WO2016157324A1 - Procédé de division/séparation de colonie cellulaire - Google Patents

Procédé de division/séparation de colonie cellulaire Download PDF

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Publication number
WO2016157324A1
WO2016157324A1 PCT/JP2015/059724 JP2015059724W WO2016157324A1 WO 2016157324 A1 WO2016157324 A1 WO 2016157324A1 JP 2015059724 W JP2015059724 W JP 2015059724W WO 2016157324 A1 WO2016157324 A1 WO 2016157324A1
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WIPO (PCT)
Prior art keywords
dividing
cell
detaching
colony
cell colony
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PCT/JP2015/059724
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English (en)
Japanese (ja)
Inventor
広斌 周
鈴木　大介
絵里乃 松本
洸 斉藤
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株式会社日立製作所
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Priority to PCT/JP2015/059724 priority Critical patent/WO2016157324A1/fr
Publication of WO2016157324A1 publication Critical patent/WO2016157324A1/fr

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    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/08—Apparatus for tissue disaggregation

Definitions

  • the present invention relates to a technique for dividing / separating cell colonies in a cell culture apparatus.
  • Induced pluripotent stem cells due to their pluripotency can unlimited growth, application to regenerative medicine and drug screening is expected.
  • iPS cells can be cultured in a suspension culture, planar culture in which the cells are adhered to feeder cells or various polymers is generally used. This expansion culture that expands the number of iPS cells requires repeated subculture. At that time, in order to culture a large colony, it is necessary to peel the cultured iPS cell colony from the adhesive surface and pulverize it to a size suitable for the next cell seeding.
  • the entire cultured iPS cell colony is immersed in a peeling enzyme, the colony is lifted from the outer periphery, and then the colony is peeled off from the culture surface and pulverized using a cell scraper.
  • the seeded cell colony created is too large, iPS cells tend to differentiate, while if the seeded cell colony is too small, the cell colony at the time of seeding has a tendency to not grow. It is important to grind to an appropriate size of about 200 to 300 ⁇ m.
  • Patent Document 1 proposes a method of exfoliating colonies with an exfoliating enzyme and then pulverizing them to a seeding size with a cutter.
  • Patent Document 2 proposes a method of dividing a cell cluster of pluripotent stem cells through a microgrid.
  • Patent Document 3 proposes a method of fragmenting cell colonies using a laser cutting pattern.
  • Patent Document 1 it is difficult to control the detachment enzyme treatment time, and there is a problem that damage to cells due to long-time treatment is large.
  • the method described in Patent Document 2 has a problem that when it is used for culturing a large amount of pluripotent stem cells, clogging of a microgrid or the like occurs and appropriate and efficient passage cannot be realized.
  • Patent Document 3 since laser light is used to provide a laser cutting pattern on the culture surface, there is a problem that the colony dividing operation becomes complicated.
  • An object of the present invention is to provide a method for dividing / separating cell colonies that solves the above-described problems, reduces the damage to the cells, and enables the cell colonies to be efficiently cultured.
  • a step of dividing a cell colony cultured on the culture surface into a desired size on the culture surface, a step of introducing a cell detachment enzyme into the divided gap after the division provides a method for dividing / separating cell colonies, which comprises a step of peeling the divided cell colonies from the culture surface after introduction of the cell detachment enzyme.
  • uniform cell colonies can be efficiently detached.
  • FIG. 6 is a plan view showing an application process of a cell colony dividing / peeling method according to Example 1.
  • FIG. It is a figure which shows one structural example of the peeling part of the cell colony division
  • FIG. It is a figure which shows an example of the front-end
  • FIG. FIG. 6 is a diagram showing a configuration example of a cell colony dividing / peeling member according to Example 2.
  • FIG. 6 is a diagram illustrating another configuration example of the cell colony dividing / peeling member according to Embodiment 2.
  • FIG. 6 is a diagram illustrating an example of a schematic configuration of a culture container according to Example 3.
  • FIG. 6 is a diagram illustrating an example of a schematic configuration of a culture container according to Example 3.
  • FIG. 4 is a diagram illustrating a schematic configuration example of a culture apparatus according to Example 3.
  • FIG. 10 is a flowchart showing an example of subculture processing by the culture device according to Example 3.
  • the cell colony dividing / peeling method of this example divides the cell colony before peeling the cell colony from the culture surface, introduces a cell peeling enzyme into the gap after division, and then peels the cell colony from the culture surface This is an example.
  • FIG. 1 is a schematic view showing an embodiment of the cell colony dividing / detaching method of the present embodiment
  • FIG. 2 is a plan view showing an application process of the cell colony dividing / detaching method of the present embodiment.
  • the cell colony dividing / peeling method of the present embodiment is applied to the cell colony 2 cultured on the cell culture surface 1.
  • FIG. 2A shows a state in which a cell colony is divided into two and a divided gap 3 is formed.
  • FIG. 2B shows a state in which the cell detachment enzyme 4 is introduced at the division interval 3.
  • FIG. 2C shows the state of the divided / detached cell colony 5 having a desired size after the introduction of the release enzyme 4.
  • the dividing / peeling member of this example is composed of a peeling enzyme introduction part 6, a plow-shaped dividing part 7, and a cell scraper-shaped peeling part 8.
  • the exfoliation enzyme introduction part 6 has an exfoliation enzyme introduction part inlet 6A for introducing / derived the cell exfoliation enzyme 4 and an exfoliation enzyme introduction part outlet 6B, and the dividing part 7 and the exfoliation part 8 are located in the vicinity of the exfoliation enzyme introduction part outlet 6B. is set up.
  • the exfoliation enzyme introduction part 6 has an exfoliation enzyme introduction part inlet 6A for introducing / derived the cell exfoliation enzyme 4 and an exfoliation enzyme introduction part outlet 6B, and the dividing part 7 and the exfoliation part 8 are located in the vicinity of the exfoliation enzyme introduction part outlet 6B. is set up.
  • the peeling enzyme 4 introduced from the peeling enzyme introduction part inlet 6A of the peeling enzyme introduction part 6 is introduced into the divided gap 3 of the divided cell colony 2 on the cell culture surface 1 from the peeling enzyme introduction part outlet 6B.
  • the cell colony 2 is cultured on the cell culture surface 1, and after the cell colony 2 becomes a predetermined size, the dividing portion of the dividing / peeling member of FIG. 7 is moved at a predetermined interval on the cell culture surface 1 to form a division gap 3 in the cell colony 2 as shown in FIG.
  • the cell detachment enzyme 4 is injected into the separation gap 3 from the detachment enzyme introduction part outlet 6B through the detachment enzyme introduction part 6 as shown in FIG.
  • the detachment part 8 of the detachment / separation member detaches, and a detachment / detachment cell colony 5 is generated as shown in FIG.
  • the time required for the release enzyme treatment can be shortened, resulting in less damage to the cells by the release enzyme. Further, since the cell colony 2 is divided into a desired size of the next seeding size before detachment, the cell colony crushing operation after detachment becomes unnecessary, and the operation time at the time of cell colony passage can be shortened.
  • FIG. 3 shows an example of the peeling part 8 of the cell colony dividing / peeling member of this example.
  • peeling of the cell colony of this example can be performed in a shorter time because the cell colony after the culture is divided and peeled off, but in order to further shorten, the peeling part 8 of the cell colony dividing / peeling member having a cell scraper shape is used. Is used. That is, the cell scraper-shaped exfoliation part 8 can exfoliate the cell colony from the culture surface after the exfoliation enzyme-treated cell colony 2 has been lifted from the outer periphery in the same manner as the cell scraper.
  • FIG. 4 is a diagram showing an example of a tip portion of the dividing portion 7 of the cell colony dividing / peeling member having a plow shape used in the cell colony dividing / peeling method of the present embodiment. If the plow-shaped division part 7 provided with such tip parts 7A and 7B is used, the cell colony 2 can be divided smoothly.
  • the cell colony dividing / peeling member of this example it is desirable to use a material that can withstand high humidity and sterilization, such as resin. Thereby, it can be adapted to sterilization such as ⁇ -ray, and can be applied to physics and chemistry use and regenerative medicine use.
  • Example 1 uniform cell colonies can be efficiently detached by dividing a cell colony into an appropriate size and then introducing a release enzyme into the dividing gap.
  • the shape of the cell culture surface is not specifically described.
  • the cell colony dividing / peeling method and the dividing / peeling member of this embodiment can be applied to each shape of the culture surface.
  • Example 2 an example of a dividing / peeling member suitable for a large culture area and capable of dividing and peeling a large number of cell colonies into a predetermined size at the same time will be described.
  • FIG. 5 is a schematic diagram showing a multiple split / peel member that integrates a plurality of split / peel members according to the second embodiment.
  • portions other than the communication unit 10 are configured to include a plurality of configurations similar to those in the first embodiment, and thus description thereof is omitted here.
  • the detachment enzyme introduction part 6 of the dividing / separation member includes one detachment enzyme introduction part inlet 6 ⁇ / b> A for introducing cell detachment enzyme, a plurality of detachment enzyme introduction part outlets 6 ⁇ / b> B, and a detachment enzyme introduction part inlet.
  • the communication part 10 which connects 6A and several peeling enzyme introduction part exit 6B is provided.
  • the dividing / peeling member of the present embodiment has the multiple structure of the first embodiment by the communication portion 10. And, since the peeling enzyme 4 can be introduced from one peeling enzyme introduction part inlet 6A and can be led out from the plurality of peeling enzyme introduction part outlets 6B through the communication part 10, a large number of cell colonies can be simultaneously formed to a predetermined size. It becomes possible to divide and peel.
  • FIG. 6 is a view showing a hollow grid type dividing / peeling member, which is a modification of the dividing / peeling member of Example 2.
  • the portions other than the hollow grid 11 corresponding to the communication portion 10 and the leg portions 11A of the hollow grid are the same as those in the first embodiment.
  • the detachment enzyme introduction part 6 of the dividing / peeling member includes one detachment enzyme introduction part inlet 6A for introducing a cell detachment enzyme and a plurality of detachment enzyme introduction part outlets.
  • the hollow grid 11 which connects 6B, the peeling enzyme introduction part inlet 6A, and the some peeling enzyme introduction part outlet 6B is provided.
  • the cell colony 2 is divided into a predetermined size by a dividing part attached to the leg part 11A of the hollow grid, and the peeling enzyme 4 is further separated from the leg part 11A of the hollow grid 11.
  • the cell colonies can be detached from the culture surface in a shorter time by injecting into the dividing gap.
  • the third embodiment is an embodiment of the cell colony dividing / peeling method, the culture container and the culture apparatus using the splitting / peeling member of each embodiment described above.
  • 7A and 7B are diagrams showing a schematic configuration example of the culture vessel of Example 3.
  • the culture vessel 12 of FIGS. 7A and 7B includes a culture surface 12A of the culture vessel corresponding to the cell culture surface 1, a lid 12B of the culture vessel, a medium inlet 13, a medium outlet 14, a mixed gas inlet 15, a mixed gas outlet 16, and
  • a culture vessel inner magnet 17 and a culture vessel outer magnet 18 are provided as a mechanism for moving the separation / separation member.
  • This embodiment is an implementation of a culture container that performs culture medium exchange to perform culture medium discharge / injection to the culture container 12, mixed gas exchange to perform mixed gas discharge / injection, cultures cell colonies, and further divides and detaches cell colonies. It is an example.
  • the splitting / peeling member has the same configuration as that of the second embodiment, and a split plow portion corresponding to the split portion and a peeler scraper corresponding to the split portion. Department. These switching operations by the culture vessel inner magnet 17 and the culture vessel outer magnet 18 will be briefly described.
  • the center of the culture vessel external magnet 18 is an axis (indicated by a dotted line), and the culture vessel external magnet 18 is rotated 180 degrees under the control of a control terminal constituting a control unit described later.
  • the culture vessel external magnet 18 is moved in the direction of the arrow (shown in white), and the culture vessel internal magnet 17 is moved in the same direction, whereby the culture surface 12A is divided.
  • the plow part and the peeling scraper part can be moved.
  • FIG. 8 is a diagram showing a schematic configuration example of the culture apparatus according to the present embodiment.
  • the culture apparatus shown in FIG. 8 is installed in an incubator 28 and is connected to a culture medium inlet 13 through a flow path, a liquid feed pump 19, a liquid supply pump 19 and a culture medium bottle 19 ⁇ / b> A connected through a flow path, a culture medium outlet 14 and a flow path.
  • the drainage pump 20 connected in the above, the drainage bottle 20A connected to the drainage pump 20 by the flow path, the mixed gas controller 21 connected to the mixed gas inlet 15 by the flow path, the exhaust gas filter 22, the separation enzyme feed A pump 23, a peeling enzyme bottle 23A connected to the peeling enzyme feeding pump 23 through a flow path, a moving mechanism 24 of the dividing / peeling member, a microscope 25, a moving mechanism 26 of the microscope 25, a temperature control unit 27, and a control signal line 29 And the control terminal 30 constituting the control unit.
  • the control terminal 30 is configured by a personal computer (PC) having a computer configuration including a normal central processing unit (CPU), a storage unit, an input / output interface unit, and the like, and the CPU executes control software.
  • PC personal computer
  • the medium feeding pump 19 the drainage pump 20, the mixed gas controller 21, the peeling enzyme feeding pump 23, the dividing / peeling member moving mechanism 24, the microscope 25, the microscope moving mechanism 26, the temperature
  • the control unit 27 is controlled through the control signal line 29.
  • a cell image is input from the microscope 25 and transmitted to the server in the control terminal 30 or via the network, and image processing is executed by various image processing software.
  • FIG. 9 is a flowchart showing an example of subculture processing by the culture apparatus according to the present embodiment.
  • the culture process is started under the control of the control terminal 30, iPS cell colonies are seeded in the culture vessel (ST1). Thereafter, conditions such as temperature and humidity are adjusted, and the cells are cultured (ST2). Subsequently, when a predetermined medium replacement time is reached, the old medium in the culture container is discharged from the culture container 12 (ST3). Thereafter, a new medium is poured into the culture vessel 12 (ST4). ST3 and ST4 are repeated until the number of cells reaches a predetermined time (ST5).
  • the medium is discharged from the culture vessel (ST6).
  • the cell colony cultured in the dividing unit 7 is divided into an appropriate size, and a peeling enzyme is introduced (ST7).
  • the cell colonies are detached at the separation unit 8 (ST8), and the divided / detached cell colonies 5 after the separation / detachment are collected (ST9).
  • ST1 to ST9 are repeated until the predetermined number of cells is reached (ST10).
  • the culture is terminated.
  • the divided / detached cell colonies obtained in the removing step are used for subculture.
  • the dividing step is performed.
  • the divided / separated cell colonies are seeded in a culture vessel and repeated subculture, and the divided When the number of exfoliated cell colonies reaches a predetermined number, the subculture of the cell colonies is terminated.
  • the culture apparatus of this example by dividing a cell colony into an appropriate size and then introducing a detaching enzyme into the dividing gap, it becomes possible to efficiently detach a uniform cell colony. Subculture can be performed.
  • the culture apparatus of the present embodiment has mechanisms such as automatic cell seeding, automatic medium exchange, automatic passage, and automatic observation, and can maintain and manage more uniform culture quality.

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Abstract

L'invention fournit un procédé de division/séparation de colonie cellulaire selon lequel une colonie cellulaire uniforme est séparée. Selon l'invention, une division/séparation de colonie cellulaire est effectuée efficacement à l'aide d'un élément de division/séparation qui possède : une partie division (7) qui divise selon une taille adéquate une colonie cellulaire (2) préalablement à sa séparation, sur une surface de culture (1) sur laquelle la colonie cellulaire est cultivée ; une partie introduction d'enzyme (6) qui introduit un enzyme de séparation (4) destiné à séparer des cellules, dans l'interstice de division après division par la partie division (7) ; et une partie séparation (8) qui sépare complètement la colonie cellulaire ainsi divisée.
PCT/JP2015/059724 2015-03-27 2015-03-27 Procédé de division/séparation de colonie cellulaire WO2016157324A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2531969Y2 (ja) * 1992-03-04 1997-04-09 住友ベークライト株式会社 セルスクレーパー
JP2008508957A (ja) * 2004-08-06 2008-03-27 サイトレイト ベースローテン フェンノートシャップ 角膜の表面から上皮層を分離するための装置
CN102120983A (zh) * 2010-01-07 2011-07-13 中国农业科学院北京畜牧兽医研究所 北京鸭表皮干细胞的分离和培养方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2531969Y2 (ja) * 1992-03-04 1997-04-09 住友ベークライト株式会社 セルスクレーパー
JP2008508957A (ja) * 2004-08-06 2008-03-27 サイトレイト ベースローテン フェンノートシャップ 角膜の表面から上皮層を分離するための装置
CN102120983A (zh) * 2010-01-07 2011-07-13 中国农业科学院北京畜牧兽医研究所 北京鸭表皮干细胞的分离和培养方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LISA M HOFFMAN, NATURE BIOTECHNOLOGY, vol. 23, no. 6, 2005, pages 699 - 708 *
MAGDALINE COSTA, CURRENT PROTOCOLS IN STEM CELL BIOLOGY, 2008, pages 1C.1.1 - 1C.1.7 *

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