EP4689052A1 - Dispositif de manipulation de liquide pour cisaillement et filtration de matière biologique - Google Patents

Dispositif de manipulation de liquide pour cisaillement et filtration de matière biologique

Info

Publication number
EP4689052A1
EP4689052A1 EP24719015.0A EP24719015A EP4689052A1 EP 4689052 A1 EP4689052 A1 EP 4689052A1 EP 24719015 A EP24719015 A EP 24719015A EP 4689052 A1 EP4689052 A1 EP 4689052A1
Authority
EP
European Patent Office
Prior art keywords
handling device
liquid handling
screening unit
hollow passage
biological matter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24719015.0A
Other languages
German (de)
English (en)
Inventor
Audrey Blanche BERGERON
Thomas Albert Cloutier
Gregory Roger Martin
Hilary Anne SHERMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Corning Inc
Original Assignee
Corning Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Corning Inc filed Critical Corning Inc
Publication of EP4689052A1 publication Critical patent/EP4689052A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • 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.
  • 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.
  • 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.
  • biological matter shearing apparatuses 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.
  • 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.
  • a screening unit of the liquid handling device are 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.
  • 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.
  • the at least one opening may be a plurality of openings.
  • the selected cut-off size for the openings may be in a range of 20-300 pm, or 50- 100 pm.
  • 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.
  • the openings of the screening unit are pores, each having an approximately identical size.
  • the pores comprise a round, oval, triangular, polygonal, or other shape.
  • the size of the pores varies within ⁇ 20 % or less of a selected cut-off size.
  • the screening unit includes a grid of crossing wires.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hollow passage of the liquid handling device comprises a tapered shape. In other aspects, the hollow passage is cylindrical.
  • 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.
  • the first passage comprises a tapered shape.
  • 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.
  • 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.
  • 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.
  • 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.
  • PS polylstyrene
  • PVC polyvinylchloride
  • PC polycarbonate
  • PEA phenylethylamine
  • PAM polyacrylamide
  • PMMA poly(methyhnethacrylate
  • PET polyethylene terephthalate
  • PTEF polytetrafluoroethylene
  • PU polyurethane
  • PE polyethylene
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • at least one side wall of the slot comprises inwardly extending protrusions.
  • the at least part of the main body is formed by molding that uses a rigid frame to shape a polymeric material.
  • 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.
  • the slot formed in the process of making the liquid handling device has a uniform distribution of inwardly extending protrusions.
  • 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.
  • 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.
  • 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.
  • 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.
  • a distal tip of the liquid handling device is immersed into the mixture without contacting any other object during shearing.
  • 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.
  • 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.
  • the method further includes the step of dispensing biological matter of a passable size out of the liquid handling device.
  • 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.
  • 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.
  • FIGS. 2A-2C are schematic top-view diagrams of 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 pipe
  • 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.
  • 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.
  • 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.
  • 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.
  • indefinite articles “a,” “an,” and the corresponding definite article “the” mean “at least one” or “one or more,” unless otherwise specified.
  • glass is meant to include any material made at least partially of glass, including glass and glass-ceramics.
  • the term “shear” refers to the action of splitting articles into smaller pieces.
  • 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.
  • 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.
  • liquid handling devices including needles, pipets and pipette tips that efficiently shear biological matter such as organoids or cell aggregates are disclosed herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the screening unit can be manufactured as an integral part of the liquid handling device.
  • the screening unit comprises an insert which can be affixed or molded along with the liquid handling device.
  • the screening unit can be separately formed and then subsequently attached to the tip of the liquid handling device.
  • 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.
  • the separately formed screening unit may be part of an attachable extension tip to a pipette tip for a pipettor.
  • the sharpness of the leading edge of the screening unit can be selected, depending upon the targeted application.
  • a leading edge having a sharper edge e.g., having an angle of the edge of less than 90 degrees
  • 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.
  • 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.
  • a screen-like structure with defined openings of a certain size 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.
  • 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.
  • 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.
  • the volume and bore size of the tips of the liquid handling device could vary for different sample volumes.
  • the tip/pipet/needle and restriction e.g. strainer
  • the tip/pipet/needle and restriction 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.
  • 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.
  • PS polylstyrene
  • PVC polyvinylchloride
  • PC polycarbonate
  • PEA phenylethylamine
  • PAM polyacrylamide
  • PMMA poly(methyhnethacrylate
  • PET polyethylene terephthalate
  • PTEF polytetrafluoroethylene
  • PU polyurethane
  • PE polyethylene
  • 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.
  • 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.
  • a liquid handling device can be molded with a larger opening, and then the tip can be mechanically reformed after removal from the mold.
  • 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.
  • 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.
  • 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.
  • the singular passage may either be tapered towards the end with the aperture, or the singular passage may have a uniform cross-sectional area.
  • the singular passage may be cylindrical.
  • the screening unit may be in-line with the aperture, or adjacent to it (i.e., just above or below aperture).
  • 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.
  • FIG. 12A 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.
  • 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.
  • 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.
  • 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.
  • each opening size is between about 50 and about 100 pm.
  • 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).
  • 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.
  • the main body and/or the screening unit is coated with a biological matter anti-stick material.
  • anti-stick materials include non-ionic hydrogels, such as a polyethylene glycol, polyacrylamide, among other compounds with alcohol functionality.
  • the main body and/or screening unit itself comprises an anti-stick material for biological matter in its composition.
  • the main body and/or screening unit is coated with a lubricant.
  • lubricants include water-associating compounds that provide slip to surfaces such as hyaluronic acid.
  • the main body and/or screening unit comprises a lubricant in its composition materials.
  • the main body and/or screening unit comprises a coating having a low friction surface material.
  • low friction surface materials include for example, silicone-based compounds and fluorinated compounds.
  • the main body and/or screening unit comprises a low friction surface material in its composition.
  • the main body and/or screening unit comprises a coating having a hydrophobic surface material.
  • hydrophobic surface materials include, for example, waxes and paraffins.
  • the main body and/or screening unit comprises a hydrophobic material in its composition.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • screening units such as those in FIGS. 3A-3D can induce additional shearing action on organoids, cell aggregates, or other biological matter.
  • 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.
  • FIG. 4A-4B a simplified process of how an elongated screening unit is reformed is depicted.
  • 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.
  • 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.
  • 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).
  • methods are disclosed herein for shearing and/filtering biological matter such as organoids or cell aggregates with a liquid handling device.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • the mixture comprises a growth medium.
  • 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.
  • part of the organoids larger than a certain size 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.
  • 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.
  • 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.
  • the shearing and/or filtering of biological matter occurs internally within the hollow passage.
  • the shearing occurs when the biological matter contact or pass through the screening unit.
  • the shearing occurs both inside the hollow passage and at the screening unit (by contacting and/or passing through it).
  • 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.
  • biological matter such as organoids or cell aggregates are sheared by physical contact with the screening unit.
  • 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.
  • 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.

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Abstract

L'invention propose un dispositif de manipulation de liquide qui cisaille une matière biologique, comprenant des agrégats de cellules (par exemple, des organoïdes) et filtre des fragments des agrégats de cellules (ou d'autres matières biologiques) d'une taille de coupure sélectionnée. En outre, l'invention propose des procédés de cisaillement de matière biologique avec le dispositif de manipulation de liquide et de criblage de fragments de la matière biologique d'une coupure sélectionnée, notamment sans mise en contact du dispositif avec un autre objet solide.
EP24719015.0A 2023-03-31 2024-03-25 Dispositif de manipulation de liquide pour cisaillement et filtration de matière biologique Pending EP4689052A1 (fr)

Applications Claiming Priority (3)

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US202363456424P 2023-03-31 2023-03-31
US202363526603P 2023-07-13 2023-07-13
PCT/US2024/021262 WO2024206199A1 (fr) 2023-03-31 2024-03-25 Dispositif de manipulation de liquide pour cisaillement et filtration de matière biologique

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EP4689052A1 true EP4689052A1 (fr) 2026-02-11

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EP (1) EP4689052A1 (fr)
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US9290731B2 (en) * 2008-12-19 2016-03-22 Stemcell Technologies Inc. Filter apparatus and filter plate system
EP2618933A1 (fr) * 2010-09-23 2013-07-31 Porex Corporation Adaptateur-filtre pour pipettes
EP2933625A4 (fr) * 2012-12-13 2015-12-02 Yamaha Motor Co Ltd Pointe d'aspiration, dispositif d'observation d'objet utilisant ladite pointe d'aspiration, et procédé permettant d'observer un objet
KR101750790B1 (ko) * 2015-04-06 2017-06-27 메디칸(주) 배양세포의 세포간 분리방법
WO2019071297A1 (fr) * 2017-10-09 2019-04-18 Griffith University Appareil et procédés d'élimination de fluide d'une culture cellulaire
JP7501365B2 (ja) * 2018-09-11 2024-06-18 日産化学株式会社 分離デバイスおよびそれを用いて分離対象物を分離する方法

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