EP4689052A1

EP4689052A1 - Liquid handling device for shearing and filtration of biological matter

Info

Publication number: EP4689052A1
Application number: EP24719015.0A
Authority: EP
Inventors: Audrey Blanche BERGERON; Thomas Albert Cloutier; Gregory Roger Martin; Hilary Anne SHERMAN
Original assignee: Corning Inc
Current assignee: Corning Inc
Priority date: 2023-03-31
Filing date: 2024-03-25
Publication date: 2026-02-11
Also published as: WO2024206199A1; CN121039269A

Abstract

A liquid handling device is provided that shears biological matter, including cell aggregates (e.g., organoids) and filters out fragments of the cell aggregates (or other biological matter) of a selected cut-off size. Further, methods are provided for shearing biological matter with the liquid handling device and for screening out fragments of the biological matter of a selected cut-off, including without contacting the device with another solid object.

Description

LIQUID HANDLING DEVICE FOR SHEARING AND FILTRATION OF BIOLOGICAL MATTER

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §120 of U.S. Provisional Application Serial No. 63/456,424 filed on March 31, 2023, and U.S. Provisional Application Serial No. 63/526,603 filed on July 13, 2023, the content of both of which is relied upon and incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to liquid handling devices and filtration devices. More specifically, aspects of this disclosure are related to a liquid handling devices for filtration and shearing of cells or cells aggregates, methods of making thereof, and methods of shearing biological matter such as groups of cells or tissue or organoids, such as to gather samples thereof.

BACKGROUND

[0003] Patient derived organoids are of increasing interest for research and personalized medicine applications due to the ability of organoids to retain genetic information as the organoids are grown in vitro. An important step in the process of culturing and scaling up patient derived organoids requires shearing the organoids into smaller fragments during passaging. Ideally, these smaller fragments are sized are small enough to enable efficient scale up, but large enough to likely contain epithelial stem cells, as only fragments containing stem cells will grow. Current methods for shearing involve using a small pipet, pipettor tip, a blunt needle, or a pre-narrowed glass Pasteur pipette and holding the pipette/tip/needle against the inside of a tube or cell culture device wall while dispensing to provide mechanical shear stress. However, the efficacy of such method is low because of user-dependent factors, such as how tightly the pipette/tip/needle is held against the tube, the pressure that is exerted by the pipette/tip/needle against the tube, and/or the bore size of the pipette/tip/needle. Such methods also produce a large size distribution of fragments that are not-well controlled. Further with such methods, in addition to breaking apart larger fragments, such methods can also unnecessarily and disadvantageous^ shear or dissociate fragments that are already at or below the optimal passaging size. Accordingly, there is a need for a liquid handling device (and a method of using one) for breaking apart cell aggregates, organoids, or other biological matter that improve efficiency and size control of the resultant fragments.

SUMMARY

[0004] The present application discloses a liquid handling device for fdtration and shearing of cells or cells aggregates for improved efficiency, recovery percentage and size control, and methods of making and using such liquid handling devices.

[0005] In some aspects, biological matter shearing apparatuses are disclosed herein such as a liquid handling device such as a pipet, pipette tip, serological pipet, or needle that contains a screening unit configured to induce shear forces on groups of cells, tissue, an organoid, and the like. Various embodiments of the screening unit of the liquid handling device are provided.

[0006] In accordance with the present disclosure, there is provided a liquid handling device for shearing and/or filtration of biological matter (e.g., organoids or other cells aggregates, tissue fragments) that includes a main body having a hollow passage, an aperture sized to permit liquid containing biological matter to flow into and/or out of the hollow passage, and a screening unit. The screening unit may be positioned inside the hollow passage, at or proximate to the distal end of the hollow passage where the aperture is, or externally to the distal end of the hollow passage and aperture by attaching the screening unit to the distal end of the main body and fluidly connecting the screening unit to the hollow passage. In use, at least some of the biological -matter containing liquid passes through the screening unit at least once. The screening unit includes at least one opening that fdters out biological matter larger than a selected cut-off size (a certain size) and/or shears the biological matter into smaller fragments such that at least some of the biological matter is no larger than the selected cut-off size. In some embodiments, the at least one opening may be a plurality of openings. In some embodiments, the selected cut-off size for the openings may be in a range of 20-300 pm, or 50- 100 pm.

[0007] In some aspects, the liquid handling device has an operational range, where a flowing rate of the liquid/biological matter mixture while passing through the screening unit within the operational range is in a range of 0.1-5 m/s.

[0008] In some aspects, the openings of the screening unit are pores, each having an approximately identical size. In some aspects, the pores comprise a round, oval, triangular, polygonal, or other shape. In one or more aspects of the disclosure, the size of the pores varies within ±20 % or less of a selected cut-off size.

[0009] In some aspects, the screening unit includes a grid of crossing wires. In some further aspects, at least one of the crossing wires includes a leading edge configured to cut or shear or dissociate biological matter into fragments capable of passing through the grid.

[0010] In some aspects, the opening is a slot that comprises two elongated sides aligned with each other and two shortened sides aligned with each other, wherein the length ratio of the elongated sides and the shortened sides is larger than 10: 1. In some aspects, the length ratio of the elongated side and the shortened sides is instead between 3: 1 and 12: 1. In one or more aspects, the length of the shortened sides is no larger than the selected cut-off size. The structure of slots is beneficial in that the limited length of the shortened sides facilitates the shearing of the organoid (or other biological matter), while the elongated sides help the liquid flow through the opening smoothly.

[0011] In some aspects, at least one side wall of the slot has uniformly distributed, inwardly extending protrusions. The deployment of protrusions on the side wall strengthens the shearing effect on the organoids.

[0012] In some aspects, the inwardly extending protrusions positioned on one elongated side in the side wall of the slot are configured to contact with the adjacent protrusions positioned on the other elongated side in the side wall of the slot. By the contact among the adjacent protrusions, small pores are therefore created to facilitate the shearing of organoids to a certain size.

[0013] In some aspects the hollow passage of the liquid handling device comprises a tapered shape. In other aspects, the hollow passage is cylindrical.

[0014] In some aspects, the hollow passage comprises a first passage connected to the aperture and a second passage adjacent to the first passage, wherein the cross-section area of the first passage is smaller than the cross-section area of the second passage. In some aspects, the first passage comprises a tapered shape.

[0015] In some aspects, the screening unit is positioned inside the first passage. The velocity of any liquid that flows through the pipette is higher inside the first passage than inside the second passage, due to smaller cross-section area of the first passage, such that the compressive stress applied onto the surface of the organoid (or other biological matter) by the screening unit is greater if the screening unit is positioned inside the first passage. Therefore, the deployment of screening unit inside the first passage is beneficial for improving the effect of shearing.

[0016] In some aspects, the screening unit is positioned within the hollow passage, at or proximate to the distal end of the hollow passage where the aperture is, or external to the distal end of the main body such that the screening unit and the hollow passage are fluidly connected. [0017] In some aspects, the screening unit is integrated continuously with the inside wall the of the main body. In some aspects, the screening unit is formed separately and then attached to the main body of the liquid handling device.

[0018] In some aspects, the material of the main body of the liquid handling device that surrounds the hollow passage, the screening unit, or both, includes PS (polylstyrene), PVC (polyvinylchloride), PC (polycarbonate), PEA (phenylethylamine), PAM (polyacrylamide), PMMA (poly(methyhnethacrylate), PET (polyethylene terephthalate), PTEF (polytetrafluoroethylene), PU (polyurethane), PE (polyethylene), PP (polypropylene), PVA (polyvinyl alcohol), silicone or glass, or a combination thereof.

[0019] In some aspects, the main body and/or the screening unit of the liquid handling device includes at least one anti-stick material, lubricant material, low-friction material, or hydrophobic material, or a combination thereof. In some aspects, the main body and/or the screening unit are coated with at least one anti-stick material, lubricant material, low-friction material, or hydrophobic material, or a combination thereof. In other aspects, the main body and/or screening unit are made from at least one anti-stick material, lubricant material, low- friction material, or hydrophobic material, or a combination thereof.

[0020] In some aspects, the screening unit filters out biological matter larger than a selected cut-off size and shears biological matter larger than the selected cut-off size into at least one fragment that is no larger than the selected cut-off size.

[0021] In some aspects, a method of making a liquid handling device for biological matter comprises the steps of forming a main body having a hollow passage and an aperture, and reforming at least part of the main body by applying a force from an outer peripheral surface of the main body to form a slot in one portion of the hollow passage that filters out biological matter larger than a selected cut-off size or that shears the biological matter into a fragment no larger than the selected cut-off size. In some embodiments, at least one side wall of the slot comprises inwardly extending protrusions. In some embodiments, the at least part of the main body is formed by molding that uses a rigid frame to shape a polymeric material. In some embodiments, the main body is made from polymeric material and the force is applied in a condition that the at least part of the main body is heated in a temperature higher than the glass transition temperature of the polymeric material.

[0022] In some aspects, the slot formed in the process of making the liquid handling device has a uniform distribution of inwardly extending protrusions.

[0023] In some aspects, a method for shearing biological matters includes the following steps. A mixture having liquid and biological matter is provided. A liquid handling device that includes a main body, a hollow passage, an aperture, and a screening unit is inserted into the mixture. A portion of the mixture is drawn into the hollow passage. In some embodiments, the portion of the mixture that is drawn into the hollow passage is drawn in through the aperture and screening at or in the hollow passage. Alternatively, the portion of the mixture that is drawn into the hollow passage is expelled from the hollow passage through the aperture and screening unit. The biological matter of a passable size travels past the screening unit, and biological matter of a non-passable size is sheared by the screening unit into one or more pieces of biological matter having a passable size which travels past the screening unit. The screening unit may be disposed at or proximate to the aperture, disposed within the hollow passage, or disposed external to the hollow passage and the aperture such than the hollow passage and screening unit are fluidly connected. In one embodiment of the method, a distal tip of the liquid handling device remains contactless with any solid object during the step of drawing in a portion of the mixture. In another embodiment, a distal tip of the liquid handling device is immersed into the mixture without contacting any other object during shearing.

[0024] In one aspect, the method further includes the step of collecting in the hollow passage at least some of the biological matter which passes through the screening unit.

[0025] In one aspect, the method further includes the step of agitating a distal end of the liquid handling device in the mixture before the step of drawing in the mixture into the hollow passage.

[0026] In one aspect, the method further includes the step of dispensing biological matter of a passable size out of the liquid handling device.

[0027] In one aspect, the method includes the steps of both drawing in a portion of the mixture into the hollow passage through the aperture and past the screening unit and expelling from the hollow passage a drawn in portion of the mixture through the aperture and past the screening unit. [0028] Additional features and advantages of the aspects disclosed herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the disclosed aspects as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0029] It is to be understood that both the foregoing summary and the following detailed description present aspects intended to provide an overview or framework for understanding the nature and character of the claimed aspects. The accompanying drawings are included to provide further understanding and are incorporated into and constitute a part of this specification. The drawings illustrate various aspects of the disclosure, and together with the description serve to explain the structures and operations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A-1C are schematic side-view diagrams of embodiments of liquid handling devices with screening units, according to some aspects of the present disclosure. FIG. 1A illustrates an embodiment of a liquid handling device with a screening unit positioned away from a distal end of an aperture of the liquid handling device. FIG. IB illustrates a different embodiment of a liquid handling device with a screening unit positioned away from a distal end of an aperture of the pipette. FIG. 1C illustrates yet another embodiment of a liquid handling device with a screening unit positioned at the aperture of the liquid handling device. [0031] FIGS. 2A-2C are schematic top-view diagrams of screening units, according to some aspects of the present disclosure. FIG. 2A illustrates a schematic of a screening unit having openings shaped as round pores, the screening unit being a separate unit that may be attached to the distal end of the main body of a liquid handling device. FIG. 2B illustrates a schematic of a screening unit having a mesh configuration and the screening unit is integratedly extended from the main body of the liquid handling device. FIG. 2C illustrates a schematic of a screening unit having openings in a fan-shape configuration and the screening unit is integratedly extended from the liquid handling device.

[0032] FIG. 3A is schematic side-view diagram of part of a liquid handling device, according to some aspects of the present disclosure. FIG. 3B is a schematic bottom-view diagram of a screening unit having an elongated shape according to the liquid handling device in FIG. 3A. FIGS. 3C-3D are schematic bottom views of screening units, according to some aspects of the liquid handling device in FIG. 3 A.

[0033] FIGS. 4A-4B are schematic cross-sectional diagrams of a simplified process for how a slot-shaped opening with inwardly extending protrusions is formed by reforming the main body. FIG. 4A illustrates the pre-reforming configuration of a screening unit, while FIG. 4B illustrates the post-reforming configuration.

[0034] FIGS. 5A-5C are schematic diagrams from a side view of steps in a process for how organoids or cell aggregates are sheared when passing through the screen unit of a liquid handling device, according to some aspects of the present disclosure.

[0035] The drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar.

DETAILED DESCRIPTION

[0036] In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration several specific embodiments of devices, systems and methods. It is to be understood that other embodiments are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. [0037] In the following detailed description, like reference characters designate like or corresponding parts throughout the several views shown in the figures. Directional terms as used herein — for example up, down, top, bottom, out of, outside, inside, lower and upper — are made only with reference to the figures as drawn and are not intended to imply absolute orientation. In addition, whenever a group is described as comprising at least one of a group of elements and combinations thereof, it is understood that the group may comprise, consist essentially of, or consist of any number of those elements recited, either individually or in combination with each other. Similarly, whenever a group is described as consisting of at least one of a group of elements or combinations thereof, it is understood that the group may consist of any number of those elements recited, either individually or in combination with each other. Unless otherwise specified, a range of values, when recited, includes both the upper and lower limits of the range as well as any ranges therebetween. It also is understood that the various features disclosed in the specification and the drawings can be used in any and all combinations. [0038] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0039] As used herein, the indefinite articles “a,” “an,” and the corresponding definite article “the” mean “at least one” or “one or more,” unless otherwise specified.

[0040] As used herein, the term “glass” is meant to include any material made at least partially of glass, including glass and glass-ceramics.

[0041] As used herein, the term “shear” refers to the action of splitting articles into smaller pieces.

[0042] As used herein, the term “integrate continuously” or “integratedly extended” refers to a seamless blending or incorporation of the article A and article B, where there is a smooth and uninterrupted transition from article A to article B, such that article A and article B cannot decouple when liquid flows through the junction area of article A and article B.

[0043] As used herein, the term “uniformly” refers that various amount of article A is evenly spread or distributed across the entire surface of article B without any obvious clustering or variations in density. It does not mean that the distribution of article A must be strictly uniform, but the distribution can be generally uniform.

[0044] In one aspect of the present disclosure, liquid handling devices including needles, pipets and pipette tips that efficiently shear biological matter such as organoids or cell aggregates are disclosed herein. In some embodiments, a pipet may be a serological pipet that comprises a screening unit for shearing biological matter such as organoids and cell aggregates and filtering out fragments of the biological matter of certain sizes. In some embodiments, the liquid handling device may be a syringe and needle that comprises a screening unit for shearing biological matter such as organoids and cell aggregates and filtering out fragments of the biological matter of certain sizes. In some embodiments, a pipette may be a pipette configured for a pipette tip that comprises a screening unit for shearing biological matter such as organoids or cell aggregates and filtering out fragments of the biological matter of certain sizes. [0045] In one set of embodiments, the distal tip of a liquid handling device comprising a screening unit may be angled. In another set of embodiments, the distal tip of the liquid handling device comprising a screening unit may be flat or rounded.

[0046] In one set of embodiments, the screening unit comprises a grid-like strainer structure. In another set of embodiments, the screening unit comprises a plurality of pores. In yet another set of embodiments, the screening unit comprises a slot-like structure with optional, inwardly extending protrusions.

[0047] The liquid handling devices with screening units of the present disclosure generate biological matter fragments (including organoid fragments and other cell aggregate fragments) that are more uniform in size compared to the liquid handling devices of the present disclosure without screening units. Similarly, a higher percentage of fragments that are at or below the selected cut-off size of the screening unit are recovered when the screening unit is employed. This is advantageous, especially for organoids, for down stream processing of the fragments, which require more uniformly sized fragments that are less damaged than traditional methods. [0048] In some embodiments, the screening unit can be manufactured as an integral part of the liquid handling device. In some embodiments, the screening unit comprises an insert which can be affixed or molded along with the liquid handling device. In some embodiments, the screening unit can be separately formed and then subsequently attached to the tip of the liquid handling device. In one embodiment, the separately formed screening unit is fluidly connected to or integral to a main body having a hollow passage that attaches to the main body and hollow passage of the liquid handling device. For example, the separately formed screening unit may be part of an attachable extension tip to a pipette tip for a pipettor.

[0049] In some aspects, the sharpness of the leading edge of the screening unit can be selected, depending upon the targeted application. In some embodiments, a leading edge having a sharper edge (e.g., having an angle of the edge of less than 90 degrees) may improve the shearing ability of the liquid handling device.

[0050] In use, varying pressures can be applied or maintained while drawing in (i.e., sucking in, applying a vacuum pressure, or aspirating), and then dispensing, such as when using a pipettor, pipet aid, syringe, or other liquid handler, such that larger pieces or fragments of biological matter such as organoid fragments or tissue samples are dissociated by mechanical shear stress induced by fluid flowing into or out of the device and the organoid or tissue or group of cells (or other biological matter) engaging with the screening unit in a controlled manner.

[0051] In some embodiments, a screen-like structure with defined openings of a certain size (e.g. 200 pm) can be implemented, so that fragments smaller than the size of the opening could go past the restriction areas unsheared and therefore pass through and into the device unbroken, while larger fragments (groups of cells, organoids, tissue, etc.) can be dissociated or sheared as they pass through the screen.

[0052] Such liquid handling devices can help eliminate the current variabilities in how the user might hold distal tip of the liquid handling device against the tube. Instead, with the device and/or method disclosed herein, the shearing would be more controlled and well defined by the type of restriction, or strainer feature, within or on the device itself, as the user would hold the distal end of the device within a suspension in the tube without contacting a wall or other solid object, thereby eliminating the need to hold the device against the tube or cell culture device wall, for example.

[0053] Various embodiments of liquid handling devices with strainers containing different sized openings may be prepared in order to selectively obtain different fragment sizes (or ranges in fragment sizes) depending upon which sample types and applications are being sampled. Additionally, the volume and bore size of the tips of the liquid handling device could vary for different sample volumes. Optionally, the tip/pipet/needle and restriction (e.g. strainer) may be constructed having one or more materials having low-stick or low attachment properties, such as to prevent the attachment or entrapment of sticky organoid fragments and/or cells by the device and/or strainer; such material, or coating, can thereby help reduce a loss in the quantity or variety of sampled material.

[0054] In aspects, the material of the main body of the liquid handling device that surrounds the hollow passage, the screening unit, or both, includes PS (polylstyrene), PVC (polyvinylchloride), PC (polycarbonate), PEA (phenylethylamine), PAM (polyacrylamide), PMMA (poly(methyhnethacrylate), PET (polyethylene terephthalate), PTEF (polytetrafluoroethylene), PU (polyurethane), PE (polyethylene), PP (polypropylene), PVA (polyvinyl alcohol), silicone or glass, or a combination thereof.

[0055] Any organoid, other cell aggregate, or tissue fragments that remain within the device after initial pipetting of the cell suspension could be rinsed out with culture media. [0056] As an alternative (or addition) to directly molding, insert molding or attaching a mesh or grid to provide a strainer function, a smooth or patterned slot design may be produced to restrict flow in such a way as to generate the required shear forces upon aspirating the suspension into the shearing device. In some embodiments, a liquid handling device can be molded with a larger opening, and then the tip can be mechanically reformed after removal from the mold.

[0057] Referring now to FIG. 1 A, liquid handling device 1 used for filtration of cells or cells aggregates is illustrated. A hollow passage 11 is formed inside the main body 10 while a screening unit 12 is positioned inside the hollow passage 11. An aperture 13 is formed on the bottom (distal end) of the main body 10, which allows a liquid to flow into or out of the hollow passage 11. FIG. IB illustrates another aspect of a liquid handling device of the present disclosure. Liquid handling device 1 has a hollow passage 11 comprising a first passage 14, a screening unit 12, and a second passage 15. The second passage 15 has a uniform cross- sectional area, and a first passage 14 having a tapered shape. The cross-sectional area of the first passage 14 is smaller than that of the second passage 15. The screening unit 12 is positioned inside the first passage 14. FIG. 1 C illustrates yet another aspect of a liquid handling device of the present disclosure. Liquid handling device 1 has a hollow passage 11 formed inside the main body 10, with a screening unit 12 positioned at aperture 13, which is formed on the bottom of the main body 10.

[0058] In some embodiments, the screening unit is disposed within the hollow passage and spaced away from the aperture of the liquid handling device. In some embodiments, the screening unit is disposed within the hollow passage and at the aperture of the liquid handling device. In some embodiments, the screening unit is disposed as an attachment to the distal end of the main body, and the screening unit is fluidly connected to the hollow passage of the main body.

[0059] In aspects where there is a singular passage in the main body of the liquid handling device (e.g., FIGS. 1A, 1C), the singular passage may either be tapered towards the end with the aperture, or the singular passage may have a uniform cross-sectional area. In one embodiment, the singular passage may be cylindrical. In aspects where the screening unit is positioned at the aperture, the screening unit may be in-line with the aperture, or adjacent to it (i.e., just above or below aperture). [0060] Referring now to FIG. 2A-2C, top views of some aspects of screening unit 12 are depicted. FIG. 2A illustrates a screening unit 12 with a combination of round pores 21, each pore being an opening that allows liquid to flow through. Although the pores in FIG. 12A are depicted as round, in other embodiments, the pores may be oval, triangular, polygonal, or other shape, and in some embodiments, the pores may be elongated in one or more direction. FIG. 2A illustrates the screening unit 12 as a separate structure that can be attached tightly on the side wall 20 of the main body of the liquid handling device. However, the screening unit 12 may instead be integratedly extended from the side wall 20 of the main body of the liquid handling device. FIG. 2B illustrates a mesh-like screening unit 12, with a combination of rectangular pores 21, each pore being an opening. The mesh structure is integratedly extended from the side wall 20 of the main body of the liquid handling device. However, in some aspects, the mesh-like screening unit is instead a separate structure attached tightly on the side wall of the main body of the liquid handling device such that the hollow passage of the main body and the interior of the separate structure are fluidly connected. FIG. 2C illustrates another aspect of a screening unit 12 with a combination of fan-shaped pores 21, each pore being an opening. The structure of the screening unit 12 is integratedly extended from the side wall 20 of the main body of the liquid handling device. However, in some aspects, the screening unit 12 is instead a separate structure attached tightly on the side wall 20 of the main body of the liquid handling device.

[0061] In some aspects, the screening unit of the present disclosure have a selected (chosen) or defined opening size (e.g. 200 pm). Each opening size may be in the range of 20-300 pm, 20-250 pm, 20-200 pm, 20-150 pm, 20-100 pm, 50-100 pm, 20-70 pm, 20-40 pm, 40-300 pm, 40-250 pm, 40-200 pm, 40-150 pm, 40-100 pm, 40-70 pm, 70-300 pm, 70-250 pm, 70-200 pm, 70-150 pm, 70-100 pm, 100-300 pm, 100-250 pm, 100-200 pm, 100-150 pm, 150-300 pm, 150-250 pm, 150-200 pm, 200-300 pm, 200-250 pm or 250-300 pm. In one embodiment, each opening size is between about 50 and about 100 pm.

[0062] In some aspects, the opening is provided with pores having identical size. “Identical” here means the size of the pores is generally the same but not exactly the same. In one or more aspects of the disclosure, the size of the pores varies within ±20 %, within ±15 %, within ±10 %, within ±5 %, or within less than 5%, of a selected cut-off size. Liquid handling devices of different strainer sizes can be provided and selected depending on the desired obtained sample size (e.g. desired organoid fragment size). [0063] In some embodiments, the screening unit comprises a grid of crossing wires. In some embodiments, at least one of the crossing wires comprises one or more sharp or angled portions. In some embodiments, the at least one crossing wire comprises a leading edge configured to cut or shear or dissociate biological matter (including cell aggregates and organoids) into pieces capable of passing through the pores of the screening unit.

[0064] In some embodiments, at least part of the main body and/or the screening unit is coated with a biological matter anti-stick material. Non-limiting examples of anti-stick materials include non-ionic hydrogels, such as a polyethylene glycol, polyacrylamide, among other compounds with alcohol functionality. In some embodiments, the main body and/or screening unit itself comprises an anti-stick material for biological matter in its composition. In some embodiments, the main body and/or screening unit is coated with a lubricant. Nonlimiting examples of lubricants include water-associating compounds that provide slip to surfaces such as hyaluronic acid. In some embodiments, the main body and/or screening unit comprises a lubricant in its composition materials. In some embodiments, the main body and/or screening unit comprises a coating having a low friction surface material. Non-limiting examples of low friction surface materials include for example, silicone-based compounds and fluorinated compounds. In some embodiments, the main body and/or screening unit comprises a low friction surface material in its composition. In some embodiments, the main body and/or screening unit comprises a coating having a hydrophobic surface material. Non-limiting examples of hydrophobic surface materials include, for example, waxes and paraffins. In some embodiments, the main body and/or screening unit comprises a hydrophobic material in its composition. In embodiments with a coating, the coating may be only on the portion of the main body or screening unit that contacts the liquid containing the organoids or cell aggregates (or other biological matter), or alternatively, the entire screening unit may be coated. Further, in some embodiments, the coating or composition may include a combination of these materials. Although examples of suitable materials are listed, it should be understood that any suitable material that decreases the sticking of cells to the screening unit may be used.

[0065] Referring now to FIG. 3A, another aspect of a liquid handling device 1 is illustrated. In FIG. 3 A, liquid handling device 1 has a main body 10, a hollow passage 11, an aperture 13 at the distal end of the hollow passage 11 where the aperture is, and a screening unit 12 at the end of hollow passage 11 . A bottom view of the screening unit 12 of the liquid handling device 1 in FIG. 3A is depicted in FIG. 3B. The screening unit 12 is positioned in a portion of the hollow passage 11 where the enclosure of the side wall of the hollow passage 11 forms a slotlike opening, which consists of two elongated sides 23 and two shortened sides 24. The length ratio of the elongated sides and the shortened sides is 12: 1. In some aspects, the length ratio of the elongated sides and the shortened sides is 15: 1. In one or more aspects, the length ratio of the elongated sides and the shortened sides is 20: 1. In some aspects, the length ratio of the elongated sides and the shortened sides is from about 3: 1 to about 12: 1, from about 3: 1 to about 10: 1, from about 3: 1 to about 8: 1, from about 3: 1 to about 6: 1, from about 5: 1 to about 12: 1, from about 7: 1 to about 12: 1, from about 9: 1 to about 12: 1, or from about 5: 1 to about 10: 1.

[0066] Referring now to FIG. 3C, another aspect of screening unit 12 is illustrated. Based on the structure of screening unit 12 in FIG. 3B, a plurality of inwardly extending protrusions 22 are provided on the side wall of the hollow passage 11 in the area of the screening unit 12. The protrusions 22 do not contact each other. However, in one or more aspects, such as the one in FIG. 3D, the protrusions 22 positioned on one elongated side 23 contact with the adjacent protrusions 22 positioned on the other elongated side 23, which forms a plurality of pores to facilitate the shearing of organoids or other cell aggregates or biological matter.

[0067] The plurality of protrusions extending inwardly towards the center of the screening unit may take on any shape and size that creates pores of the desired size. For example, the protrusions may be rounded, triangular, or polygonal (including but not limited to squares, rectangles, rhomboids, pentagons, hexagons, and octagons). The portion of the protrusion that contacts the target liquid may be smooth, textured, or patterned.

[0068] The elongated nature of screening units such as those in FIGS. 3A-3D can induce additional shearing action on organoids, cell aggregates, or other biological matter. In embodiments having elongated screening units, such screening units can be either molded to their final form, or they can be thermo-mechanically reformed from a precursor form to provide the narrow or thin slot widths.

[0069] Referring now to FIG. 4A-4B, a simplified process of how an elongated screening unit is reformed is depicted. Prior to reforming, the first step (not shown) is to form a screening unit using having a first shape, which is a precursor shape to the final elongated screening unit shape. FIG. 4A illustrates a top view of an example of a precursor shape structure of screening unit 12a. In FIG. 4A, the shape of the cross-section of the screening unit 12a is round, where a plurality of inwardly extending protrusions 22a is uniformly distributed on the side wall of the hollow passage in the portion of screening unit 12a. In the process of reforming, two pressors 31 are squeezed toward each other (according to the direction of arrows shown in FIG. 4A) on the outer periphery of the screening unit 1 la such that the screening unit 12a is reformed into a second, elongated screening unit shape as shown in FIG. 4B. In some aspects, the screening unit is made from polymeric material and it is heated to a temperature higher than its glass transition temperature when reforming. The cross-section of screening unit 12/? after deformation is in a structure of a rectangular shape with elongated length and shortened width. The width of the slot-like screening unit 126 and distribution of inwardly extending protrusions define the desired size of the organoid or cell aggregates or other biological matter after shearing. Therefore, the distribution density and shape of the protrusions, together with the width of the slot-like screening unit can be tuned according to different requirement for shearing of the organoids or cell aggregates (or other biological matter).

[0070] In one aspect of the present disclosure, methods are disclosed herein for shearing and/filtering biological matter such as organoids or cell aggregates with a liquid handling device. In one embodiment, liquid containing the biological matter is drawn in (sucked up) into the hollow passage of the liquid handling device and passes through the screening unit, and then is expelled from the hollow passage back through the screening unit into a separate container. This process may be repeated such that all of the passable sized fragments are collected in the separate container. The collection of passable sized fragments in the separate container are more uniform in size than if shearing was performed with the same liquid handling device without the screening unit. Also, a higher percentage of fragments that are at or below the selected cut-off size of the screening unit are present in the collection in the separate container than if the shearing was performed with the same liquid handling device without the screening unit.

[0071] In another embodiment, liquid containing the biological matter is drawn in (sucked up) into the hollow passage of the liquid handling device and passes through the screening unit, and then is expelled from the hollow passage into a separate container without passing back through the screening unit. Alternatively, liquid containing the biological matter is drawn in (sucked up) into the hollow passage of the liquid handling device without passing through the screening unit, and then is expelled from the hollow passage through the screening unit and into a separate container. In either of these embodiments, the process may be repeated such that all of the passable sized fragments are collected in the separate container. Again, the uniformity and percentage recovery of the biological matter at or less than a selected cut-off size of the screening unit is greater than if shearing was performed with the same liquid handling device without the screening unit.

[0072] In some aspects of the present disclosure, the methods for shearing and/or filtering the biological matter is performed without contacting the device with another solid object (such as a vial, dish, or other container). For example, the distal tip of the liquid handling device may be submerged in a container having biological matter in a liquid, and then the mixture of biological matter/liquid is drawn up into the hollow passage of a main body of a liquid handling device without the distal tip of the liquid handling device coming into contact with the container. In some embodiments, during the process of shearing, the tip of the liquid handling device remains contactless with any solid object (such as a vial, dish, or other container) during the shearing process. In some embodiments, the tip of the liquid handling device does not touch any solid object during the shearing, and only contacts the mixture of liquid and organoids or cell aggregates. This is advantageous as it eliminates the user variability inherent in placing the device against another object. Variability in pressure, angle, and choice of the solid object are problematic for achieving uniform shearing. In addition, this contactless method allows for using these liquid handling devices with automation.

[0073] Referring now to FIG. 5A-5C, a process for shearing organoids or cell aggregates (and other biological matter) in the liquid handling device is depicted. The direction of the arrow represents the flowing direction of a liquid containing organoids (cell aggregates). In some embodiments, the mixture comprises a growth medium. As shown in FIG. 5 A, the organoids 4 la contained in a liquid are firstly sucked (drawn in) into a liquid handling device comprising a screening unit 12 in a hollow passage 11 within the main body 10 of the liquid handling device. After passing through the screening unit 12 as shown in FIG. 5B, part of the organoids larger than a certain size (the size of the openings in the screening unit 12) are sheared into smaller fragments. The liquid with the organoids 41/? passes back through the screening unit 12 for another time when it is ejected out of the liquid handling device as depicted in FIG. 5C. The organoids are further sheared in this action. The process from FIG. 5A to FIG. 5C can be repeated for several times until a desired size distribution of organoids is achieved. Any organoids (or other cell culture aggregates) that are not sheared to small enough sizes to pass through the screening unit on a first round of passes, may pass through in later rounds of passes after the larger fragments have sheared to a small enough size. In some embodiments, the fragments that have passed through the screening unit and so contained inside the hollow passage are then ejected into a collection container for further use. This can be performed for each passage that is performed.

[0074] In some aspects, the liquid handling device has an operational range, where a flowing rate of the organoid-carrying liquid while passing through the screen unit within the operational range is in a range of 0.1-5 m/s, 0.5-5 m/s, 1-5 m/s, 2-5 m/s, 3-5 m/s, 4-5 m/s, 0.1-4 m/s, 0.5-4 m/s, 1-4 m/s, 2-4 m/s, 3-4 m/s, 0.1-3 m/s, 0.5-3 m/s, 1-3 m/s, 2-3 m/s, 0. 1-2 m/s, 0.5-2 m/s, 1- 2 m/s, 0. 1-1 m/s, 0.5-1 m/s or 0.1-0.5 m/s.

[0075] In some embodiments, the shearing and/or filtering of biological matter, including the organoids or cell aggregates, occurs internally within the hollow passage. In other embodiments, the shearing occurs when the biological matter contact or pass through the screening unit. In some embodiments, the shearing occurs both inside the hollow passage and at the screening unit (by contacting and/or passing through it). In some embodiments, the drawing in or aspirating of the liquid comprising the biological matter is of sufficient strength to shear the biological matter of non-passable size independently of physical contact with the screening unit. In some embodiments, biological matter such as organoids or cell aggregates are sheared by physical contact with the screening unit. In any of these embodiments, the screening unit acts a filter by allowing the passage of biological material smaller than a selected cut-off size of the openings in the screening unit and preventing passage of biological material larger than the selected cut-off size. The screening unit simultaneously shears some of the biological matter larger than the selected cut-off size into at least one fragment, which can then pass through the screening unit.

[0076] In some embodiments, the method of shearing biological matter such as organoids or cell aggregates further comprises agitating the liquid handling device in the liquid/biological matter mixture before the sucking (drawing in) the mixture into the liquid handling device. In some embodiments, the method comprises agitating the liquid handling device in the mixture while sucking the mixture into the liquid handling device. In some embodiments, the method comprises agitating the liquid handling device in the mixture after the sucking the mixture into the liquid handling device.

[0077] Additionally, any examples set forth in this specification are illustrative, but not limiting, and merely set forth some of the many possible aspects of the claimed disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in the field, and which would be apparent to those skilled in the art, are within the spirit and scope of the disclosure.

Claims

CLAIMS What is claimed is:

1. A liquid handling device for biological matter, comprising: a main body comprising a hollow passage; an aperture sized to permit a liquid comprising biological matter to flow into or out of the hollow passage; a screening unit positioned inside the hollow passage such that at least some of the liquid passes through the screening unit; wherein the screening unit comprises an opening that fdters out biological matter larger than a selected cut-off size or that shears the biological matter into fragments no larger than the selected cut-off size; wherein the selected cut-off size is in a range of 20-300 pm.

2. The liquid handling device of claim 1, wherein the liquid handling device has an operational range, where a flowing rate of the liquid while passing through the screen unit within the operational range is in a range of 0. 1-5 m/s.

3. The liquid handling device of any of claims 1-2, wherein the opening comprises pores having identical size.

4. The liquid handling device of claim 3, wherein the pores comprise a round, oval, triangular, or polygonal shape.

5. The liquid handling device of claim 1, wherein the opening is a slot comprising two elongated sides aligned with each other and two shortened sides aligned with each other, wherein the length ratio of the elongated sides and the shortened sides is larger than 10: 1.

6. The liquid handling device of claim 1, wherein the opening is a slot comprising two elongated sides aligned with each other and two shortened sides aligned with each other, wherein the length ratio of the elongated sides and the shortened sides is between 3 : 1 and

7. The liquid handling device of any of claims 1, 2, 5 or 6, wherein at least one side wall of the opening comprises inwardly extending protrusions.

8. The liquid handling device of claim 1, wherein the hollow passage comprises a first passage connected to the aperture and a second passage adjacent to the first passage, wherein the cross-section area of the first passage is smaller than the cross-section area of the second passage.

9. The liquid handling device of claim 1, wherein the hollow passage comprises a tapered shape.

10. The liquid handling device of claim 1, wherein the hollow passage is cylindrical and extends along a central or longitudinal axis.

11. The liquid handling device of claim 1 , wherein the screening unit is positioned inside the hollow passage.

12. The liquid handling device of claim 1, wherein the screening unit positioned at or proximate to the distal end of the hollow passage where the aperture is.

13. The liquid handling device of claim 1, wherein the screening unit is positioned external to the hollow passage and the aperture, and is positioned such that the hollow passage and screening unit are fluidly connected.

14. The liquid handling device of claim 1, wherein the screening unit is integrated continuously with an inside wall the of the main body.

15. The liquid handling device of claim 1, wherein the screening unit is attached to the main body.

16. The liquid handling device of claim 1, wherein the material of the main body and screening unit comprises PS, PVC, PC, PEA, PAM, PMMA, PET, PTEF, PU, PE, PP, PVA, silicone or glass, or a combination thereof.

17. The liquid handling device of claim 1, wherein the main body, the screening unit, or both, comprise at least one anti-stick material, lubricant material, low -friction material, or hydrophobic surface material, or a combination thereof.

18. The liquid handling device of claim 17, wherein the at least one material is a coating.

19. The liquid handling device of claim 1, wherein the screening unit comprises a grid of crossing wires.

20. The liquid handling device of claim 19, wherein at least one of the crossing wires comprises a leading edge configured to cut or shear or dissociate biological matter into fragments capable of passing through the grid.

21. The liquid handling device of claim 1, wherein the biological material comprises cell aggregates or organoids, or both.

22. The liquid handling device of claim 1, wherein the screening unit filters out biological matter larger than the selected cut-off size and shears biological matter larger than the selected cut-off size into at least one fragment no larger than the selected cut-off size.

23. A method for making a liquid handling device for biological materials, comprising: forming a main body having a hollow passage and an aperture; reforming at least part of the main body by applying a force from an outer peripheral surface of the main body to form a slot in one portion of the hollow passage that filters out biological matter larger than a selected cut-off size or that shears the biological matter into a fragment no larger than the selected cut-off size.

24. A method for making the liquid handling device of claim 23, wherein at least one side wall of the slot comprises inwardly extending protrusions.

25. A method for making a liquid handling device of claim 23, wherein the at least one part of the main body is formed by molding that uses a rigid frame to shape a polymeric material.

26. A method for making a liquid handling device of claim 23, wherein the at least one part of the main body is made from a polymeric material and the force is applied in a condition that the at least one part of the main body is heated in a temperature higher than the glass transition temperature of the polymeric material.

27. A method of shearing biological matter, comprising the steps of: providing a mixture comprising a liquid and biological matter; inserting a liquid handling device into the mixture, the liquid handling device comprising a main body, a hollow passage, an aperture, and a screening unit; and drawing in a portion of the mixture into the hollow passage through the aperture and past the screening unit, or expelling from the hollow passage a drawn in portion of the mixture through the aperture and past the screening unit; wherein biological matter of a passable size travels past the screening unit; and wherein biological matter of a non-passable size is sheared by the screening unit into one or more pieces of biological matter having a passable size which travels past the screening unit.

28. The method of claim 27, wherein the screening unit is disposed at or proximate to the aperture.

29. The method of claim 27, wherein the screening unit is disposed within the hollow passage

30. The method of claim 27, wherein the screening unit is external to the hollow passage and the aperture, and the hollow passage and screening unit are fluidly connected.

31. The method of any of claims 28-29, further comprising the step of collecting in the hollow passage at least some of the biological matter which passes through the screening unit.

32. The method of claim 27, wherein a distal tip of the liquid handling device remains contactless with any solid object during the step of drawing in a portion of the mixture.

33. The method of claim 27, wherein a distal tip of the liquid handling device is immersed in the mixture without contacting any other object during shearing.

34. The method of claim 27, further comprising the step of agitating a distal end of the liquid handling device in the mixture before the step of drawing in the mixture.

35. The method of any of claims 28-29, further comprising the step of dispensing biological matter of a passable size out of the liquid handling device.

36. The method of any of claim 27, wherein the method comprises the steps of both drawing in a portion of the mixture into the hollow passage through the aperture and past the screening unit and expelling from the hollow passage a drawn in portion of the mixture through the aperture and past the screening unit.