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WO2025094147A1 - Procédés et appareil de lavage de cellules cryoconservées - Google Patents

Procédés et appareil de lavage de cellules cryoconservées Download PDF

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Publication number
WO2025094147A1
WO2025094147A1 PCT/IB2024/060837 IB2024060837W WO2025094147A1 WO 2025094147 A1 WO2025094147 A1 WO 2025094147A1 IB 2024060837 W IB2024060837 W IB 2024060837W WO 2025094147 A1 WO2025094147 A1 WO 2025094147A1
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WO
WIPO (PCT)
Prior art keywords
receptacle
liquid
orientation
cells
angle
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
PCT/IB2024/060837
Other languages
English (en)
Inventor
Kok Siang Tan
Chyan Ying KE
Namyong Kim
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.)
Curiox Biosystems Co Ltd
Original Assignee
Curiox Biosystems Co Ltd
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 Curiox Biosystems Co Ltd filed Critical Curiox Biosystems Co Ltd
Publication of WO2025094147A1 publication Critical patent/WO2025094147A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • C12M45/22—Means for packing or storing viable microorganisms

Definitions

  • This application relates to methods, devices, and apparatus for washing cryopreserved cells.
  • Separation of biological cells is a critical step in many biological processes and assays.
  • Conventional methods for separating biological cells include subjecting the biological cells in a liquid to a large acceleration, such as by using a centrifuge. The centrifugal force causes the biological cells to travel to the bottom of a tube and form a precipitate, allowing a remaining liquid called supernatant to be separated from the precipitate.
  • the mechanical force applied on the biological cells may affect the biological cells. For example, too much centrifugal force will cause lysis of biological cells.
  • a method includes maintaining a receptacle with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; and, subsequent to maintaining the receptacle in the first orientation for the first period of time, placing the receptacle in a second orientation distinct from the first orientation.
  • the second orientation has a second angle distinct from the first angle with respect to the vertical axis.
  • the method also includes aspirating the first liquid in the receptacle while leaving the cells in the receptacle; dispensing a second liquid into the receptacle; and aspirating the second liquid in the receptacle while leaving the cells in the receptacle.
  • an apparatus includes a receptacle holder for holding a receptacle; a tilting device coupled with the receptacle holder for placing the receptacle holder in a first orientation at a first time and placing the receptacle holder in a second orientation distinct from the first orientation at a second time distinct from the first time; one or more liquid handling devices; one or more processors; and memory storing one or more programs for execution by the one or more processors.
  • the one or more programs include instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the cells in the receptacle.
  • the second orientation has a second angle distinct from the first angle with respect to the vertical axis.
  • a method includes computer-readable storage medium storing one or more programs for execution by one or more processors in communication with a tilting device and one or more liquid handling devices.
  • the one or more programs including instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle holder in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the receptacle while leaving the
  • Such methods, apparatus, and computer readable storage medium may replace conventional methods, apparatus, and computer readable storage medium. In some cases, such methods, apparatus, and computer readable storage medium may complement conventional methods, apparatus, and computer readable storage medium.
  • Figure l is a perspective view of a plate in accordance with some embodiments.
  • Figure 2 is a cross-sectional view of the plate shown in Figure 1.
  • Figure 3 illustrates a tube in accordance with some embodiments.
  • Figures 4A-4M illustrate devices for separating biological cells or one or more portions thereof in accordance with some embodiments.
  • Figure 5 illustrates experimental results obtained by using the methods described herein.
  • first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another.
  • a first channel could be termed a second channel, and, similarly, a second channel could be termed a first channel, without departing from the scope of the embodiments.
  • the first channel and the second channel are both channels, but they are not the same channel.
  • a first period of time could be termed a second period of time, and, similarly, a second period of time could be termed a first period of time, without departing from the scope of the embodiments.
  • the first period of time and the second period of time are both periods of time, but they are not the same period of time.
  • FIG 1 is a perspective view of a receptacle (e.g., plate 100) in accordance with some embodiments.
  • the plate 100 has atop surface 120 and abottom surface 130 opposite to the top surface 120.
  • a plurality of wells 112 (e.g., wells 112-1 through 112-8) is defined in the plate 100.
  • the plate 100 includes a first portion 140 that corresponds to a bottom of the plurality of wells 112 and a second portion 150 that corresponds to one or more walls of the plurality of wells 112.
  • the plate 100 is formed integrally.
  • the plate 100 is formed by attaching two or more portions together (e.g., by bonding separately formed first and second portions 140 and 150).
  • the plurality of wells 112 is arranged in an array (e.g., 2-by-3 array, 2-by-4 array, 3-by-4 array, 4-by-6 array, 6-by-8 array, 8-by-12 array, 16-by-24 array, 32-by-48 array, etc.).
  • a respective well 112 is a cylindrical well (e.g., a cross-section of the respective well 112 along a plane substantially parallel to the plate 100 has a shape of a circle). Also shown in Figure 1 is line II-II, from which the cross-sectional view of Figure 2 is taken.
  • FIG. 2 is a cross-sectional view of the plate 100 shown in Figure 1.
  • a respective well 112 has a width (W) of 2 mm - 170 mm (e.g., 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm, 85 mm, 90 mm, 95 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, or any interval between two of the aforementioned values, such as 5 mm - 8 mm).
  • W width
  • Figure 2 also shows a first axis 202 defined by a first channel (e.g., well 112-1) and a second axis 204 defined by a second channel (e.g., well 112-2).
  • first axis 202 is parallel to a longitudinal direction of the first channel (e.g., well 112-1).
  • first axis 202 passes through a center of the first channel.
  • second axis 204 is parallel to a longitudinal direction of the second channel (e.g., well 112-2).
  • the second axis 204 passes through a center of the second channel.
  • the first axis 202 is parallel to a height-wise direction of the first channel (e.g., well 112-1).
  • the second axis 204 is parallel to a height-wise direction of the second channel (e.g., well 112-2).
  • the first axis 202 is parallel to the second axis 204.
  • the first axis 202 is nonparallel to the second axis 204 (e.g., the first axis 202 is at a predefined non-zero angle from the second axis 204, such as 1 degree, 5 degrees, 10 degrees, 15 degrees, 20 degrees, or between any two angles selected from the aforementioned angles).
  • Figures 1 and 2 illustrate a plate with a plurality of wells
  • a receptacle having only a single well or channel e.g., a tube
  • a single well or channel e.g., a tube
  • Figure 3 illustrates a tube 300 in accordance with some embodiments.
  • the tube 300 includes a tube wall 310, which defines a channel 302.
  • the channel 302 has, or defines, an axis 318.
  • the axis 318 is parallel to the longitudinal direction of the channel 302.
  • Figure 3 also illustrates that the tube 300 includes a solution 320 containing biological cells 330 or particles in some cases.
  • the solution 320 and the biological cells 330 are not part of the tube 300.
  • Figures 4A-4M illustrate an apparatus 400 for separating biological cells or one or more portions thereof in accordance with some embodiments.
  • the receptacle holder 410 is in a second orientation.
  • the second orientation is a substantially vertical orientation as shown in Figure 4A.
  • the receptacle holder 410 is deemed to be in a substantially vertical orientation when a channel defined by a receptacle to be held by the receptacle holder 410 defines an axis that is in a substantially vertical orientation.
  • the receptacle holder 410 (e.g., a tube holder) is deemed to be in a substantially vertical orientation when a channel defined by the receptacle holder 410 (e.g., a channel into which the tube is to be inserted) defines an axis that is in a substantially vertical orientation.
  • a channel defined by the receptacle holder 410 e.g., a channel into which the tube is to be inserted
  • the first axis 318 defined by the channel 302 of the receptacle 300 or a channel of the receptacle holder 410 is substantially vertical (e.g., the axis 318 is substantially parallel to a vertical direction 440).
  • the receptacle holder 410 (e.g., a plate holder) is deemed to be in a substantially vertical orientation when a base surface of the receptacle holder 410 is substantially perpendicular to the vertical direction 440.
  • Figure 4A also shows one or more processors 412 and memory 414 storing one or more programs for execution by the one or more processors 412.
  • the one or more programs include instructions that cause the one or more processors to send one or more signals or instructions to devices in communications with the one or more processors (e.g., the tilting device 420, an aspirator, a dispenser, etc.).
  • Figure 4A shows that the receptacle holder 410 holder is to be rotated or tilted by the tilting device 420.
  • Figure 4B illustrates that the receptacle holder 410 is a first orientation.
  • the first orientation is a non-vertical orientation as shown in Figure 4B.
  • the receptacle holder 410 is deemed to be in a non-vertical orientation when the channel defined by the receptacle to be held by the receptacle holder 410 defines an axis that is in a non-vertical orientation.
  • the receptacle holder 410 (e.g., a tube holder) is deemed to be in a non-vertical orientation when a channel defined by the receptacle holder 410 (e.g., a channel into which the tube is to be inserted) defines an axis that is in a non- vertical orientation (e.g., the axis has a non-zero angle with respect to the vertical direction).
  • a channel defined by the receptacle holder 410 e.g., a channel into which the tube is to be inserted
  • the first axis 318 defined by the channel 302 of the receptacle 300 or a channel of the receptacle holder 410 is non-vertical (e.g., the axis 318 is non-parallel to the vertical direction 440).
  • the receptacle holder 410 (e.g., a plate holder) is deemed to be in a non-vertical orientation when a base surface of the receptacle holder 410 is nonperpendicular to the vertical direction 440.
  • Figure 4C shows that the receptacle holder 410 is maintained in the first orientation.
  • the tilting of the channel defined by the receptacle 310 speeds up settling of the biological cells or particles.
  • Figure 4C also shows that the receptacle holder 410 is to be rotated or tilted back by the tilting device 420.
  • Figure 4D illustrates that the receptacle holder 410 is in the second orientation.
  • the biological cells 330 or particles are settled down to a bottom of the receptacle.
  • the biological cells 330 or particles may have settled in a non-symmetric manner (e.g., the biological cells 330 or particles may pile more on one end than an opposing end when the receptacle is positioned in a vertical orientation).
  • a top surface defined by the settled biological cells 330 or particles may be non-perpendicular to the vertical direction while the receptacle holder 410 or the receptacle 300 is in the vertical orientation as shown in Figure 4D.
  • Figure 4E illustrates that, in some embodiments, one or more mechanical impulses are provided to the receptacle 310 or the receptacle holder 410.
  • the one or more mechanical impulses cause the biological cells 330 or particles to redistribute.
  • the redistribution of the biological cells 330 or particles causes a top surface defined by the settled biological cells 330 or particles to be substantially perpendicular to the vertical direction while the receptacle holder 410 or the receptacle 300 is in the vertical orientation as shown in Figure 4E.
  • Figure 4F illustrates an aspirator 450 or its tip.
  • the aspirator 450 aspirates at least a portion of the solution 320 (and any substances not settled down with the biological cells 330 or particles, such as non-cellular substances).
  • Figure 4G illustrates that a portion (e.g., a large portion) of the solution 320 is aspirated. A remaining portion 320-2 of the solution 320 with the biological cells 330 is left in the receptacle 300.
  • the aspirated portion 320-1 is also illustrated in the inset of Figure 4G to show that the aspirated portion 320-1 has no or a low concentration of biological cells whereas the remaining portion 320-2 has a high concentration of biological cells.
  • Figure 4H illustrates a dispenser 460 or its tip.
  • the dispenser 460 is distinct from the aspirator 450.
  • the dispenser 460 dispenses a different solution (e.g., a wash buffer), which is mixed with the remaining portion of the solution 320 to form a mixed solution 470.
  • a different solution e.g., a wash buffer
  • the aspiration of a portion of a solution in the receptacle and dispensing of additional solution are repeated to further decrease the concentration of any substances not settled down with the biological cells 330 or particles, such as non-cellular substances.
  • the dispenser 460 or another dispenser provides one or more reagents.
  • Figure 41 illustrates the aspirator 450 or its tip.
  • the aspirator 450 aspirates at least a portion of the solution 470 (and any substances not settled down with the biological cells 330 or particles, such as non-cellular substances).
  • Figure 4J illustrates that a portion (e.g., a large portion) of the solution 370 is aspirated. A remaining portion 470-2 of the solution 470 with the biological cells 330 is left in the receptacle 300.
  • the aspirated portion 470-1 is also illustrated in the inset of Figure 4J to show that the aspirated portion 470-1 has no or a low concentration of biological cells whereas the remaining portion 470-2 has a high concentration of biological cells.
  • Figure 4K illustrates the dispenser 460 or its tip.
  • the dispenser 460 dispenses a solution (e.g., an incubation buffer), which is mixed with the remaining portion of the solution 470 to form a mixed solution 480.
  • a solution e.g., an incubation buffer
  • Figure 4L illustrates the aspirator 450 or its tip.
  • the aspirator 450 aspirates at least a portion of the solution 480.
  • Figure 5 illustrates experimental results obtained by using the methods described herein.
  • Figure 5 also illustrates, on the right side, the viability of thawed PBMC washed by the centrifuge-based method and the centrifuge-free washing. The results show that the viability of thawed PBMC after a washing step is comparable between centrifuge-based washing and centrifuge-less washing.
  • a method includes maintaining a receptacle with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time (e.g., Figures 4B and 4C); and subsequent to maintaining the receptacle in the first orientation for the first period of time, placing the receptacle in a second orientation distinct from the first orientation (e.g., Figure 4D).
  • the second orientation has a second angle distinct from the first angle with respect to the vertical axis.
  • the method also includes aspirating the first liquid in the receptacle while leaving the cells in the receptacle (e.g., Figures 4F-4G); dispensing a second liquid into the receptacle (e.g., Figure 4H); and aspirating the second liquid in the receptacle while leaving the cells in the receptacle (e.g., Figures 4I-4J).
  • aspirating the first liquid in the receptacle while leaving the cells in the receptacle e.g., Figures 4F-4G
  • dispensing a second liquid into the receptacle e.g., Figure 4H
  • aspirating the second liquid in the receptacle while leaving the cells in the receptacle e.g., Figures 4I-4J.
  • the first angle is a non-zero angle.
  • the second angle is less than the first angle.
  • the method includes, prior to maintaining the receptacle with the first liquid in the first orientation, placing in the first orientation the receptacle that had been in a third orientation having a third angle distinct from the first angle with respect to the vertical axis (e.g., Figure 4A).
  • the second angle and the third angle are substantially identical.
  • the method includes, prior to maintaining the receptacle with the first liquid in the first orientation: placing cryopreserved cells into the receptacle; and heating the cryopreserved cells.
  • the method includes, prior to maintaining the receptacle with the first liquid in the first orientation, dispensing the first liquid containing cells that had been cryopreserved into the receptacle.
  • the method includes, prior to maintaining the receptacle with the first liquid in the first orientation: placing cryopreserved cells into the receptacle; and dispensing the first liquid into the receptacle.
  • the first liquid includes an incubation buffer.
  • the method includes repeating the dispensing of the second liquid and the aspiration of the second liquid.
  • the method includes dispensing a third liquid into the receptacle (e.g., Figure 4K); and aspirating the third liquid in the receptacle with the cells for transporting the cells (e.g., Figures 4L and 4M).
  • the third liquid includes an incubation buffer.
  • the first liquid has a greater volume than the second liquid.
  • an apparatus includes a receptacle holder (e.g., receptacle holder 410) for holding a receptacle (e.g., receptacle 310); a tilting device (e.g., tilting device 420) coupled with the receptacle holder for placing the receptacle holder in a first orientation at a first time and placing the receptacle holder in a second orientation distinct from the first orientation at a second time distinct from the first time; one or more liquid handling devices (e.g., aspirator 450 and/or dispenser 460); one or more processors (e.g., processor 412); and memory (e.g., memory 414) storing one or more programs for execution by the one or more processors.
  • a receptacle holder e.g., receptacle holder 410
  • a tilting device e.g., tilting device 420
  • liquid handling devices e.g., aspirator 450
  • the one or more liquid handling devices include a dispenser.
  • the one or more liquid handling devices include a pipette for aspirating and dispensing liquid.
  • a (non-transitory) computer-readable storage medium storing one or more programs for execution by one or more processors in communication with a tilting device and one or more liquid handling devices, the one or more programs including instructions for: causing the tilting device to maintain the receptacle holder with a first liquid containing cells that had been cryopreserved in a first orientation having a first angle with respect to a vertical axis for a first period of time; causing the tilting device to, subsequent to maintaining the receptacle in the first orientation for the first period of time, place the receptacle holder in a second orientation distinct from the first orientation; causing the one or more liquid handling devices to aspirate the first liquid in the receptacle while leaving the cells in the receptacle; causing the one or more liquid handling devices to dispense a second liquid into the receptacle; and causing the one or more liquid handling devices to aspirate the second liquid in the re

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Procédé de lavage de cellules cryoconservées comprenant le maintien d'un récipient avec un premier liquide contenant des cellules ayant été cryoconservées dans une première orientation, et le placement ultérieur du récipient dans une deuxième orientation. Le procédé consiste également à aspirer le premier liquide dans le récipient tout en laissant les cellules dans le récipient ; à distribuer un deuxième liquide dans le récipient ; et à aspirer le deuxième liquide dans le récipient tout en laissant les cellules dans le récipient. L'invention concerne également un appareil de lavage de cellules cryoconservées et un support de stockage lisible par ordinateur stockant des instructions pour le lavage de cellules cryoconservées.
PCT/IB2024/060837 2023-11-02 2024-11-02 Procédés et appareil de lavage de cellules cryoconservées Pending WO2025094147A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363595749P 2023-11-02 2023-11-02
US63/595,749 2023-11-02

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WO2025094147A1 true WO2025094147A1 (fr) 2025-05-08

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130210130A1 (en) * 2010-04-21 2013-08-15 Octane Biotech, Inc. Automated cell culture system
WO2020028406A1 (fr) * 2018-07-30 2020-02-06 Curiox Biosystems Pte Ltd. Procédés, dispositifs et appareil pour laver des échantillons contenant des cellules
US20200181551A1 (en) * 2018-12-06 2020-06-11 Hackensack Meridian Health Center For Discovery and Innovation Pumpless platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation
US20210301239A1 (en) * 2020-03-30 2021-09-30 Kabushiki Kaisha Yaskawa Denki Cell production apparatus, cell production method, computer-readable storage medium, and cell production system
CN115279884A (zh) * 2020-01-13 2022-11-01 奥瑞生物技术有限公司 一种用于处理细胞的设备和方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130210130A1 (en) * 2010-04-21 2013-08-15 Octane Biotech, Inc. Automated cell culture system
WO2020028406A1 (fr) * 2018-07-30 2020-02-06 Curiox Biosystems Pte Ltd. Procédés, dispositifs et appareil pour laver des échantillons contenant des cellules
US20200181551A1 (en) * 2018-12-06 2020-06-11 Hackensack Meridian Health Center For Discovery and Innovation Pumpless platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation
CN115279884A (zh) * 2020-01-13 2022-11-01 奥瑞生物技术有限公司 一种用于处理细胞的设备和方法
US20210301239A1 (en) * 2020-03-30 2021-09-30 Kabushiki Kaisha Yaskawa Denki Cell production apparatus, cell production method, computer-readable storage medium, and cell production system

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