 5 years with a single dispensing unit. Sterile and viable production is therefore out of the question. A much faster 3D bioprinting technology will be required for this.

[0018] The invention is therefore based on the object of providing a method and a device for dispensing isolated biological cells, which enables the printing of complex cell structures in a short time.

[0019] The object is achieved by a method according to claim 1 and a device according to claim 11. The method according to the invention for dispensing isolated biological cells comprises the following steps:

[0020] - Providing a cell suspension consisting of a carrier fluid and biological cells,

[0021] - Providing a dispensing device with a carrier in which a channel for passing the cell suspension is formed, wherein the channel extends in a longitudinal direction L and has a circumferential channel wall with at least one lateral opening with an extension D in the longitudinal direction, and wherein the channel has a cell trap adjacent to the opening,

[0022] - generating a flow of the cell suspension in the channel,

[0023] - capturing a single cell with a diameter dz D from the flowing cell suspension in the cell trap in such a way that the location of the captured cell, relative to the longitudinal direction L, is at the same height as the lateral opening,

[0024] - generating a pressure surge in the flowing cell suspension within the channel in the vicinity of the lateral opening, which results in a lateral force acting on the trapped cell in the direction of the opening, whereby the cell is ejected from the channel through the lateral opening, overcoming the surface tension.

[0025] The device according to the invention for dispensing isolated biological cells from a cell suspension comprises: - a carrier in which a channel is formed for passing the cell suspension, wherein the channel extends in a longitudinal direction L and has a circumferential channel wall with at least one lateral opening with an extension D in the longitudinal direction L,

[0026] - a cell trap in the channel adjacent to the lateral opening, which is designed to trap a single cell with a diameter dz D such that the location of the trapped cell, relative to the longitudinal direction L, is at the same level as the lateral opening,

[0027] - and means for generating a pressure surge in the flowing cell suspension within the channel in the vicinity of the lateral opening, which results in a lateral force acting in the direction of the opening on a cell trapped in the cell trap, which force is sufficient to eject the cell from the channel through the lateral opening while overcoming the surface tension.

[0028] The invention is based on the finding that, in order to achieve sufficient pressure rates, cells must be available to the dispensing device at an adequately high rate. To this end, the invention provides for an uninterrupted flow of the cell suspension in the channel. Furthermore, the additive construction of microstructures in complex organ tissues, which can be achieved, for example, based on a digitally stored 3D cell atlas of the organ, requires that individual cells of different cell types also be made available, while the requirement for a high pressure rate must not be compromised. This requirement is also referred to hereinafter as "cell-on-demand."

[0029] The present invention precisely fulfills these requirements by ensuring, prior to droplet dispensing, that individual cells from a continuously flowing cell suspension are held at a spatially defined location—namely, at the same height as the lateral opening—for the next dispensing process by means of a cell trap. The only decisive factor for the frequency of sequential printing processes is then that the cell traps are repopulated at a sufficient rate. This repopulating rate, in turn, depends on the capture efficiency of the cell trap, the flow velocity, the cell suspension, and the cell concentration in the carrier fluid. These parameters can be adjusted and optimized relatively easily.

[0030] The term "lateral" means perpendicular to the longitudinal direction L of the channel and indicates that the opening is arranged in the channel wall. The channel wall encloses the channel on all sides and, for example, in a rectangular channel, is formed from a bottom wall, two side walls, and a top wall. The channel is preferably formed in a carrier in the form of a groove that is closed off with a lid on its open side. Both the carrier material and the lid material preferably contain silicon, glass, or a temperature-stable polymer. Such arrangements are known as microfluidic chips. In addition to the dispensing device, other functional fluidic structures can also be arranged on or in such a carrier or chip.

[0031] The channel typically has lateral dimensions of 20 pm to 500 pm, preferably 50 pm to 250 pm. The channel preferably has a rectangular cross-section with an aspect ratio of channel width to channel depth >1. The channel width is then preferably 25 pm to 500 pm and particularly preferably 50 pm to 100 pm. The channel depth is preferably 20 pm to 100 pm. If the channel is in the form of a groove, the groove width in the direction of the support plane has the larger lateral dimension than the groove depth perpendicular to the support plane. The lateral opening is then preferably located in the bottom wall of the groove. The channel or groove depth is advantageously dimensioned such that no two cells are located one above the other in this direction in the channel. This almost completely rules out the accidental compression of two cells by means of a dispensing process and also achieves a high capture probability.

[0032] The lateral opening represents the mouth of an ejection channel into the channel for passing the cell suspension. The ejection channel extends laterally relative to the longitudinal direction L, but not necessarily perpendicular to it. The opening can be circular or have another shape, such as a slit or an oblong hole. The decisive factor for the positioning of the cell trap relative to the opening is always its longitudinal extension.

[0033] A "cell trap" is formed by a barrier designed to prevent a cell from the cell suspension from flowing further with the rest of the cell suspension. The unique feature of the cell trap is that it is designed to capture "a single cell," meaning exactly one cell, and allow further cells in the remaining suspension to pass through as long as the cell trap is occupied by that single cell. This ensures the defined dispensing of individual cells without "misprints."

[0034] "In proximity" means in close proximity spatially. A single cell is trapped in the cell trap adjacent to the lateral opening if its location, longitudinally speaking, is at the same height as the lateral opening. "At the same height" does not refer to a vertical direction, but always to the longitudinal direction of the channel. "Trapping" is understood to mean the (temporary) retention or deceleration of a cell from the cell suspension, which, as a result of the trapping, does not leave its location at the same height as the opening, while the remaining cell suspension continues to flow relative to it in the channel. The extent of the location, longitudinally speaking, therefore corresponds to the extent of the lateral opening.In other words, the arrangement according to the invention provides that the projection of the trapped cell with the diameter dz perpendicular to the channel wall and in the direction of the lateral opening is always within the extent D of the opening in relation to the longitudinal direction. Preferably, the extent of the lateral opening in the longitudinal direction in relation to the diameter of the trapped cell is D > 1.5 ■ dz. This ensures that the cell can be ejected from the channel in a straight line and over a short distance without being exposed to major shear. Preferably, the extent D of the opening in relation to the longitudinal direction is therefore large enough for a cell to pass through and small enough to form a stable interface with the environment due to the surface tension of the cell suspension and to keep the drop size sufficiently small so that the cells are close enough to one another after dispensing to be able to communicate.

[0035] The dispensing device according to the invention preferably has a plurality of lateral openings and associated cell traps of the type described above. This allows not only rapid sequential printing, but also parallel, simultaneous, or temporally overlapping printing at the locations of the multiple openings. A plurality of openings can be provided in one channel.

[0036] Furthermore, the dispensing device according to the invention preferably has a plurality of such channels. A plurality of openings can then be distributed across the various channels. If multiple channels, each with multiple openings, are provided, an array of dispensing nozzles can be formed. In this way, the printing speed of the dispensing device can be multiplied by parallel dispensing processes. A dispensing device with such an array of multiple channels, each with multiple openings, is then preferably developed into a print head, wherein the print head can further comprise one or more of the following features: a reservoir for the cell suspension, an inlet and outlet from and to the reservoir, a distribution structure to the multiple channels, electrical interfaces, a structure or apparatus for mixing the suspension to prevent the cells from sinking, or sensors for monitoring the individual cell traps.

[0037] Where reference is made here to a substrate onto which the cells are deposited by dispensing, this means, for example, an already existing layer of cells or a prefabricated, biocompatible scaffold.

[0038] The method and device according to the invention can also be used for other applications in which individual biological cells or microparticles must be rapidly and precisely placed in extremely small volumes of liquid. Examples include the filling of microtiter plates with individual cells or a defined number of cells surrounded by microdroplets with a very small or precisely determinable volume of liquid.

[0039] The pressure pulse is generated in the cell suspension within the channel in the vicinity of the lateral opening. Therefore, the means for generating the pressure pulse do not necessarily have to be located within the channel itself. What is crucial is the direction in which the pressure pulse acts on the trapped cell and the intensity with which this occurs—namely, the lateral force acting on the trapped cell that is sufficient to eject the cell from the channel through the lateral opening, overcoming the surface tension of the cell suspension.

[0040] The pressure surge is therefore preferably generated on the channel side opposite the opening and particularly preferably at the same height as the lateral opening in the longitudinal direction.

[0041] Accordingly, the means for generating the pressure surge are preferably located on the channel side opposite the opening. Particularly preferably, the means for generating the pressure surge are located at the same height as the lateral opening in the longitudinal direction.

[0042] It is important to achieve the smallest possible distance between the means for generating the pressure surge and the captured cell as well as from the lateral opening in order to eject the cell from the channel as efficiently, quickly, straightly and over the shortest possible path and thus to further minimize the drop volume of the carrier liquid ejected together with the cell.

[0043] In the case of a rectangular channel with an aspect ratio of >1, the distance between the means for generating the pressure surge and the lateral opening is preferably equal to the channel depth. The distance between the means for generating the pressure surge and the trapped cell is preferably 5 pm to 100 pm, more preferably 5 pm to 60 pm, and most preferably 5 pm to 20 pm. A minimum distance of approximately 5 pm is required to form a liquid buffer between the means for generating the pressure surge and the trapped cell, which protects the cells from damage. The maximum distance is limited to 100 pm or less in order to transfer sufficient momentum for the ejection to the cell with the least possible work.

[0044] The lateral opening is preferably designed such that the location of the captured cell is as close as possible to the liquid meniscus of the suspension in the lateral opening; more preferably, the distance between the location of the captured cell and the liquid meniscus is > 0 pm and not more than 10 pm. The position of the meniscus can be adjusted in a manner known per se by the geometry of the opening or the ejection channel, the wettability of the surface of the carrier material in the region of the opening or in the ejection channel, and as a function of the pressure difference between the suspension and the environment outside the carrier into which the opening or the ejection channel opens. In the case of a relative overpressure in the suspension compared to the environment for the purpose of transporting the same, it can be provided that the meniscus migrates, for example, to the outlet of the ejection channel.At a relative negative pressure, the suspension also serves the purpose of transporting the cell, moving to the inlet, i.e., the lateral opening, and thus directly to the location of the trapped cell. At a negative pressure, the suspension is sucked through the channel, while at a positive pressure, it is pushed.

[0045] The ejection channel should preferably be no longer than one to one and a half times the cell diameter, and most preferably not exceed 30 μm. This value ensures that the volume in the ejection channel is as small as possible to minimize the droplet volume of carrier fluid ejected along with the cell.

[0046] Furthermore, the pressure surge is preferably generated by briefly heating the carrier liquid locally, causing a gas bubble to expand in the cell suspension. This process is essentially known from printing technology as bubble jet printing. However, it was by no means obvious to transfer this technology to cell printing, as heat development is generally considered harmful when handling cell material. On the one hand, the aforementioned minimum distance of 5 pm and thus the liquid buffer counteracts damage. On the other hand, the inventors have recognized that the brief formation of the gas bubble on the channel side opposite the opening does not result in a significant increase in the temperature of the carrier liquid at the cell surface. Brief is preferably understood to mean a heating pulse duration of 2 to 50 ps, ​​particularly preferably 20 to 35 ps.During this period, an energy input into the suspension in the order of magnitude of 1 to a few 10 ■ 10' occurs. 5J. Even during ejection, the cell-containing droplet cools rapidly (<500 ps) to the temperature of the ambient air. The high surface-to-volume ratio of the droplet, as well as heat dissipation via evaporation, contribute significantly to this. As soon as the droplet lands on the substrate, it takes on the substrate temperature within seconds. The first heat shock phenomena in cellular metabolism, when heated to 43°C, only occur after 1.5 hours. With an expected maximum temperature increase of the droplet of 24 K, heat shock can be completely avoided by printing the suspension at an initial temperature of 19°C or by maintaining an elevated temperature for less than 1.5 hours. The above-mentioned processes take place in the range of seconds at most, so the latter condition is always met anyway. Short-term local heating can advantageously be achieved using resistance heating or a light pulse.The light pulse is preferably generated by a laser, which is at least partially absorbed directly by the suspension or by an absorber layer arranged along the channel wall, or by both. Accordingly, the means for generating the pressure pulse preferably comprise either a resistance heater or a laser and, optionally, an absorber layer, with the resistance heater or the absorber layer preferably being arranged on the channel side opposite the opening.

[0047] In an alternative embodiment, the pressure surge is generated by means of a piezo actuator. The piezo actuator can be integrated directly into the channel wall, preferably on the channel side opposite the opening, and generate a pressure surge directly in the channel. In an alternative configuration, a pressure chamber can be provided which provides a larger surface area into which the piezo actuator is integrated. The pressure chamber preferably opens into the channel on the channel side opposite the opening, with the outlet opening being smaller than the surface area occupied by the piezo actuator. The pressure chamber thus forms a concentrator which concentrates the pressure surge onto the outlet opening. The means for generating the pressure surge accordingly preferably comprise at least one piezo actuator and optionally a pressure chamber with an outlet opening.The term “piezo actuator” also refers to a stack of several individual piezo actuators.

[0048] In an alternative embodiment, the pressure surge is generated by briefly introducing a pressurized medium into the channel. Accordingly, the means for generating the pressure surge preferably comprise a source of a pressurized medium, a line connecting the source to the channel, and a high-speed microvalve integrated into the line.

[0049] The capture of the cell from the cell suspension in the cell trap occurs by means of a physical, optical, electrical, acoustic, or chemical barrier or by means of a combination of several of these barriers.

[0050] The cell trap is accordingly preferably formed from a physical, optical, electrical, acoustic, or chemical barrier or from a combination of several of these barriers.

[0051] Particularly preferred are physical barriers that hold the cell in place in a contact-based and / or hydrodynamic manner. For example, the cell can be held in the channel by a structure that the trapped cell cannot overcome. The structure can in turn be designed such that it deflects the remaining suspension passing through. These are, for example, barriers that protrude into the channel. However, the structure can also be designed such that it does not deflect the remaining suspension passing through. These are, for example, depressions formed in the channel wall, each of which can accommodate exactly one cell. In a minimalist embodiment of the invention, the depressions could be formed by the opening or the ejection channel itself. In addition to these contact-based barriers, the cells can also be trapped hydrodynamically by a structure that creates suitable vortices in the channel.In particular, the contact-based physical barriers but also the hydrodynamic barriers can use the effect of gravity to support the process, such as sedimentation of the cells within the channel.

[0052] Alternatively, optical barriers are preferred, such as those used for laser trapping. Such traps are known per se from Bryan Lincoln, Stefan Schinkinger, Kort Travis, Falk Wottawah, Susanne Ebert, Frank Sauer, Jochen Guck; Reconfigurable microfluidic integration of a dual-beam laser trap with biomedical applications; Biomed Microdevices (2007) 9703-710 DOI 10.1007s10544-007-9079-x. In this type of trap, optically diverging laser beams are aligned across opposite side walls of the channel, with holding forces acting on dielectric objects in the electromagnetic field thus generated.

[0053] Acoustic barriers are also preferred. This type of cell trap is also known per se, for example, from Yang, Shujie; Tian, ​​Zhenhua; Wang, Zeyu; Rufo, Joseph; Li, Peng; Mai, John et al.: Harmonie acoustics for dynamic and selective particle manipulation (2022). Nat. Mater. DOI: 10.1038 / s41563-022-01210-8. Two wave fields are aligned at right angles to each other, thereby forming a "landscape" of pressure fluctuations consisting of oscillating high- and low-pressure areas. The low-pressure areas are the points where cells or particles accumulate. These areas are spatially so small that only one cell fits into such a "wave trough." Cells can thus be isolated and trapped, creating an acoustic cell trap.

[0054] For all cell trap types, the holding force must be sufficient to hold the cell at its location, at least temporarily, against the force of the flowing suspension without damaging the cell. This is counteracted by the interest in achieving the largest possible volume flow to ensure the fastest possible repopulation of the cell traps.

[0055] The cell suspension preferably contains cells of different sizes, with cell capture occurring selectively according to cell size. This allows different cells to be captured at a defined position in the channel using a suspension and dispensed onto a substrate in a targeted manner by appropriately controlling the means for generating a pressure pulse.

[0056] Generating a flow comprises passing the cell suspension through the channel, wherein at least a portion of the cell suspension is recirculated and passed through the channel again.

[0057] According to an advantageous embodiment of the method, the cell is ejected from the channel as a result of the pressure surge together with a portion of the carrier liquid, forming a droplet, wherein the volume of the droplet, including the cell, is not more than 100 pL, preferably not more than 70 pL, and particularly preferably not more than 40 pL, depending on the application. Furthermore, the volume of the droplet, including the cell, is not less than 1 fL, preferably not less than 100 fL, and particularly preferably not less than 2 pL, depending on the application. All of the aforementioned dimensions (distance of the means for generating the pressure surge from the trapped cell and from the lateral opening, distance between the location of the trapped cell and the liquid meniscus, longitudinal extent of the opening, and length of the ejection channel) contribute to achieving these values.

[0058] In organ printing, the cell diameter typically ranges from 5 pm to 20 pm. In these cases, in the order of 10 pL, it is ensured that the droplet diameter is no larger than twice the cell diameter. This ensures that the cells can be placed at a sufficiently close distance from one another, thus ensuring a sufficiently high printing resolution of a maximum of 10 pm. This, in turn, ensures that the intercellular communication necessary for cell communication is possible. However, if bacteria, for example, are dispensed using the method according to the invention, much smaller droplet volumes in the range of fL are desirable.

[0059] Again, in the case of organ pressure, the extension D of the opening in the longitudinal direction is preferably at least 5 pm, particularly preferably at least 7 pm and preferably at most 30 pm, particularly preferably at most 20 pm.

[0060] The device further advantageously comprises means for generating a flow of the cell suspension in the channel.

[0061] Further advantages and features of the invention are explained below by way of example with reference to the figures. Figure 1 shows a schematic side view of a first embodiment of a device in a first snapshot;

[0062] Figure 2 shows the schematic side view of the first embodiment in a second snapshot;

[0063] Figure 3 shows the schematic side view of the first embodiment in a third snapshot;

[0064] Figure 4 is a schematic plan view of a second embodiment of the device according to the invention;

[0065] Figure 5 is a schematic plan view of a third embodiment of the device according to the invention;

[0066] Figure 6 is a perspective view of the device according to the invention;

[0067] Figures 7a a sequence of a section of Figure 4 with six different snapshots to 7f and

[0068] Figure 8 is a schematic plan view of a fourth embodiment of the device according to the invention.

[0069] The device 10 according to the invention for dispensing isolated biological cells from a cell suspension, in its simplest embodiment as shown in Figure 1, comprises a carrier 12 in which a channel 14 is formed for passing the cell suspension, and a cover 13 with which the groove is closed. The channel extends in a longitudinal direction L and is circumferentially delimited by a channel wall 16. The term channel wall is used herein in the singular, regardless of its cross-sectional shape. If the channel, as shown here by way of example, is quadrangular and in particular rectangular, then the channel wall consists of a bottom wall, two side walls, and a top wall. Quadrangular channels can be produced comparatively easily by manufacturing the carrier 12 with a groove that is closed in a joining step by means of a cover 13.Alternatives can be, for example, eroded channel structures with any cross-sectional shape or drilled channel structures with a circular channel cross-section and a circular-cylindrical channel wall in a one-piece carrier. A lateral opening 18 with a diameter D is provided in the channel wall 16. The opening 18 forms the entrance to an ejection channel 19 extending transversely to the longitudinal direction L, which is also referred to as a nozzle. In the case shown, the ejection channel 19 is a cylindrical bore through the carrier material, which emerges from the carrier 12 at its underside and has a length equal to the thickness of the carrier 12 from the bottom wall 16 to its underside. Since the opening 18 is circular, the diameter D is identical to the extension D of the opening in the longitudinal direction L.Alternative nozzles preferably have a conical shape tapering outwards from the channel or a slot shape running in the circumferential direction relative to the channel wall 16 or more precisely in the bottom wall perpendicular to the longitudinal direction L.

[0070] A suspension flows in the channel, forming a flow that essentially coincides with the longitudinal direction L of the channel. The cell suspension contains a large number of cells 20, which move essentially in the longitudinal direction with the flow. This is illustrated by the flow direction arrows 21. The cells 20 have a diameter dz D. The opening is therefore large enough for a cell to pass through. On the other hand, the diameter D of the opening is small enough to retain the flowing cell suspension in the channel 16 due to its surface tension. Due to its surface tension, the cell suspension forms a meniscus in the ejection channel, as shown here. Depending on the wettability of the carrier material and the dimensions of the ejection channel 19, this meniscus can also be formed immediately adjacent to the opening 18 instead of the end of the ejection channel 19 remote from the channel.

[0071] Furthermore, a cell trap 22, here in the form of a physical, contact-based barrier, is provided in the channel 14 adjacent to the lateral opening 18. The cell trap 22 has a size and shape suitable for capturing exactly one cell 20 with a diameter dz adjacent to the lateral opening, i.e., preventing it from flowing further along with the suspension. The cell trap 22 is arranged relative to the opening 18 such that the location of the captured cell 20, relative to the longitudinal direction L, is at the same height as the lateral opening 18, so that the cell can be ejected from the channel 14 in a straight line through the opening 18.

[0072] All other cells 20 in the suspension no longer find space in the cell trap and are carried along by the flow in the channel as long as the cell trap 22 is occupied by the individual cell 20. This state is shown in the snapshot in Figure 1. Between the cell trap 22 and the channel wall 16 on the side of the lateral opening 18 there is a passage 23 for the carrier liquid of the cell suspension. The passage 23 is too narrow to allow a cell 20 to pass through. This creates a flow through the cell trap 22 or under the barrier that holds the cell 20 in the cell trap 22. Furthermore, the device 10 has means for generating a pressure surge in the flowing cell suspension in the form of a resistance heating element 24. The resistance heating element 24 itself is integrated into the peripheral wall, i.e.embedded in the carrier material, on the channel side opposite the lateral opening 18 and, with respect to the longitudinal direction L, also at the same height as the lateral opening 18. The resistance heater is set up to generate a short heating pulse with which the carrier liquid of the cell suspension is locally heated. As a result, a gas bubble 26 forms for a very short time on the channel side opposite the lateral opening 18, which generates a pressure surge in the flowing cell suspension, which results in a force acting in the direction of the opening 18 on the cell 20 trapped in the cell trap 22. This process step is shown in the snapshot in Figure 2. The pressure surge is dimensioned by controlling the heating current so that the force acting on the cell 20 is sufficient to overcome the surface tension and to force the cell through the lateral opening 18 orthe ejection channel 19 ejects the cell from the channel 14 at a preselected speed and deposits it on a substrate 30. The cell is ejected together with a portion of the carrier liquid to form a droplet 28. The shape and size of the channel 14, the lateral opening 18, and the ejection channel 19, as well as the position of the opening 18, the cell trap 22, and the resistance heating element 24, are preferably dimensioned such that the diameter of the droplet is no more than twice the diameter of the cell.

[0073] At the end of the ejection process, the droplet constricts and breaks off, allowing it to be deposited in a defined manner onto a substrate 30 together with the cell within it. Limiting the droplet diameter to a maximum of twice the cell diameter dz defines the printing resolution and ensures that the distance between the cells 20 deposited on the substrate 30 is sufficiently small to ensure intercellular communication between neighboring cells.

[0074] The second embodiment of the device according to the invention according to Figure 4 differs from the embodiment described above by a somewhat different shape of the cell traps 22. These are designed here in plan view as pocket-shaped barriers open on both sides with respect to the longitudinal direction L, wherein the upstream-facing opening is dimensioned such that the cell 20 to be dispensed fits therein and the downstream-facing opening is significantly smaller and prevents the cell from flowing further and at the same time forms a passage 23 for the carrier liquid of the cell suspension, the flow of which holds the cell 20 in the cell trap 22.

[0075] A further difference in this embodiment is that the channel 14 has a plurality of lateral openings 18 in the channel wall and a corresponding number of associated cell traps 22. Furthermore, Figure 4 shows an electrode 32 for connecting an electrical line on each side of the resistance heating element 24. This arrangement of resistance heating element 24 and electrodes 32 is shown only once to simplify the figure. In fact, such an arrangement is provided in the vicinity of each individual lateral opening. Using such a configuration, the ejection of trapped cells can occur at multiple locations simultaneously or with a time delay, with or without relative movement between the channel 14 and the substrate 30, in order to increase the printing speed. For this purpose, a print control is used to optimize the printing process, preferably further depending on the desired print image.

[0076] Finally, this device provides a return 34 of the suspension after passing through the channel section with the cell traps 22 and the openings 18. The return is only indicated schematically here. It can be designed in many different ways.

[0077] The flow velocity of the cell suspension in channel 14 can be increased by means of a control system up to a certain upper limit proportional to the decrease in cell density, so that the availability of trapped cells and thus the repopulation rate of the cell traps remains constant. Alternatively and / or additionally, the cell suspension in channel 14 or in the return line can be enriched with new cells. The concentration of cells remaining after n pressure pulses can be calculated from the initial "cell fill level" and, if a threshold is exceeded, automatically trigger an appropriate action (refilling or cartridge replacement) or signal a need for action.

[0078] The third embodiment of the device according to the invention shown in Figure 5 differs firstly in that the channel 14 is folded once here, so that it has two channel sections 36, 37 through which fluid flows in opposite directions. Consequently, the longitudinal direction L of the channel is reversed. The two channel sections 36, 37 are separated from each other by a separating web 38 of the carrier 12, but not completely. In the separating web, there are open pockets on both sides transverse to the longitudinal direction, which form a fluidic cross-connection between the two channel sections 36 and 37. These pockets form the cell traps 22. They have an opening adjacent to the first channel section 36 upstream, into which the cell 20 to be dispensed fits, and an opening adjacent to the second channel section downstream, which is significantly smaller and prevents the cell from flowing further into the second channel section 37.At the same time, the smaller opening again forms a passage 23 for the carrier liquid of the cell suspension. The cross connection initially generates a flow component in the channel 14 transverse to the longitudinal direction L, so that cells 20 are deflected towards the cell traps 22. If one of the cell traps 22 is occupied by a cell 20, the flow of the carrier liquid holds the cell 20 in the cell trap 22. At the same time, the cell 20 blocks this cell trap 22, so that subsequent cells flow past it essentially unhindered along the longitudinal direction L. This device also provides for a return 34 of the suspension into the first channel section 36 after passing through the second channel section 37.

[0079] Figure 6 once again shows a single cell trap 22 similar to that shown in Figure 4 in a perspective section of the channel 14. The channel 14 has a flat rectangular cross-section and is defined by a channel wall 16 comprising a bottom wall 16a, two side walls 16b and 16c, and a ceiling wall (not shown). The side walls show that the barrier forming the cell trap 22 extends across the entire depth of the channel. The channel has a depth that only allows a single-layer cell flow. Therefore, two cells cannot flow one above the other through the channel, and the cells must pass the occupied cell trap 22 laterally, which not only prevents double occupancy of each cell trap but also accidental double cell printing, since two cells cannot fit one above the other in the channel.

[0080] Figures 7a to 7f show a section of Figure 4 in a sequence with six different snapshots to illustrate the method according to the invention. In Figure 7a, a cell 20 flows in the longitudinal direction L towards an unoccupied cell trap 22, in the vicinity of which, here below, the opening 18 is located. Furthermore, in the vicinity of the lateral opening, here above, a resistance heating element 24 is located, as previously described. Figure 7b shows that a first cell 20c occupies the cell trap 22 and prevents the deposition of following second cells 20d by the first cell 20c spatially filling the trap and blocking the passage 23, thus preventing the flow through the cell trap 22. The following second cells 20d therefore typically flow past this cell trap 22 along the trajectory 21 shown in Figures 7b and 7c.Figure 7d shows the formation of a gas bubble 26 following a brief activation of the resistance heating element 24, which generates a pressure surge and expels the first cell 20c from the cell trap through the lateral opening 18. The cell trap 22 is then repopulated by a new first cell 20c, see Figures 7e and 7f.

[0081] Finally, Figure 8 shows a fourth embodiment of the device according to the invention, which is designed for dispensing cells 20a, 20b of different sizes. Size-selective dispensing is achieved by means of different lateral openings 18a and 18b with diameters D1 and D2 and cell traps 22a and 22b of different sizes, each of which is matched to the diameter d1 and d2 of the cells 20a and 20b to be dispensed: D1 > d1 and D2 > d2. The cell traps are again each designed as pocket-shaped barriers open on both sides. The upstream-facing openings of the large cell traps 22a are designed to accommodate a large cell 20a with a diameter d1.The downstream-facing openings or passages 23a of the large cell traps 22a have a passage width w1, which is dimensioned to prevent the large cells from flowing further, while allowing the small cells with a diameter of d2 to pass through: d1 > w1 > d2. The upstream-facing openings of the small cell traps 22b are designed to accommodate only one small cell 20b. Large cells cannot find a hold therein. The downstream-facing openings or passages 23b of the small cell traps 22b have a passage width w2, which is dimensioned to prevent the small cells 20b from flowing further: d2 > w2.

[0082] List of reference symbols

[0083] 10 Dispensing device

[0084] 12 carriers

[0085] 13 lids

[0086] 14 channel

[0087] 16 Canal wall

[0088] 16a floor wall

[0089] 16b side wall

[0090] 16c side wall

[0091] 18 Opening

[0092] 18a large opening

[0093] 18b small opening

[0094] 19 Ejection channel

[0095] 20 cells

[0096] 20a large cell

[0097] 20b small cell

[0098] 20c first cell

[0099] 20d second cell

[0100] 21 Flow direction arrow, trajectory

[0101] 22 Cell trap, barrier, bag

[0102] 22a large cell trap, barrier

[0103] 22b small cell trap, barrier

[0104] 23 Passage

[0105] 23a large passage

[0106] 23b small passage

[0107] 24 resistance heating element

[0108] 26 Gas bubble

[0109] 28 drops

[0110] 30 Substrat

[0111] 32 Contact

[0112] 34 Repatriation

[0113] 36 first canal section

[0114] 37 second canal section

[0115] 38 Separator d1 large cell diameter d2 small cell diameter D opening diameter

[0116] D1 large opening diameter

[0117] D2 small opening diameter

[0118] L Longitudinal direction of the channel w1 large passage width w2 small passage width

Claims

Patent claims 1 . A method for dispensing isolated biological cells (20), comprising: - Providing a cell suspension consisting of a carrier liquid and biological cells (20), - Providing a dispensing device (10) with a carrier (12) in which a channel (14) is formed for passing the cell suspension, wherein the channel (14) extends in a longitudinal direction (L) and has a circumferential channel wall (16) with at least one lateral opening (18) with an extension (D) in the longitudinal direction (L), and wherein the channel (14) has a cell trap (22) in the vicinity of the opening (18), - generating a flow of the cell suspension in the channel (14), - capturing a single cell (20) with a diameter dz D from the flowing cell suspension in the cell trap (22) in such a way that the location of the captured cell (20) in relation to the longitudinal direction (L) is at the same height as the lateral opening (18), - generating a pressure surge in the flowing cell suspension within the channel (14) in the vicinity of the lateral opening (18), which results in a force acting on the trapped cell (20) in the direction of the opening (18), whereby the cell (20) is ejected from the channel (14) through the lateral opening (18).

2. Method according to claim 1, characterized in that the pressure surge is generated on the channel side opposite the lateral opening (18), in particular at the same height as the lateral opening (18).

3. Method according to claim 1 or 2, characterized in that the pressure surge is generated by means of a brief local heating of the carrier liquid, whereby a gas bubble (26) expands in the cell suspension.

4. Method according to claim 3, characterized in that the short-term local heating is carried out by means of a resistance heater (24) or by means of a light pulse.

5. Method according to claim 4, characterized in that the light pulse is generated by means of a laser and is at least partially absorbed by the suspension or in an absorber layer arranged along the channel wall or in both.

6. Method according to claim 1 or 2, characterized in that that the pressure surge is generated by means of a piezo actuator.

7. Method according to one of the preceding claims, characterized in that the capture of the cell (20) from the cell suspension in the cell trap (22) takes place by means of a physical, optical, electrical, acoustic or chemical barrier or by means of a combination of several of these barriers.

8. Method according to one of the preceding claims, characterized in that the suspension contains cells (20) of different sizes and that the capture of the cell (20) is carried out selectively according to the cell size.

9. Method according to one of the preceding claims, characterized in that the generation of a flow comprises passing the cell suspension through the channel (14), wherein at least a part of the cell suspension is recirculated and passed through the channel (14) again.

10. Method according to one of the preceding claims, characterized in that the cell (20) is ejected from the channel (14) through the lateral opening (18) as a result of the pressure surge together with a part of the carrier liquid to form a drop (28), wherein the volume of the drop (28) is not more than 100 pL, preferably not more than 70 pL, particularly preferably not more than 40 pL and further preferably not less than 1 fL, preferably not less than 100 fL and particularly preferably not less than 2 pL.

11. Device (10) for dispensing isolated biological cells (20) from a cell suspension, comprising: - a carrier (12) in which a channel (14) is formed for passing the cell suspension, wherein the channel (14) extends in a longitudinal direction (L) and has a circumferential channel wall (16) with at least one lateral opening (18) with an extension (D) in the longitudinal direction (L), - a cell trap (22) in the channel (14) adjacent to the lateral opening (18), which is arranged to trap a single cell (20) with a diameter dz D of the shape that the location of the trapped cell (20) with respect to the longitudinal direction (L) is at the same height as the lateral opening (18), - and means for generating a pressure surge in the flowing cell suspension within the channel (14) in the vicinity of the lateral opening (18), which results in a force acting in the direction of the opening (18) on a cell (20) trapped in the cell trap which is sufficient to eject the cell (20) from the channel (14) through the lateral opening (18).

12. Device (10) according to claim 11, characterized in that the means for generating the pressure surge are arranged on the channel side opposite the lateral opening (18), in particular at the same height as the lateral opening (18) with respect to the longitudinal direction (L).

13. Device (10) according to claim 11 or 12, characterized in that the extent (D) of the opening in the longitudinal direction (L) is at least 5 pm, preferably at least 7 pm and at most 30 pm, preferably at most 20 pm.

14. Device (10) according to one of claims 12 or 13, characterized by means for generating a flow of the cell suspension in the channel (14).

15. Device (10) according to one of claims 12 to 14, characterized in that the cell trap (22) is formed from a physical, optical, electrical, acoustic or chemical barrier or from a combination of several of these barriers.

16. Device (10) for carrying out the method according to one of claims 1 to 10.

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