Microfluidic device and method for separating objects

Abstract

A microfluidic device for separating objects from a liquid suspension, the microfluidic device comprising: at least one fluid inlet and at least one fluid outlet; at least one fluidic channel fluidically connecting the at least one fluid inlet to the at least one fluid outlet; a plurality of passive fluid-dynamic trapping sites arranged along the at least one fluidic channel, each trapping site configured to trap a defined number of objects from a liquid suspension flowing along the at least one fluidic channel; and a plurality of nozzle channels, wherein each nozzle channel is in fluid communication with one of the trapping sites and fluidically connects the trapping site with an associated nozzle orifice. A force can be applied simultaneously to the trapped objects and to a liquid column in the nozzle channel, such that a liquid aliquot containing the defined number of objects is ejected from each nozzle orifice.

Description

Technical Field

[0001] This disclosure relates to microfluidic devices and methods for separating objects, such as single cells, particles, spheres, or organelles, suspended in a liquid suspension. In particular, this disclosure relates to microfluidic devices and methods that allow the separated objects to be transferred to a receiving portion in a parallel manner. Background Technology

[0002] Single-cell isolation, sorting, and processing are becoming increasingly important in the field of single-cell analysis. Several methods exist in the prior art for the disposal of microfluidic systems used for the isolation, sorting, and processing of cells, cell clusters, and particles. For example, [1] provides a broad overview and system classification of single-cell / particle processing. [2] presents a comprehensive review of existing techniques for microfluidic separation of cells / particles. Similar techniques can also be used for larger biological particles, such as spheroids. CA 2 805 909 A1 discloses a highly parallel solution for the culture of single spheroids by hanging drop method. [3] presents a method for the controlled assembly of spheroids from a cell suspension and their subsequent transfer to a microplate (MWP). [4] describes a miniaturized version of the hanging drop method and a printing technique for transferring single spheroids from a suspension to a receiving portion. [5] presents a method for single spheroid deposition specifically for 3D bioprinting applications. [6] discloses the use of micropore arrays for micropore trapping and DNA (deoxyribonucleic acid) damage analysis. [7] Discloses single-cell isolation using modular single-cell pipettes for RNA (ribonucleic acid) sequencing. [8] Teaches cell isolation using affinity methods.

[0003] [9] Discloses an inverted open microwell for cell capture, cell aggregate formation, and parallel recovery of live cells. The substrate includes microwells, wherein the upper end of the microwell is connected via a fluid channel, and the lower end of the microwell is open. Dielectrophoresis is used during the delivery of the cell suspension to control cell entry into the microwell and to force the formation of cell aggregates to ensure cell-cell contact and interaction. Cells are captured at the air-fluid interface at the bottom edge of the open microwell and analyzed by microscopy. After analysis, live cells are recovered from multiple microwells onto multiple wells of a standard microtiter plate by blowing air through the fluid channel.

[0004] A detailed analysis of existing techniques demonstrates that cells, particles, spheroids, or tumor-like bodies suspended in liquids can be separated using a variety of methods. These techniques are applicable in principle to all entities within these liquids, regardless of their size and nature.

[0005] For simplicity, the term "object" or "cell" will be used in the following text to refer to the object under consideration. The term "object" or "cell" should imply any kind of solid or soft object suspended in a liquid, regardless of its size (provided it can be handled using the microfluidic devices disclosed herein) or whether it has biological properties.

[0006] Although there are many available methods for processing single cells to place them in a controlled manner into the receiving unit, current technologies do have at least one or even several different drawbacks, such as low cell viability, limited throughput, low single-cell efficiency, and closed systems that do not allow the transfer of separated cells to other receiving units (e.g., to the MWP).

[0007] Another drawback of the available technologies is the need for expensive laboratory equipment to handle cells suspended in liquids, such as fluorescently activated cell sorting systems, pipetting robots, single-cell printers, and similar instruments.

[0008] Therefore, the improved method will help provide single-cell processing in terms of simple manual and automated operation, low cost, high cell viability, high throughput and high single-cell efficiency. Summary of the Invention

[0009] The purpose of this disclosure is to provide a microfluidic apparatus and method that allows for the efficient separation of objects such as cells, cell clusters, or particles and allows for the parallel transfer of the separated objects into a receiving portion such as the pores of a microplate.

[0010] This objective is achieved by a microfluidic device according to a first aspect of the invention and a method according to a second aspect of the invention.

[0011] Examples of this disclosure provide a microfluidic device comprising: at least one fluid inlet and at least one fluid outlet; at least one fluid channel connecting the at least one fluid inlet to the at least one fluid outlet; a plurality of passive microfluidic trapping sites arranged along the at least one fluid channel; and nozzle channels associated with each trapping site. The passive microfluidic trapping sites are configured to trap a defined number of objects from a liquid suspension flowing along the at least one fluid channel. Each nozzle channel in the nozzle channel is in fluid communication with an associated nozzle orifice.

[0012] The present disclosure provides a method for operating such a microfluidic device. The method includes enabling flow of a liquid suspension containing objects from at least one inlet through at least one fluid channel, whereby a capture site is occupied by a defined number of objects, and a liquid column is formed in a nozzle channel. The method further includes mounting the microfluidic device on a receiving plate comprising a plurality of receiving portions, such that each nozzle orifice engages with one of the receiving portions, and simultaneously applying force to the captured objects and the liquid column in the nozzle channel, such that an aliquot of liquid containing a defined number of objects is ejected from each nozzle orifice.

[0013] Therefore, the examples of this disclosure are based on the discovery that multiple passive microfluidic capture sites can be used to separate a limited number of objects, such as cells, cell clusters or particles like spheroids or organelles, in preparation for simultaneous transfer of the separated objects to various receiving sites. The use of microfluidic capture sites allows for the arrangement of a large number of capture sites in a microfluidic device. Because the microfluidic capture sites are configured to capture a limited number of objects, such as single cells, capture requires neither individual monitoring nor individual control. Because each capture site includes an associated nozzle channel, the captured object can be transferred to the respective receiving site through the nozzle channel. Due to the arrangement of the nozzle channels and the actuation perpendicular to the fluid channels, the captured object is discharged from the nozzle along with a small amount of liquid contained only in the nozzle. Therefore, this disclosure allows for the highly parallel separation of objects and the highly parallel transfer of objects to various receiving sites without any additional active intervention, sensing, or control mechanisms.

[0014] In the examples, the microfluidic device may include a plurality of fluid channels arranged parallel to each other, wherein a plurality of microfluidic capture sites having associated nozzle channels are arranged along each fluid channel, wherein the plurality of nozzle orifices are arranged in a two-dimensional array, which preferably corresponds to the arrangement of orifices in a microwell plate. The microfluidic device may be adapted to be placed on a microwell plate as a cover, or may include a retainer adapted to hold the microwell plate. Thus, the examples of this disclosure allow for the transfer of separated objects to a receiving portion of a microwell plate in a parallel and easy manner.

[0015] In the example, the microfluidic device includes a removable seal covering the nozzle orifice to prevent leakage of the liquid suspension during object separation. In the example, the fluid outlet leads to a waste reservoir formed within the microfluidic device, allowing excess liquid suspension to be contained by the microfluidic device.

[0016] In the example, the microfluidic device includes a microfluidic chip having opposing first and second main surfaces, wherein at least one fluid channel extends parallel to the first and second main surfaces, and wherein a nozzle channel extends perpendicular to the at least one fluid channel, and preferably, perpendicular to both the first and second main surfaces. In such an example, fluid acceleration substantially perpendicular to the at least one fluid channel connecting the respective capture sites and substantially parallel to the nozzle channel can be generated to eject free-flying droplets containing objects from each nozzle orifice.

[0017] In one example, the microfluidic device includes a force applicator configured to apply force to an object trapped in a passive microfluidic capture site and a liquid column in a nozzle channel to eject a liquid droplet containing the object through a nozzle orifice. In another example, the force applicator is configured to apply a force such that a liquid acceleration is generated in each nozzle channel in a direction parallel to the nozzle channel. In yet another example, the force applicator is configured to apply forces to the object and the liquid column in parallel, i.e., to simultaneously and at the same level to all capture sites and nozzle channels. In yet another example, the force applicator includes a centrifuge mechanism that causes a centrifugal force to be applied to the object and the liquid column in the nozzle channel to eject a droplet containing the object through a nozzle orifice. In yet another example, the force applicator includes a displaceable wall and an actuator disposed on the side of the nozzle channel opposite to the nozzle orifice, the actuator being configured to displace the displaceable wall to eject a droplet containing the object through the nozzle orifice. In the example, the force applicator includes a drive mechanism configured to apply acceleration or deceleration to the microfluidic device to generate inertial momentum on the object and the liquid column, thereby driving the liquid containing the object away from the nozzle orifice. Using such a force applicator, fluid acceleration in the liquid column within the nozzle channel can be generated simultaneously and at the same level, through which the corresponding droplet containing the object is ejected.

[0018] In the examples of this disclosure, a pipette tip is used to supply a liquid suspension to the inlet of at least one fluid channel. In the case of multiple fluid channels and associated inlets, each flow channel may be supplied with the same or different liquid suspensions by a single pipette tip. The flow of the liquid suspension through at least one fluid channel may be supported by the hydrostatic pressure of the liquid column inside the pipette tip or by an active drive mechanism, such as a liquid pump or the like. Attached Figure Description

[0019] Examples of this disclosure will be described using the accompanying drawings, in which:

[0020] Figure 1A and Figure 1B A schematic top view and cross-sectional view of the microfluidic device are shown;

[0021] Figure 2 It is a schematic cross-sectional view of a microfluidic device including a force applicator and a receiving plate;

[0022] Figure 3 This is a schematic diagram of a microfluidic device inserted into a centrifuge;

[0023] Figure 4 This is a schematic perspective view of an example of a microfluidic device including a microporous plate holder;

[0024] Figure 5 yes Figure 4 A schematic cross-sectional view showing details of the microfluidic device;

[0025] Figure 6 This is a three-dimensional view of an example of a microplate;

[0026] Figure 7 It has pipette tips and microplates. Figure 4 A schematic perspective view of an example of a microfluidic device;

[0027] Figure 8 yes Figure 7 A detailed, enlarged illustration;

[0028] Figure 9 This is a schematic cross-sectional view of an example of a capture site with an associated nozzle channel during cell capture; and

[0029] Figure 10 yes Figure 9 A schematic cross-sectional view of the capture site during cell transfer. Detailed Implementation

[0030] In the following description, examples of this disclosure will be described in detail using the accompanying drawings. It should be noted that one or more identical elements having the same function are provided with the same or similar reference numerals, and repeated descriptions of elements provided with the same or similar reference numerals are generally omitted. Therefore, the descriptions provided for elements with the same or similar reference numerals are interchangeable. In the following description, several details are set forth to provide a more complete explanation of the examples of this disclosure. However, it will be apparent to those skilled in the art that other examples may be implemented without these specific details. In other instances, well-known structures and apparatuses are shown in block diagram form rather than in detail in order to avoid obscuring the examples described herein. Furthermore, unless specifically stated otherwise, features of the different examples described herein can be combined with each other.

[0031] This disclosure addresses the problem of separating objects from a suspension and transferring them to various receiving portions, such as the wells of a microplate (MWP), such that a controlled number of objects, e.g., exactly one object, are placed in each receiving portion. The term "object" as used herein should imply any kind of solid or soft object suspended in a liquid, regardless of its size (provided it can be handled using the microfluidic device disclosed herein) or whether it is biological. Examples of "objects" are single cells, cell clusters (e.g., spheroids, organelles, and tumor-like structures), and particles. Examples relate to microfluidic devices that can be operated using conventional laboratory equipment, such as pipettes and laboratory centrifuges. Examples relate to methods for operating such devices.

[0032] Microfluidic devices can be manufactured as single-use articles using injection molding or similar low-cost manufacturing techniques to enable hygienic or even aseptic operation. The separation and transfer of objects to a receiving unit can be achieved in a highly parallel manner using microfluidic devices, enabling, for example, the separation and transfer of 96, 384, or 1536 objects at a time. Therefore, this disclosure improves cell handling in terms of cost and throughput, while providing cell viability and single-cell efficiency similar to currently available best methods. Thus, examples of this disclosure are particularly suitable for applications requiring high throughput and high cell viability for single-cell separation, such as monoclonal cell line development or in vitro diagnostic applications utilizing spheroids or tumor-like organisms.

[0033] As used herein, the term microfluidic device refers to a device comprising a structure suitable for handling liquid volumes ranging from picoliters to milliliters. The fluid structure of a microfluidic device may have dimensions suitable for handling such liquid volumes.

[0034] Figure 1A A schematic top view of an example of a microfluidic device including a substrate 10 in which a fluid structure is formed. Figure 1BA cross-sectional view is shown. The substrate 10 may be formed of a single layer or multiple layers. The substrate 10 may be formed of a polymeric material or any other suitable material. The substrate 10 may include one or more layers. The fluid structure includes a fluid inlet 12, a fluid outlet 14, and a fluid channel 16 fluidly connecting the fluid inlet 12 to the fluid outlet 14. A plurality of passive microfluidic trapping sites 20 are arranged along the fluid channel 16. Although a single fluid channel 16 and four trapping sites 20 are shown in Figure 1, it is self-evident that other examples may include a different number of fluid channels and trapping sites. The microfluidic device includes a plurality of nozzle channels 22, each nozzle channel 22 being associated with one of the trapping sites 20. More specifically, each nozzle channel 22 is arranged in fluid communication with the associated trapping site 20 such that an object, such as a single cell, trapped in the trapping site 20 is positioned at one end of the nozzle channel 22. In other words, each trapping site 20 includes a nozzle channel 22. Each nozzle channel 22 includes a nozzle orifice 24 at its other end.

[0035] The opposite upper and lower surfaces of substrate 10 can be referred to as the main surfaces of the microfluidic device because these surfaces are the largest surfaces of the microfluidic device. Fluid channels 16 extend in a direction parallel to the main surfaces. Nozzle channels 22 extend in a direction perpendicular to both fluid channels 16 and the main surfaces. Fluid channels 16 have protrusions 16a at the location of the capture point 20.

[0036] exist Figure 1A and Figure 1BIn the illustrated example, each passive microfluidic trapping site 20 includes an obstacle 30 located within a main flow path parallel to the fluid channel 16. Each obstacle 30 includes a concave surface representing a recess 32. The size of the recess 32 is adapted to the size of a predetermined number of objects to be trapped, such as the size of a single cell or the size of a cell cluster or a specific particle. The fluid channel 16 and the passive microfluidic trapping sites 20 are configured such that objects flowing along the channel are guided to each passive microfluidic trapping site 20 as long as the corresponding trapping site 20 is not occupied by the predetermined number of objects, and the main flow path is guided around the corresponding passive microfluidic trapping site when the trapping site is occupied by the predetermined number of objects. In the illustrated example, if the predetermined number of objects are arranged in the corresponding trapping site, the main flow path is guided around the obstacle via a protrusion 16a. In the example, the obstacle may have orifices, a mesh, or a mesh structure to block objects, but allow liquid to pass through when not occupied by the predetermined number of objects. In this scenario, the main fluid flow is directed toward the obstacle, and the object is delivered to the capture site more efficiently by hydrodynamic forces compared to the example shown in the accompanying figures. Therefore, this type of capture site is also referred to as a passive hydrodynamic capture site. Compared to general microfluidic capture sites that do not utilize hydrodynamic forces to guide the object to the capture site, hydrodynamic capture sites can, on average, result in faster capture site occupancy because the object is directed to the capture site. In particular, hydrodynamic capture sites can also operate in a space environment, whereas many microfluidic capture techniques rely on gravity to capture cells and therefore cannot function in space. In other examples, the capture site can be formed from any other type of passive microfluidic capture site described herein and any other type of passive microfluidic capture site known according to the art, which are adapted to capture a limited number of objects, such as single cells, at the appropriate location.

[0037] In this example, a cover may be disposed on the main surface of the substrate in which the fluid channel 16 is formed. The cover may include a location at the fluid inlet 12 or an opening forming the fluid inlet. In this example, a waste reservoir 40 may be formed in the substrate 10, such as… Figure 1B The microfluidic device may include a seal 50 that seals the nozzle orifice 24, as shown schematically in dashed lines. Figure 1B It is shown in dashed lines.

[0038] During operation, a liquid suspension containing the objects to be separated is supplied to inlet 12, for example, via a pipette tip. The liquid suspension flows along fluid channel 16. As the liquid suspension flows along the fluid channel, a predetermined number of objects are captured at each passive hydrodynamic capture point 20. Furthermore, a liquid column is formed in nozzle channel 22. Excess liquid suspension may flow into waste reservoir 40. During this operation, nozzle orifice 24 may be sealed by seal 50. Additionally, leakage from nozzle orifice 24 is prevented by the surface tension at the lower end of nozzle channel 22, i.e., by the meniscus formed at nozzle orifice 24.

[0039] The microfluidic device can be placed on the receiving plate, such as a microporous plate, after or before the above capture process. If a seal 50 is provided, it is removed before placing the microfluidic device on the receiving plate.

[0040] Figure 2 A microfluidic device is shown mounted on a receiving plate 60, which includes a receiving portion 62 such that a nozzle orifice 24 mates with the receiving portion 62. It is not necessary for a single nozzle to mate with a single receiving portion. In the example, two or more nozzles can be arranged such that two or more nozzles mate with the same receiving portion to deliver two or more objects to that receiving portion. In such an example, the number of nozzles can be greater than the number of receiving portions. Figure 2 A force applicator 70 is also shown. In the illustrated example, the force applicator 70 includes a displaceable wall 72 disposed on the surface of a substrate 10 in which a fluid channel 16 is formed. The force applicator 70 also includes a pressure chamber 74 and a pressure generator 76 in fluid communication with the pressure chamber 74 to generate overpressure in the pressure chamber 74. The generation of overpressure causes a portion of the displaceable wall 72 to displace into a fluid structure formed on the surface of the substrate 10 on which the displaceable wall 72 is formed. Thus, forces are simultaneously applied to the liquid column in the nozzle channel 22 and to the object trapped in the trapping point 20. As a result, liquid acceleration is generated parallel to the nozzle channel, and liquid droplets containing the object are ejected from the nozzle orifice 24 into the receiving portion 62. Thus, the object is ejected into the receiving portion in parallel.

[0041] In other examples, the force applicator includes a centrifuge used to rotate the microfluidic device and the receiving plate, such that the acceleration of the liquid in the nozzle channel is caused by centrifugal force. Figure 3A centrifuge 80 is shown, comprising a rotor 82 supported by a shaft 84. The shaft 84 can be rotated by a motor 86, allowing the rotor 82 to rotate about a rotation axis 88. Further schematically shown is an assembly of a microfluidic device 90 mounted on top of a receiving plate 60. The microfluidic device 90 is an example of any microfluidic device described herein. This assembly can be rotated by a oscillating device (in... Figure 3 (Schematably shown via a swing axis 92) is connected to the rotor, such that as the rotor 82 rotates, the assembly can swing from a horizontally loaded orientation to... Figure 3 In the vertical orientation shown, centrifugal force acts parallel to the nozzle channel to propel droplets containing the object from the nozzle orifice into the receiving portion. In other examples, the component can be inserted into the rotor in a vertical orientation, eliminating the need for a oscillating mechanism.

[0042] Examples of this disclosure are based on microfluidic capture techniques for cells, in which the captured object is subsequently ejected through adjacent nozzle channels. Examples of microfluidic cell capture are described in [2] and US 10,351,894 B2, which provide examples of specific capture techniques. Passive microfluidic capture utilizes a microfluidic structure that generates a flow profile that supplies cells suspended in a liquid to a location in the channel called a “capture site.” Once a capture site is occupied by a specific object, such as a cell or cell cluster, the flow field in passive capture is altered so that other objects cannot occupy the same capture site. In active capture, once a capture site has been occupied, the flow field is actively altered by an external mechanism controlled by an operator or an automated detection system. Thus, the main difference between active and passive capture is that active capture requires additional technical means and energy in addition to the liquid flow supplying the captured object, while passive microfluidic capture does not. In both cases, other objects will not be directed to the same capture site. Therefore, each capture site is occupied by only a single object, such as a single cell. This fundamental principle can be achieved through many different microfluidic designs and devices. Therefore, microfluidic cell capture is a well-known technique according to existing technology. The examples disclosed herein utilize passive microfluidic cell capture to separate cells or cell clusters from a liquid suspension.

[0043] A drawback of some currently available microfluidic capture devices is that the separated objects cannot be transferred to separate receiving units, such as MWPs, which are not part of the microfluidic device. Therefore, cells are typically analyzed inside the device and / or released as a large suspension for collection as a cell aggregate outside the device. This disclosure addresses the problem of simultaneously transferring captured objects from a microfluidic capture device with multiple capture sites to multiple external receiving units while maintaining cell separation, such that a limited number of objects, e.g., exactly one object, are transferred from each capture site of the microfluidic capture device to each receiving unit.

[0044] To achieve this performance, a microfluidic (capture) device, which may be a microfluidic chip according to this disclosure, may have an opening associated with each passive hydrodynamic capture site, referred herein as a nozzle channel and nozzle orifice, which is open or can be open to the environment and is large enough to allow the captured object to pass through. The fluid design of the passive hydrodynamic capture site may be of any type as known according to the prior art or developed in the future. For example, for an overview of examples, refer to [2].

[0045] Passive microfluidic trapping sites are characterized by the absence of external, energy-consuming actuation mechanisms (such as dielectrophoresis (DEP) or any other external means like additional hydrodynamic pressure, mechanical force, light, etc.) to force objects into the trapping site and / or control their entry into the trap. In any case, passive microfluidic trapping sites rely on forces present in the trapping chip or environment. For example, gravity is often used to trap cells in micropores. As cells flow through pores of appropriate size, gravity pulls the cells into the pores, for example, see [2]. Figure 10Global external forces acting throughout the microfluidic device, such as gravity, centrifugal force, electricity, or similar external forces acting on an object anywhere within the microfluidic device, can support passive capture. If the diameter and length of the nozzle are approximately equal to, or at least not twice the diameter of, the object to be captured, only one object can enter the nozzle, and the nozzle itself can serve as the capture site. Alternatively, surface forces exerted by biomolecules immobilized at the capture site can be used to capture cells by chemical interactions between immobilized ligands and targeted receptors on the cell surface (referred to as “affinity capture”), see, for example, Figure 1 in [8]. A very common approach to passive capture is to utilize energy provided by the flow, which is used to transport the object along the fluid channel and also to force the object into the capture site, see again [2]. As described above, this approach is referred to as hydrodynamic capture. This approach requires neither additional external energy nor environmental forces such as gravity. Fluid dynamic capture works solely through a specific fluid dynamic flow field—designed by microfluidic structures that form fluid channels and capture sites—that guides objects into the trap and changes significantly when the trap is occupied.

[0046] In the example, the passive microfluidic trapping site is a passive hydrodynamic trapping site or trap, which is a special case of a passive microfluidic trap that relies on a hydrodynamic force applied by the transport flow to effectively guide an object into the trap. The hydrodynamic trapping force is generated by shaping the flow field in a specific way such that the main flow path is directed toward the trapping site as long as the trapping site is not occupied by an object or a limited number of objects. Once an object has been trapped, the flow field changes due to the presence of the object, which creates additional resistance to the flow when the object is fixed at the trapping site. Due to this additional resistance, the flow field changes, and the main flow path is now directed around the trapping site while still maintaining a certain force on the trapped object to keep it at the trapping site. Various different examples of such passive hydrodynamic traps are known according to the prior art, see, for example, [2]. Figure 5 , Figure 8 and Figure 9 In the example, the capture site in this disclosure can be formed by a passive hydrodynamic trap known in the art. The change in the flow field before and after the capture of the object is caused by channel walls and barriers of a certain geometry, which result in the characteristic effects of: a) the flow field preferably guiding the object to the capture site; and b) in most cases, once the capture site is occupied, the flow field will change to guide the flow around the occupied capture site and toward the next empty trap.

[0047] In this example, at least some of the passive microfluidic trapping sites can be configured to function according to the micropore trapping principle. In such an example, a nozzle channel can be used as a trapping site without any additional obstructions. The nozzle channel can include a cross-sectional dimension (e.g., diameter) and length larger than the size of the object to be trapped but less than twice the size of the object. In operation, the object can be driven into the nozzle channel by gravity. In such an example, [the following can be omitted] Figure 1A , Figure 1B and Figure 2 Obstacle 30 is shown in the diagram.

[0048] In the example, the flow of the liquid suspension through the fluid channel is caused by the driving pressure acting on the liquid suspension supplied to the inlet. When the microfluidic capture device is loaded with an object, i.e., the object is separated at the corresponding capture site, the driving pressure for the flow of the liquid suspension through the fluid channel at the capture site must not exceed the capillary pressure of the nozzle channel if the nozzle orifice is not closed. Otherwise, liquid may leak from the nozzle orifice. This can be prevented by using a seal to cover the nozzle orifice to prevent liquid from flowing through the nozzle channel during the capture process. Therefore, capture can be achieved using both open and sealed nozzle orifices, depending on the magnitude of the driving pressure relative to the capillary pressure in the nozzle channel.

[0049] In the example, the driving pressure can be established by hydrostatic pressure, slow centrifugation, or any other suitable method. Once the capture process is complete and each capture site is occupied by an object or a predetermined number of objects, the flow stops, the seals on the nozzle orifices (if present) are removed, and the microfluidic capture device is placed on the receiving section such that each receiving section mates with a predetermined number of nozzle orifices, for example, each receiving section mates with one nozzle orifice.

[0050] To transfer an object into the receiving portion, a force causing liquid acceleration can be applied substantially perpendicular to, i.e., substantially parallel to, the nozzle channel connecting the various capture sites, to propel liquid from each nozzle orifice containing the object into the corresponding receiving portion. In the example, the actuation force can be established by centrifugation of the microfluidic capture device located on top of the receiving portion, such that the centrifugal force is directed from the nozzle orifice towards the receiving portion. This is particularly advantageous when the object will be transferred into the MWP, as in this case, existing MWP centrifuges can be used, which are readily available in most laboratories. Alternatively, the force for accelerating the liquid inside the nozzle can be applied by mechanical displacement of the outer surface of the microfluidic capture chip. Alternatively, a drive device can be provided for generating the inertial momentum of the liquid inside the nozzle by rapid deceleration (such as that disclosed in DE 19 913 076 A1 for printing microarrays) or rapid acceleration or any other suitable means. For characteristics of a drive device configured to achieve such inertial momentum by acceleration or deceleration, refer to the teachings of DE 19 913 076 A1. Therefore, in the example, fluid displacement can be applied to the nozzle channel, or the microfluidic device can accelerate or decelerate along the direction of the nozzle channel to generate inertial forces on the liquid column and the object. The amount of liquid transferred along with the individual objects depends on the magnitude and duration of the actuation force, as well as other parameters such as the size of the nozzle channel and whether the inlet and / or outlet of the microfluidic capture chip are sealed when the actuation force is applied.

[0051] In other examples, the transfer of the object can be achieved in different ways. In one example, the aliquot sample is not ejected as a free-flying droplet. In another example, a pendant can be generated at the nozzle orifice, which, upon reaching a certain size, contacts the bottom of the associated receiver, transferring a portion of the liquid and the object to the receiver. In yet another example, the nozzle orifice can contact another liquid, allowing the object to be ejected directly into that liquid without forming droplets.

[0052] This disclosure does not rely on a particular type and design of passive microfluidic trapping mechanism, nor on a particular actuation mechanism to first separate the object and then eject it from the nozzle orifice. This disclosure can work with any kind of corresponding microfluidic trapping mechanism and any kind of actuation mechanism capable of ejecting liquid from a nozzle. Without losing sight of the general concept, other examples of apparatus and methods according to this disclosure are described below for clarity only. Preferred examples may have particularly advantageous features for transferring single cells into each orifice of the MWP.

[0053] like Figure 4As shown, an example of the microfluidic device of this disclosure may include a flat rectangular microfluidic chip 100. The microfluidic chip 100 may have a MWP size, for example, approximately 80 mm × 120 mm, and a thickness of approximately 1 mm to 4 mm. The larger surfaces will be referred to as the main surfaces of the chip, and specifically as the “top” side and “bottom” side of the chip. The chip 100 may be made of a transparent plastic material. In the example shown, the chip 100 may be supported by a support member 102. The support member 102 and the chip 100 may be formed as a single piece. The support member 102 may be configured as a holder for the MWP, wherein an example of the MWP 104 is as follows: Figure 6 As shown in the diagram. More specifically, the support 102 includes a cavity 106 located below the chip 100, the cavity 106 being configured to receive the MWP 104. Features such as a tilting bend 108 may be provided to ensure that the MWP 104 is properly oriented within the cavity 106.

[0054] Chip 100 includes eight fluid channels 110 extending parallel to and parallel to the top and bottom sides of chip 100. The fluid channels 110 may be formed within chip 100 such that the top and bottom sides of the channels are closed. In an example, the fluid channels 110 may be formed in the top side of chip 100 and may be covered by a cap (not shown). It should be noted that in other examples, the number of fluid channels may be different. The number of fluid channels 110 may correspond to the number of receptacles in one direction of the MWP, wherein, in the example shown, MWP 104 includes an array of eight receptacles 112 in one direction multiplied by twelve receptacles 112 in another direction. Each fluid channel 110 connects a fluid inlet 114 to a fluid outlet 116, and thus represents a microfluidic connection channel between the corresponding inlet 114 and outlet 116. A plurality of capture sites 118 are arranged along each fluid channel 110. The number of capture sites 118 can correspond to the number of MWPs in the other direction, i.e., twelve in the example shown. Of course, the number of capture sites may be different in other examples.

[0055] Figure 5 A magnified view showing details of chip 100 is provided. Figure 5As shown, each capture site 118 may include a protrusion 110a of a corresponding fluid channel 110 and an obstruction 120 located along the main flow path of the corresponding fluid channel 110. A nozzle channel 122 is formed below each capture site 118. In other examples, the nozzle channel may form part of the capture site. The nozzle channel 122 extends substantially perpendicular to the fluid channel 110. This may include nozzle channels 122 extending precisely perpendicular (90 degrees) to the fluid channel 110 and nozzle channels 122 extending at an angle of 85 to 95 degrees relative to the fluid channel 110. The lower end of the nozzle channel 122 forms a nozzle orifice 124 in the bottom side portion of the chip 100. The chip 100 may be formed of more than one layer, such as two layers, wherein the fluid structure may be formed in different layers. For example, the fluid channel 110 may be constructed in the first layer, and the nozzle channel 122 may be constructed in the second layer. The obstruction 120 may be formed in or on the second layer.

[0056] See Figure 4 The inlet 114 and outlet 116 can be located on the top or bottom side of the chip, or at the “edge” of the chip, which is any side other than the top or bottom side. In the example, the inlet 114 is located on the top side of the chip 100 to facilitate the supply of a liquid suspension to the fluid channel 110. Eight inlets 114 spaced 9 mm apart may be present to accommodate pipette tips of an 8-channel manual pipette that can be used to fill the inlets 114. Figure 7 Eight pipette tips 130 of this manual pipette, arranged to fill inlet 114, are shown. The pipette tips 130 can be disposable. Figure 7 As shown, the inlet 114 can be sized such that when the chip 100 is positioned upright with the inlet 114 facing upwards, the pipette tip 130 can be inserted into the inlet 114 to create a fluid reservoir tightly connected to the chip and achieve a certain hydrostatic height. Figure 4 In this configuration, the MWP 104, having a receiving portion 112, is positioned within the cavity 106 of the support 102 such that the receiving portion 112 is aligned with the capture point 118. More specifically, each nozzle orifice 124 faces one of the receiving portions.

[0057] One of the microfluidic connection channels 110 leads from each inlet 114 to an outlet 116 located on the opposite side of the microfluidic chip 100. Along each microfluidic connection channel 110, a total of twelve capture sites 118 are arranged at 9 mm intervals to accommodate a standard 96-well MWP layout. At each capture site 118, one of the nozzle channels 122 branches from the fluid channel 110. Thus, an array of 8×12 capture sites is formed inside the chip. The nozzle channel 122 terminates at the bottom side of the chip 100, forming an array of 8×12 nozzle orifices at a 9 mm spacing. See also Figure 5 Depending on the object to be separated and transferred, the nozzle orifice 124 can have a circular shape and a diameter of 10 µm to 500 µm.

[0058] As mentioned above, there are various designs known from existing technologies for designing effective passive microfluidic trapping sites. For example... Figure 5 and Figure 6 In the sketch, for the example described herein, capture site 118 includes a concave capture structure 120.

[0059] To ensure that an object does not accidentally exit the nozzle channel 122 through the nozzle orifice 124 before the capture operation is complete, the nozzle orifice 124 can be sealed, for example, by a self-adhesive tape seal for a microplate or by pressing a rubber seal against the bottom surface. If the nozzle orifice is not sealed, the lower boundary of the nozzle channel (and therefore the trap) will be formed by a liquid meniscus spanning the nozzle orifice. This meniscus can block the object as long as the driving pressure at each capture point does not exceed the capillary pressure of the liquid meniscus inside the nozzle channel.

[0060] Now, let's explain based on the reference. Figures 4 to 7 The operation of the microfluidic device is described as an example.

[0061] like Figure 7 As shown, a liquid suspension containing the object to be separated, such as cells, cell clusters, or particles, is supplied to inlet 114, for example, via an 8-channel pipette. The disposable pipette tip 130 can be filled to a height of several millimeters or centimeters by aspiration using the pipette. The disposable tip is then snapped into the opening of inlet 114, and while the tip remains snapped in the inlet, the rest of the pipette is removed, such as... Figure 7 As shown.

[0062] The flow rate at which the liquid suspension is moved from the pipette tip 130 into the microfluidic chip 100 is provided by the hydrostatic pressure of the liquid column inside the pipette tip 130, which slowly drives the liquid suspension from the pipette tip 130 into the microfluidic chip 100. This causes the liquid to move along the microfluidic connection channel 110 and the capture site 118, such that each capture site 118 is occupied by a limited number of objects, such as a single object, over time.

[0063] After a certain period of time, the pipette tip 130 becomes empty, and then most of the liquid is discharged through the outlet, preferably into some kind of waste container. In this example, the waste container may be integrated into the microfluidic chip 100. If the supplied liquid suspension contains enough objects, the capture sites are simultaneously occupied by a single object.

[0064] Alternatively, after capturing an object, the microfluidic connection channel 110 can be flushed to remove any remaining object from the microfluidic connection channel 110, for example by adding some clearer liquid to the pipette tip.

[0065] If the nozzle orifice 124 is sealed before loading the liquid suspension, the seal is carefully removed after the capture operation and before the microfluidic device is placed on top of a receiving plate such as a MWP.

[0066] The microfluidic device is then mounted on top of the MWP. Mounting the microfluidic device on top of the MWP may include placing the MWP 104 within a cavity 106 of the support 102. In other examples, the microfluidic device may be mounted on the MWP as a cover. In the example shown, by design, 96 nozzle orifices 124 mate with individual orifices 112 of the MWP 104 such that each nozzle orifice 124 is positioned on top of its corresponding orifice 112 of the MWP 104.

[0067] Remove the pipette tip before or after placing the microfluidic device on top of the MWP. Figure 8 It shows Figure 7 The enlarged cross-section of the arrangement shows that the pipette tip 130 is still stuck in the inlet 114, and the microfluidic device has been placed on top of the MWP 104.

[0068] In the example, inlet 114 and / or outlet 116 may be carefully sealed by a belt, stopcock, or plug after the capture operation to prevent the fluid passage 110 from venting during another process.

[0069] Figure 9A schematic enlarged view of two capture sites in capture site 118 is shown, through which a corresponding object 150 is captured. The capture site 118 is configured to capture object 150 such that object 150 is positioned at the end of the nozzle channel, which is opposite to the nozzle orifice 124 or located inside the nozzle channel.

[0070] The microfluidic chip 100 and the MWP 104 assembly are then carefully placed into a centrifuge for the MWP. In this example, the microfluidic chip is designed to resemble a cover for a conventional MWP board and includes features to prevent the microfluidic chip from shifting or detaching from the edge of the MWP. The assembly is then centrifuged for a short time at a moderate frequency. The centrifuge can be a conventional centrifuge with a oscillating rotor that ensures the applied centrifugal force is always directed perpendicularly to the bottom of the microfluidic chip. Therefore, the driving force that removes the liquid column confined within the microfluidic nozzle channel from the nozzle orifice is always substantially parallel to the nozzle channel and substantially perpendicular to the microfluidic connection channel. Thus, a liquid volume 152 containing the object 150 is ejected from each nozzle orifice 124. The liquid volume can be ejected as free-flying droplets separating from the nozzle orifice, such as... Figure 10 As shown, it can also be transferred through physical contact with the receiving part or the liquid already contained therein without separation from the nozzle orifice. Figure 10 In the diagram, arrow 160 indicates that the driving force for spraying liquid 152 is substantially parallel to the nozzle channel 122. Liquid volume 152 represents the liquid aliquot sample sprayed from each nozzle orifice in the nozzle orifice.

[0071] After the object has been transferred to the MWP, the microfluidic chip can be discarded, and the MWP containing a single object, such as a single cell, can be further processed. For example, the transferred object can be analyzed while it is being transferred to the MWP.

[0072] Examples of this disclosure allow for the rapid and efficient separation of suspended objects, such as cells, including their subsequent highly parallel transfer to an external receiving portion, such as an orifice of a microfluidic capture chip. In these examples, expensive equipment for fluid control is unnecessary because either hydrostatic pressure provided by a filled pipette tip or centrifugal force provided by a conventional laboratory centrifuge can be used. The examples allow for the transfer of highly viable single cells encapsulated in free-flying droplets with low shear force and without subjecting living cells to an electric field. Therefore, examples of this disclosure allow for the non-contact transfer of single objects from a microfluidic capture chip to a receiving portion using simple, standard laboratory equipment, such as a centrifuge and pipettes.

[0073] In this example, it is not necessary to monitor whether a limited number of objects, such as individual cells, are captured, because the capture site is configured to capture only a limited number of objects. However, the example provides the possibility of analyzing the captured objects within the microfluidic chip for a considerable period of time, for example, by microscopy, which can provide additional advantages for analytical and / or regulatory compliance purposes. Therefore, the example allows for optional analysis and classification of the captured objects (e.g., by microscopic imaging) before transfer to the receiving section.

[0074] For regulatory compliance purposes, the example provides the possibility of observing the transfer of an object from the top side of the transparent microfluidic chip to the receiving section using a camera, high-speed camera, or other sensor. Thus, the example allows for observation of cells during ejection from the nozzle and transfer to the receiving section using a specific imaging system, for example, to demonstrate the monoclonal nature of a single cell.

[0075] The example provides a method for capturing objects within a nozzle channel that has no bottom but instead has a liquid meniscus to prevent the captured objects from being discharged. If different inlets are supplied with different object types, such as different cell types, the example allows multiple different objects to be transferred in parallel and simultaneously to multiple separate receiving sections in a single run. If the nozzle orifices are arranged in a pattern such that different objects supplied to different inlets are guided to the same receiving section, the example allows a controlled number of different objects to be assembled into a single receiving section.

[0076] Therefore, the examples provide highly parallel separation and transfer of a single object by a single use item, namely the microfluidic chip according to this disclosure. This enables hygienic or even sterile conditions that can be operated by conventional laboratory equipment such as pipettes and centrifuges, without requiring any additional / expensive equipment.

[0077] According to a particular aspect, this disclosure provides a microfluidic device having at least one inlet and one outlet, the inlet and outlet being fluidly connected via at least one microfluidic connection channel characterized by a plurality of passive microfluidic capture sites. Each capture site is capable of capturing a defined number of cells, particles, or objects from a liquid suspension flowing along the microfluidic connection channel without the intervention of an operator or automated detection system. Each capture site is fluidly connected to a microfluidic nozzle channel substantially perpendicular to the microfluidic connection channel and has an exposed nozzle orifice at its other end from which liquid can exit into a receiving portion. An example provides a method for operating a microfluidic device according to a particular aspect, the method comprising the steps of: supplying a liquid suspension containing cells, particles, or objects to at least one inlet; applying pressure or flow rate to the supplied liquid to move the liquid suspension along at least one microfluidic connecting channel; waiting until a specific number of cells, particles, or objects are occupied at the capture site (and optionally flushing the microfluidic channel with a clarified liquid to remove any remaining cells, particles, or objects through an outlet); positioning the microfluidic device on top of a receiving portion intended to receive separated cells, particles, or objects, such that a nozzle engages with the corresponding receiving portion; and applying a force substantially parallel to the nozzle channel to a liquid column within the microfluidic nozzle channel in a suitable manner to spray an aliquot of liquid containing the captured cells, particles, or objects.

[0078] Further improvements to the apparatus according to a particular aspect may include at least one of the following: a drive mechanism (hydrostatic, centrifugal, etc.) for realizing liquid flow through the microfluidic connection channel and / or droplet ejection; a specific design of the passive microfluidic capture site (according to one or more examples presented in the literature); and the spatial arrangement of the microfluidic connection channel, capture site, and nozzle for cooperating with the receiving part of the MWP.

[0079] In the example, according to the prior art, the microfluidic device includes multiple passive microfluidic capture sites. These multiple passive microfluidic capture sites do not necessarily have the same properties, nor do they need to be designed for objects of the same size. Therefore, the same microfluidic device can be used to separate different objects of different sizes. The spatial arrangement of the capture sites on the chip is not required and can be adapted to any arrangement of the receiving portion. Examples may include multiple microfluidic connection channels connecting multiple inlets and outlets. Examples may include multiple multifluidic connection channels connecting multiple inlets to one outlet or connecting one inlet to multiple outlets. Other examples may include a single inlet, a single outlet, and a single microfluidic channel connecting all capture sites. In the example, the nozzle channel is perpendicular to the microfluidic connection channel, and the capture sites are connected to a nozzle orifice from which objects can be ejected. In the example, the force used to simultaneously eject the captured objects is substantially parallel to the nozzle channel; that is, the main direction of the force is parallel to the longitudinal extension of the nozzle channel (perpendicular to the cross-section).

[0080] In the examples, the microfluidic device may have a size and shape corresponding to the cap of the microporous plate. In the examples, the microfluidic device may include 96, 384, or 1536 nozzle orifices arranged in an array corresponding to the arrangement of the orifices in the microporous plate. In the examples, the microfluidic device has at least one inlet on one side and nozzle orifices on the opposite side. In the examples, the microfluidic device has at least one inlet on one side and nozzle orifices on the same side. In the examples, the microfluidic device has at least one inlet on one side at the edge of the microfluidic device. In the examples, the microfluidic device has an integrated waste reservoir to capture excess liquid flowing from the outlet. In the examples, the microfluidic device is made of a transparent polymer material. In the examples, a receiving plate is securely attached to, or can be securely attached to, the microfluidic device to be formed on a single integrated unit. In the examples, the microfluidic device is arranged relative to the receiving plate such that more than one individual nozzle orifice mates with the receiving plate.

[0081] In the examples, the size of the capture site is suitable for capturing a limited number of objects, such as only one or two objects, in the range of 10 µm to 100 µm in size. In the examples, the objects are cells or cell clusters, such as spheroids. The term capture site size refers to the internal dimensions of the structure in which a limited number of particles are captured, such as the diameter and length of a nozzle channel or the dimensions of a capture recess in an obstacle. In the examples, the capture size can be larger than the size of the single object to be captured, but less than twice the size of that object. In the examples, the size of the capture site is in the range of 15 µm to 150 µm. In the examples, the inner diameter of the nozzle channel is in the range of 15 µm and 150 µm. In the examples, the aspect ratio (length / diameter) of the nozzle channel is in the range of 1:1 to 2:1.

[0082] The microfluidic device according to this disclosure includes passive microfluidic trapping sites rather than active trapping mechanisms. [9] discloses an active trapping mechanism using dielectrophoresis. The mechanism disclosed in [9] relies on active control of cells allowed to enter the trap and requires microscopy to confirm the presence of cells inside the trap. This device is not well-suited for practical applications because it is complex, slow, and requires operator control to trap cells. This makes it unsuitable for a large number of trapping sites. Passive microfluidic traps have proven to be substantially more advantageous for a large number of trapping sites and for the highly parallel separation and transfer of single objects, such as single cells. The operation of the active trap disclosed in [9] is based on a controllable barrier that prevents cells from entering the trap via DEP, and if the barrier is closed, cells are deposited into the trap by gravity, which also serves as a nozzle for later cell transfer. This active trapping principle is significantly different from the passive microfluidic traps used in this disclosure. If a fluid dynamic trap is already in use, the actuation principle (i.e., pneumatic actuation) described in [9] for initiating the transfer from the trap to the receiver will likely not be applicable to a large number of trap sites. Of course, for larger array sizes, such as 8×12 nozzles or larger, the pneumatic actuation principle will fail because the actuation pressure cannot be equally transmitted to all trap sites due to fluid resistance, capacitance, and impedance caused by the connecting channel and the passive microfluidic trap sites. The transmission of pressure from the inlet along the connecting channel and through the trap sites will result in a delay in the pressure pulse and a reduced pressure level at the farthest nozzle. Therefore, the pneumatic drive system disclosed in [9] cannot simultaneously and at the same pressure level supply all nozzles. Because the pressure supply is between the inlet and outlet of the connecting channel, nozzles closer to the outlet experience a reduced pressure level compared to nozzles closer to the inlet. Since all nozzles are supplied from the same inlet, there is significant crosstalk between all nozzles. In devices with a large number of nozzles (e.g., 12 or 24 nozzles along a channel), the fluid resistance and inductance of the connecting channel, as well as crosstalk between nozzles, will result in unequal volumes, different injection times, and the potential failure to inject liquid from nozzles that are too far from the inlet or in cases where one nozzle dries out. Therefore, it is not straightforward to replace active capture mechanisms with passive microfluidic capture and to extend the principles described in [9] to a larger number of nozzles. The only reason the device disclosed in [9] can work is because the number of nozzles is small and the connecting channel is relatively short. Therefore, a significant improvement of the examples of this disclosure is that an actuation method is also provided that can simultaneously inject droplets from the nozzles and has the same pressure for all nozzles, even for devices with a large number of nozzles.To transfer an object into the receiving portion according to this disclosure, a fluid acceleration (actuating force) is applied substantially perpendicular to, i.e. substantially parallel to, the nozzle channels connecting the respective capture sites, to eject droplets from each nozzle orifice containing the object into the corresponding receiving portion. In a preferred embodiment, acceleration can be established by centrifuging the microfluidic capture chip located on top of the receiving portion, such that the centrifugal force is directed from the nozzle towards the receiving portion. Such fluid acceleration (actuating force) can be provided to each nozzle channel in parallel (i.e., simultaneously and at the same level) using centrifugal force, using a displaceable wall capable of shifting towards the nozzle channels, or by generating inertial momentum of the liquid inside the nozzle through rapid deceleration. Centrifugal force is particularly advantageous when the object is transferred into the MWP, as in this case, existing MWP centrifuges can be used, which are readily available in most laboratories.

[0083] Although some aspects have already been described as features within the context of the device, it is clear that such descriptions can also be considered descriptions of the corresponding features of the method.

[0084] As can be seen in the preceding detailed descriptions, various features have been combined in the examples for the purpose of simplifying this disclosure. The approach of this disclosure should not be construed as reflecting an intention to require more features than are expressly stated in each claim. Rather, as reflected in the appended claims, the subject matter of the invention may lie in fewer than all the features of a single disclosed example. Therefore, the appended claims are incorporated herein by reference, wherein each claim may exist independently as a separate example. While each claim may exist independently as a separate example, it should be noted that although a dependent claim may refer in the claim to a specific combination of one or more other claims, other examples may also include combinations of dependent claims with the subject matter of each other dependent claim, or combinations of each feature with other dependent or independent claims. Such combinations are presented herein unless stated otherwise. Furthermore, even if a claim is not directly dependent on an independent claim, it is intended to include the features of that claim in any other independent claim.

[0085] The examples described above are for illustrative purposes only. It should be understood that modifications and variations of the arrangements and details described herein will be apparent to those skilled in the art. Therefore, it is intended only to limit the scope of the pending patent claims, and not to the specific details presented in the manner of description and interpretation of the examples herein.

[0086] literature

[0087] [1]J. Riba, S. Zimmermann, P. Koltay, Technologies for AutomatedSingle Cell Isolation In: Handbook of Single Cell Technologies 2018, SpringerNature, T. S. Santra, F.-G. Tseng, T. S. Santra, F.-G. Tseng, ISBN: 978-981-10-4857-9

[0088] [2]Luan, Q., Macaraniag, C., Zhou, J., & Papautsky, I. (2020).Microfluidic systems for hydrodynamic trapping of cells and clusters.Biomicrofluidics, 14(3), 031502.

[0089] [3]Birchler, A., Berger, M., Jäggin, V., Lopes, T., Etzrodt, M.,Misun, P. M., ... & Frey, O. (2016). Seamless combination of fluorescence-activated cell sorting and hanging-drop networks for individual handling andculturing of stem cells and microtissue spheroids. Analytical chemistry, 88(2), 1222-1229.

[0090] [4]L. Gutzweiler, S. Kartmann, K. Troendle, L. Benning, G.Finkenzeller, R. Zengerle, P. Koltay, B. Stark, S. Zimmermann, Large scaleproduction and controlled deposition of single HUVEC spheroids forbioprinting applications, 2017 Biofabrication, Band: 9(2).

[0091] [5]Mekhileri, N. V., Lim, K. S., Brown, G. C. J., Mutreja, I., Schon,B. S., Hooper, G. J., & Woodfield, T. B. F. (2018). Automated 3D bioassemblyof micro-tissues for biofabrication of hybrid tissue engineered constructs.Biofabrication, 10(2), 024103.

[0092] [6]David K.Wood、David M.Weingeist、Sangeeta N.Bhatia and BevinP.Engelward, Single cell trapping and DNA damage analysis using microwellarrays, PNAS June 1, 2010 107(22)10008-10013;

[0093] [7]Zhang, K., Gao, M., Chong, Z., Li, Y., Han, X., Chen, R., & Qin,L. (2016). Single-cell isolation by a modular single-cell pipette for RNA-sequencing. Lab on a Chip, 16(24), 4742-4748.

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[0095] [9]Massimo Bocchi et.al.,“Inverted open microwells for cell trapping,cell aggregate formation and parallel recovery of live cells, Lab Chip, 2012,12, 3168-3176。

Claims

1. A microfluidic device, comprising: At least one fluid inlet (12, 114) and at least one fluid outlet (14, 116). At least one fluid passage (16, 110) fluidly connects the at least one fluid inlet (12, 114) to the at least one fluid outlet (14, 116). Multiple passive hydrodynamic capture sites (20, 118) are arranged along the at least one fluid channel (16, 110), each passive hydrodynamic capture site (20, 118) being configured to capture a single object (150) from a liquid suspension flowing along the at least one fluid channel (16, 110). Each passive hydrodynamic capture site includes a nozzle passage (22, 122) in fluid communication with the associated nozzle orifice (24, 124). The at least one fluid channel (16, 110) and the passive fluid dynamic capture site (20, 118) are configured to operate as a passive fluid dynamic capture site by guiding the main flow path toward an obstacle (30, 120) as long as the passive fluid dynamic capture site (20, 118) is not occupied by a single object (150), and to guide the main flow path around the obstacle (30, 120) of the corresponding passive fluid dynamic capture site (20, 118) when the passive fluid dynamic capture site (20, 118) is occupied by a single object (150).

2. The microfluidic device according to claim 1, wherein, At least some of the passive hydrodynamic trapping sites (20, 118) include the obstruction located within the main flow path, wherein the obstruction includes a trapping recess (32) whose size is adapted to the size of a single object (150).

3. The microfluidic device according to claim 1 or 2, wherein, At least some of the passive hydrodynamic trapping sites (20, 118) include protrusions (16a, 110a) of the fluid channels (16, 110), wherein if a single object (150) is arranged in the passive hydrodynamic trapping site (20, 118), the main flow path is guided around the obstacle via the protrusions (16a, 110a).

4. The microfluidic device according to claim 1, wherein, The microfluidic device includes a microfluidic chip (10, 100) having opposite first and second main surfaces, wherein at least one fluid channel (16, 110) extends parallel to the first and second main surfaces, and wherein the nozzle channel (22, 122) extends perpendicular to the at least one fluid channel (16, 110).

5. The microfluidic device according to claim 1, comprising a plurality of fluid channels arranged in parallel with each other, wherein, Multiple passive hydrodynamic trapping sites with associated nozzle channels are arranged along each fluid channel, wherein the multiple nozzle orifices are arranged in a two-dimensional array.

6. The microfluidic device according to claim 4, wherein the microfluidic device is adapted to be disposed as a cover on a microporous plate, or includes a retainer (102) adapted to retain the microporous plate.

7. The microfluidic device of claim 1, comprising a removable seal (50) covering the nozzle orifice (24, 124).

8. The microfluidic device according to claim 1, wherein, The fluid outlet (14, 116) leads to a waste reservoir (40) formed in the microfluidic device.

9. The microfluidic device of claim 1, further comprising a force applicator configured to simultaneously and at the same level apply force to an object (150) trapped in the passive hydrodynamic trapping site (20, 118) and a column of liquid in the nozzle channel (22, 122) to simultaneously eject a liquid droplet (152) containing the object (150) through the nozzle orifice (24, 124).

10. The microfluidic device according to claim 9, wherein, The force applicator includes any of the following: Centrifuge (80) configured to apply centrifugal force to the object (150) and the liquid column in order to eject liquid droplets (152) containing the object (150) through the nozzle orifice (24, 124). A movable wall (72) and actuators (74, 76) are provided, the movable wall (72) being disposed on the side of the nozzle passage (22, 122) opposite to the nozzle orifice (24, 124), and the actuators (74, 76) being configured to displace the movable wall (72) to eject a liquid droplet (152) containing the object (150) through the nozzle orifice (24, 124), or A drive device configured to apply acceleration or deceleration to the microfluidic device to generate an inertial torque on the object (150) and the liquid column to drive the liquid droplet (152) containing the object (150) away from the nozzle orifice (24, 124).

11. A method for operating a microfluidic device according to claim 1, the method comprising: A liquid suspension containing the object (150) is made to flow from the at least one fluid inlet (12, 114) through the at least one fluid channel (16, 110), whereby the passive hydrodynamic trapping site (20, 118) is occupied by a single object (150) and a liquid column is formed in the nozzle channel (22, 122). The microfluidic device is placed on a receiving plate (60, 104) including a plurality of receiving portions (62, 112) such that the nozzle orifice (24, 124) mates with the receiving portion (62, 112). as well as Simultaneously, force is applied to the captured object (150) and the liquid column in the nozzle channel (22, 122), such that liquid droplets (152) containing a single object (150) are ejected from each nozzle orifice (24, 124).

12. The method according to claim 11, wherein, Applying force to the captured object (150) and the liquid column in the nozzle channels (22, 122) includes rotating the microfluidic device and the receiving plate to apply centrifugal force to the liquid column and the object (150), or displacing a movable wall (72) disposed on the side of the nozzle channels (22, 122) opposite to the nozzle orifices (24, 124) to apply fluid displacement to the nozzle channels (22, 122), or accelerating and decelerating the microfluidic device along the direction of the nozzle channels (22, 122) to generate inertial force on the liquid column and the object (150).

13. The method of claim 11, further comprising supplying the liquid suspension to each fluid channel (16, 110) using a pipette tip (130), wherein, The flow of the liquid suspension through the at least one fluid channel (16, 110) is supported by the hydrostatic pressure of the liquid column inside the pipette tip (130).

14. The method of claim 11, further comprising removing the seal (50) covering the nozzle orifice (24, 124) before placing the microfluidic device on the receiving plate (60, 104).

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