WO2014017481A1 - PROCÉDÉ DE DÉTACHEMENT DE CELLULES PAR CHOIX DE RÉGION ET PROCÉDÉ DE CULTURE AINSI QUE PROCÉDÉ DE REPIQUAGE DE CELLULES METTANT EN œUVRE CE PROCÉDÉ DE DÉTACHEMENT - Google Patents

PROCÉDÉ DE DÉTACHEMENT DE CELLULES PAR CHOIX DE RÉGION ET PROCÉDÉ DE CULTURE AINSI QUE PROCÉDÉ DE REPIQUAGE DE CELLULES METTANT EN œUVRE CE PROCÉDÉ DE DÉTACHEMENT Download PDF

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WO2014017481A1
WO2014017481A1 PCT/JP2013/069906 JP2013069906W WO2014017481A1 WO 2014017481 A1 WO2014017481 A1 WO 2014017481A1 JP 2013069906 W JP2013069906 W JP 2013069906W WO 2014017481 A1 WO2014017481 A1 WO 2014017481A1
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cells
cell
culture
passage
present
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English (en)
Japanese (ja)
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慎一 五味
成則 尾▲崎▼
智瑛 倉員
伸 川真田
直希 西下
ちえみ 竹中
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東京エレクトロン株式会社
公益財団法人先端医療振興財団
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Publication of WO2014017481A1 publication Critical patent/WO2014017481A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces
    • C12N2521/10—Sound, e.g. ultrasounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2539/00—Supports and/or coatings for cell culture characterised by properties

Definitions

  • the present invention relates to a method for selectively detaching cells from an adhesive surface of a culture vessel, particularly a closed culture vessel.
  • the present invention also relates to a cell culture method and a cell passage method using this cell detachment method.
  • the open culture is a culture using a normal dish in which the medium is exchanged and passaged by opening the lid of the dish. Open culture is often used for research cell culture because it is inexpensive and easy to operate. However, there is a high possibility of contamination with bacteria and cells. There are many safety issues such as high risk of infection for workers, and it is difficult to say that it is a perfect equipment for cell culture for treatment.
  • closed culture In order to deal with such problems, closed culture has been attempted. In closed culture, a closed culture vessel having a minimum opening is used, and a device for greatly reducing the incidence of contamination and the risk of infection has been devised. In addition, closed culture vessels and the like that have been devised to reduce the amount of medium without reducing the number of cells that can be cultured are commercially available, and are useful when using expensive reagents or rare samples.
  • pluripotent stem cells such as embryonic stem cells (ES cells) and inducible pluripotent stem cells (iPS cells)
  • ES cells embryonic stem cells
  • iPS cells inducible pluripotent stem cells
  • cells that have started differentiation in the middle of the culture have adverse effects on surrounding cells. And should be promptly removed from the culture vessel.
  • cells that have started to differentiate should be removed promptly in order to increase the yield and purity of undifferentiated cells.
  • the cells that have started differentiation are removed by, for example, opening the dish lid, inserting an aspirator or micropipette into the dish, and sucking and removing the cells that have started differentiation (non-patented). Reference 1).
  • the culture system is in contact with the outside world, there is a concern about the occurrence of contamination in the open culture.
  • closed culture although the possibility of contamination is low, an aspirator cannot be inserted into the culture vessel, and it is difficult to selectively remove cells that have started differentiation during the culture.
  • the present invention relates to a method of detaching cells from an adhesive surface of an open or closed culture vessel in a region-selective and non-contact manner, and a cell culturing method and a passage method using the method.
  • the purpose is to provide.
  • the present inventors have found that cells can be selectively detached from the adhesive surface inside the culture container by applying high-frequency vibration from the outside of the culture container.
  • the present inventors have also found that pluripotent stem cells can be favorably cultured even in closed culture by selectively removing cells that have started differentiation by high-frequency vibration. Further, the present inventors have found that cells are peeled off in a sheet form while maintaining cell-cell adhesion by high-frequency vibration. Furthermore, the present inventors have found that undifferentiated pluripotent stem cells exfoliated into a sheet can be cultured well in an undifferentiated state even after passage. The present invention has been made based on these findings.
  • a method of selectively detaching cells from an adhesive surface by high-frequency vibration (2) The method according to (1) above, wherein the adhesive surface is an adhesive surface of a closed culture vessel. (3) The method according to (1) or (2) above, wherein the cells to be detached are cells that are unnecessary for culture and / or passage. (4) The method according to (1) or (2) above, wherein the cells to be detached are cells necessary for passage. (5) A cell culturing method comprising a step of removing cells unnecessary for culturing and / or passage, wherein the step of removing cells is performed by the method described in (3) above.
  • a cell passage method comprising a step of recovering cells necessary for passage, wherein the step of recovering cells is performed by the method described in (4) above.
  • a cell subculturing method comprising a step of removing cells unnecessary for culture and / or passage and a step of collecting cells necessary for passage, the step of removing cells Is performed by the method described in (3) above, and the step of recovering cells is performed by the method described in (4) above.
  • the method according to (4), (6) or (7) above, wherein the cells required for passage are undifferentiated cells or appropriately differentiated cells.
  • the cells by applying high-frequency vibration from the outside of the culture vessel toward the adhesion surface, the cells can be detached from the adhesion surface easily and region-selectively.
  • the risk of contamination can be reduced in both open and closed cultures, and it is advantageous in that cells can be selectively and easily detached particularly in closed cultures.
  • the present invention is also advantageous in that, by appropriately removing cells that have started differentiation by the method of the present invention, undifferentiated pluripotent stem cells can be easily cultured even in closed culture. . Further, according to the present invention, undifferentiated pluripotent cells can be detached from the adhesive surface in a sheet form while maintaining the cell-cell adhesion.
  • the peeled sheet-like undifferentiated pluripotent stem cells are advantageous in that they can be cultured well in an undifferentiated state.
  • the present invention it is advantageous in that cells can be peeled off from the adhesive surface in a sheet form without using expensive culture equipment or the like that has been subjected to special surface processing.
  • the detached cell sheet can be advantageously used for medical applications such as transplantation.
  • a pluripotent stem cell sheet it is advantageous in that it can be separated into cell clusters of an appropriate size and used suitably for passage, differentiation induction and / or production of a genetically modified animal.
  • FIG. 1A is a diagram showing an adhesive surface after peeling when cells are peeled by enzyme treatment.
  • FIG. 1B is a diagram showing an adhesive surface after peeling when cells are peeled using an ultrasonic cleaner.
  • FIG. 1C is a diagram showing an adhesion surface when an iPS cell colony that has started differentiation is selectively detached using an ultrasonic crusher.
  • FIG. 1D is a diagram showing an adhesion surface when an undifferentiated iPS cell colony is selectively detached using an ultrasonic crusher.
  • FIG. 2A is a diagram showing that cells are peeled into a sheet when an ultrasonic crusher is used.
  • FIG. 2B is a diagram showing a phase contrast microscopic image of iPS cells after subculture using cells peeled into a sheet shape using an ultrasonic crusher.
  • FIGS. 2C to G show stained images by alkaline phosphatase staining (FIG. 2C), Oct3 / 4 (FIG. 2D), Nanog (FIG. 2E), SSEA3 in iPS cells after 10 subcultures under feederless conditions, respectively. (FIG. 2F) and immunostained images showing the expression of TRA-1-60 (FIG. 2G).
  • the “cell” used in the present invention is not particularly limited as long as it is a cell applicable to adhesion culture, and can be a mammalian cell such as a primate cell or a rodent cell, preferably a human, monkey, mouse Rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow or goat cell or cells derived therefrom.
  • the cells used in the present invention can also be used without particular limitation regardless of the organ or tissue from which they are derived.
  • nerve cells for example, nerve cells, muscle cells, heart cells, lung cells, kidney cells, hepatocytes, epithelial cells, endothelium Cells or somatic cells such as fibroblasts, these stem cells, germ cells or pluripotent stem cells, preferably pluripotent stem cells.
  • pluripotent stem cells used in the present invention, embryonic stem cells (ES cells), inducible pluripotent stem cells (iPS cells or induced pluripotent stem cells), Muse cells (Multilineage-differentiating Stress Enduring Cell), embryonic Examples include pluripotent stem cells such as tumor cells (EC cells) or embryonic germ stem cells (EG cells), preferably ES cells or iPS cells.
  • the pluripotent stem cells used in the present invention are more preferably pluripotent stem cells from mammals such as primates or rodents, and even more preferably ES cells or iPS from mammals such as primates or rodents A cell, most preferably a human ES cell or a human iPS cell.
  • a cell that grows by forming a monolayer on the adhesive surface or a cell that grows by forming a multilayer may be used.
  • the “adhesion surface” used in the present invention means the surface of a culture vessel to which cells are adhered, and is an adhesion surface of an open culture vessel or an adhesion surface of a closed culture vessel.
  • the “adhesion surface” used in the present invention is not particularly limited, but is a surface suitable for cell culture, and preferably a high-frequency vibration conductive surface suitable for cell culture.
  • the high-frequency vibration conductive surface suitable for cell culture is preferably the surface of a cell culture membrane, more preferably the surface of a gas permeable membrane for cell culture.
  • the gas permeable membrane for cell culture is not particularly limited, but a membrane that is permeable to oxygen and / or carbon dioxide can be used, such as a polystyrene membrane, a collagen membrane, or ethylene. A film made of vinyl acetate can be used.
  • Examples of the culture vessel provided with a gas permeable membrane for cell culture include OptiCell (trademark) (manufactured by Nunk), MultiLife (trademark) (manufactured by Takara Bio) and cell culture bag (trademark) (manufactured by ASTEC). Are already on the market.
  • the “adhesion surface” used in the present invention does not necessarily have to be coated, but is preferably coated with a cell adhesion from the viewpoint of improving the cell adhesion efficiency.
  • gelatin for example, gelatin, collagen, laminin, entactin, hyaluronic acid, Matrigel (trademark) (made by BD), CELLstart (trademark) (made by Invitrogen), pronectin (trademark) (Manufactured by Wako Pure Chemical Industries, Ltd.), an adhesive oligopeptide, an extracellular matrix (ECM) coating such as polylactic acid, polyglycolic acid, polylysine or polyornithine, or a coating such as a polymer coating.
  • ECM extracellular matrix
  • a coating for adhesion of pluripotent stem cells gelatin, collagen, Matrigel (trademark) (manufactured by BD), CELLstart (trademark) (manufactured by Invitrogen) or Pronectin (trademark) (manufactured by Wako Pure Chemical Industries, Ltd.)
  • a surface coating is preferably used.
  • ECM coating from the viewpoint of improving the adhesion of the ECM to the dish surface, it is preferable to perform a treatment that precharges the dish surface.
  • the dish surface is previously treated with plasma or corona discharge. It is preferable to keep it.
  • the coating for cell adhesion can be performed using the method described in the instruction manual by the manufacturer or other known methods. According to the present invention, feeder cells may or may not be used.
  • cells can be detached from the cell adhesion surface of the culture vessel by applying high-frequency vibration.
  • separation of cells from the cell adhesion surface of the culture container can be performed by applying high-frequency vibration to the cell adhesion surface from the outside of the culture container (the back side of the cell adhesion surface). That is, according to the present invention, cells can be detached by applying high-frequency vibration from the outside of the culture vessel without directly touching the cells themselves (that is, in a non-contact manner).
  • the risk of occurrence of contamination in the culture system is extremely low.
  • the present invention automates the treatment for detaching cells from the cell adhesion surface of the culture vessel. It is also possible to do.
  • the cells can be selectively peeled off from the adhesive surface by selectively applying high frequency vibration. Since cell detachment can be performed regardless of whether it is an open system or a closed system by using a high-frequency vibration conductive cell culture vessel, according to the present invention, in an open system culture or a closed system culture.
  • a method of selectively detaching cells from an adhesive surface by high-frequency vibration is provided.
  • giving high-frequency vibration to a region selectively means giving high-frequency vibration having a strength required for cell detachment only to a target region, and cells do not detach. It is permissible for some degree of vibration to be transmitted outside the region.
  • area-selective cell detachment means that only cells adhered to a target area are selectively detached, and the target area is a cell adhesion of a culture vessel. Even a part of the surface may extend over the entire cell adhesion surface.
  • the frequency and energy of the “high-frequency vibration” for cell detachment according to the present invention may be in any range as long as the cell detaches from the adhesive surface.
  • the time required for cell detachment is mainly related to the frequency and energy of the high-frequency vibration, so that those skilled in the art can perform high-frequency vibration according to the desired exfoliation speed.
  • the frequency and energy can be easily determined.
  • the vibration frequency of the high-frequency vibration for cell detachment according to the present invention is not particularly limited, but is preferably a sound wave or ultrasonic region frequency, for example, 20 Hz to 10 GHz, preferably 16 kHz to 10 GHz.
  • the vibration energy of the high-frequency vibration is not particularly limited, but is preferably 1 W to 100 W, more preferably 4 W to 65 W, and most preferably 20 W to 40 W.
  • the separation of cells from the adhesive surface is not particularly limited, but can be performed using a high-frequency vibration generator.
  • a high-frequency vibration generator that can selectively apply high-frequency vibrations to the bonding surface.
  • a high-frequency vibration generator that can selectively apply high-frequency vibration to the bonding surface
  • a high-frequency vibration generator (a portion that transmits high-frequency vibration to an object) has a convex tip such as a horn or a chip.
  • the high-frequency vibration generating unit of the high-frequency vibration generating device can be appropriately selected depending on the size and shape of the adhesion surface of the cell to be peeled.
  • the high-frequency vibration generating unit can be circular, and in particular, pluripotent stem cells
  • the diameter can be 0.1 mm to 5 mm, preferably 0.1 mm to 2 mm, More preferably, it can be 0.1 mm to 0.5 mm, and the diameter can be freely selected according to the size of the cell region to be detached.
  • the high-frequency vibration generating portion has a shape other than a circle, it is possible to select one having an area equivalent to that of a circle.
  • the high-frequency vibration generator used in the present invention is not particularly limited, and examples thereof include an ultrasonic generator including a high-speed rotary blade type, a Langevin type, a piezoelectric type, and a magnetostrictive type transducer.
  • Examples of such a high-frequency vibration generator include an ultrasonic crusher (ultrasonic homogenizer).
  • ultrasonic crushers such as Taitec Corporation, NR-50M (Microtech Nichion Corporation) and Sonifier (BRANSON Corporation) are commercially available.
  • the high frequency vibration can be given as follows. That is, first, a high-frequency vibration generating portion (such as the tip of a microchip) of the high-frequency vibration generating device is brought into contact with the adhesive surface of the cell to be peeled from the outside of the culture vessel. Then, the high-frequency vibration generated from the high-frequency vibration generating unit is transmitted to the adhesion surface inside the container via the culture container (for example, a membrane), and the cells are peeled selectively from the adhesion surface. When it is desired to peel the cells in a wider area, the high-frequency vibration generator may be moved up and down and left and right on the outer surface of the container, and the cells can be peeled in the moved range.
  • a high-frequency vibration generating portion such as the tip of a microchip
  • the cells can be detached without bringing the high-frequency vibration generating portion into contact with the outside of the bonding surface.
  • the high-frequency vibration generator is brought into contact with the outside of the adhesive surface or as close as possible.
  • the generated high-frequency vibration may be transmitted more extensively than the area where the high-frequency vibration generator is in contact, but according to the present invention, even in such a case, the location where the high-frequency vibration generator is in direct contact with Cells can be detached selectively in the vicinity only in the vicinity.
  • the cells detached from the adhesion surface by applying high-frequency vibration may be removed or recovered. Therefore, according to the present invention, by applying high-frequency vibration, cells in a specific region on the adhesive surface can be selectively removed or collected selectively. Therefore, the method of the present invention is particularly useful when removing and / or recovering cells in a closed culture where it has been difficult to detach cells selectively.
  • the cell detachment method of the present invention only a specific cell group can be selectively detached and removed or collected from a plurality of types of cell groups. More specifically, for example, when performing subculture for the purpose of maintaining undifferentiated cells, cells that have started differentiation may be removed regionally or undifferentiated cells may be regionally selected. It may be recovered.
  • undifferentiated cells or cells that do not differentiate appropriately may be selectively removed, or cells that are appropriately differentiated (for example, the desired function or property) Cells are acquired in a selective manner for later application, for example, gene transfer experiments, biochemical experiments or differentiation induction experiments, drug or toxin screening, or medical use as cells for medicine or transplantation. You may use for.
  • the cells that selectively peel off are cells that are unnecessary for culture and / or passage, or cells that are necessary for passage. Therefore, in the present invention, a method for removing cells unnecessary for culturing and / or passage using the cell detachment method of the present invention, and a cell necessary for passage using the cell detachment method of the present invention are recovered. A method is provided.
  • cells that are not required for culture and / or passage are cells that have started to differentiate for the purpose of maintaining undifferentiated cells, and the cells are differentiated into a specific tissue or cell.
  • the cells necessary for passage are undifferentiated cells for the purpose of maintaining undifferentiated cells, and the cells are subcultured after being differentiated into a specific tissue or cell.
  • it is a suitably differentiated cell (eg, a cell that has differentiated and acquired the desired function or property).
  • the above is an example of the necessity of the cell based on the differentiation state of the cell, but the necessity of the cell is not particularly limited, but may be appropriately determined by those skilled in the art based on the physiological state of the cell and the form of the cell.
  • cells unnecessary for culturing and / or passage may be removed only once at a predetermined time (for example, once immediately before passage) or periodically (for example, It may be performed every 3 hours, every 6 hours, every 12 hours, every day, every 2 days) or irregularly multiple times.
  • cells unnecessary for culture and / or passage are selectively detached from the adhesion surface by high-frequency vibration and removed from the culture vessel. According to the present invention, unnecessary cells are removed during culturing or at the time of passage, so that cells unnecessary for culturing and / or passage can be mixed with cultured cells or cells to be passaged. Can be prevented.
  • cells necessary for subculture are selectively separated from the adhesive surface by high-frequency vibration and collected.
  • the cells collected in this manner are hardly damaged during detachment and can be used for subsequent subculture.
  • cells unnecessary for culture and / or passage are selectively removed from the adhesion surface by high-frequency vibration, and cells necessary for passage are selectively selected from the adhesion surface by high-frequency vibration. It can be recovered.
  • the cell detachment method of the present invention is useful for culturing pluripotent stem cells (for example, ES cells or iPS cells). That is, according to the present invention, for example, pluripotent stem cells that have started to differentiate by applying high-frequency vibrations (that is, pluripotent stem cells unnecessary for culture and / or passage) can be removed. Moreover, according to the present invention, only colonies containing pluripotent stem cells that have started to differentiate in units of individual colonies can be easily and selectively removed by high-frequency vibration. For this reason, the method for detaching cells of the present invention is particularly useful in removing pluripotent stem cells that are unnecessary for culture and / or passage. Further, according to the present invention, high frequency vibration can be applied from the outside of the culture vessel.
  • pluripotent stem cells for example, ES cells or iPS cells. That is, according to the present invention, for example, pluripotent stem cells that have started to differentiate by applying high-frequency vibrations (that is, pluripotent stem cells unnecessary for
  • the cell detachment method of the present invention is particularly useful in closed culture. Furthermore, according to the present invention, undifferentiated pluripotent stem cells left in the culture container after removing unnecessary cells by applying high-frequency vibration can be suitably cultured in an undifferentiated state. Therefore, the method for detaching cells according to the present invention can be advantageously used for removing pluripotent stem cells that have started to differentiate and for culturing pluripotent stem cells, particularly in closed culture.
  • undifferentiated cells such as undifferentiated pluripotent stem cells (that is, cells necessary for passage) can be further recovered by applying high-frequency vibration.
  • the passage of pluripotent stem cells it is not preferable to dissociate the cells to the single cell level, but cells that have been detached by applying high-frequency vibrations according to the method of the present invention Surprisingly, it maintains cell-cell adhesion and does not dissociate to the single cell level. Therefore, the cells collected using the method of the present invention can be suitably used for various subsequent applications and subcultures. Therefore, the cell detachment method of the present invention is particularly useful in the recovery of pluripotent stem cells.
  • the cell detachment method of the present invention is particularly useful in closed culture.
  • the undifferentiated pluripotent stem cells recovered and passaged using the present invention can be suitably cultured in an undifferentiated state. Therefore, the cell detachment method of the present invention is advantageous for recovering undifferentiated pluripotent stem cells from the adhesive surface, and for passage of pluripotent stem cells, particularly passage of pluripotent stem cells in closed culture. Can be used.
  • only a part of cells necessary for passage may be selectively detached and recovered, or all cells necessary for passage may be detached and recovered. .
  • Cells collected by the method of the present invention can be passaged after being appropriately divided into cell clusters of a preferred size by pipetting water flow or the like.
  • cells can be peeled off from the adhesive surface in a sheet form.
  • the sheet-like cell (cell sheet) peeled from the adhesive surface according to the present invention can be removed or collected by, for example, a normal disposable syringe or the like. Since the cell sheet obtained by the present invention has the property of quickly sinking to the lower part of the culture container, it can be easily removed or collected by tilting the culture container. Specifically, for example, the cells can be detached in a state where the culture container is set up vertically with respect to the adhesion surface, and the detached cells can be settled and collected at the lower part of the culture container.
  • the cells may be collected perpendicularly to the adhesion surface, and the detached cells may be allowed to settle at the bottom of the culture vessel.
  • the cells may be allowed to settle at the bottom of the culture container while shaking the culture container.
  • a method for preparing a cell sheet and a prepared cell sheet comprising peeling cells from an adhesive surface according to the present invention.
  • the step of peeling the cells from the adhesive surface into a sheet shape can be performed according to the present invention.
  • it is not necessary to use a special culture vessel for producing the sheet and a normal vessel can be used as long as it is a culture vessel having a high-frequency vibration-transmitting surface.
  • the present invention there is no need to insert a high-frequency vibration source such as an ultrasonic probe into the culture vessel, which is advantageous in that the risk of contamination and the like can be reduced even in open culture.
  • a high-frequency vibration source such as an ultrasonic probe
  • the present invention is further advantageous in that the use of a closed culture vessel can greatly reduce the risk of contamination and the like.
  • the cell sheet obtained by the method of the present invention retains cell-cell adhesion, the engraftment rate after transplantation to a tissue or the like is high, and it can be suitably used as a biomaterial for medical applications such as cultured skin. It is expected.
  • the cell sheet is a pluripotent stem cell sheet, it can be separated into cell clusters of an appropriate size and used suitably for passage, differentiation induction and / or production of genetically modified animals.
  • an enzyme treatment for weakening the adhesion between the cells and the adhesion surface may be performed before the cells are detached from the adhesion surface.
  • treatment with an enzyme such as trypsin or dispase is well known and can be appropriately performed by those skilled in the art.
  • Example 1 Area-selective cell detachment in closed-system culture
  • a method for selectively detaching cells in a closed-system culture was examined.
  • iPS cells established strain by Kawasada Laboratory, Cell Evaluation Group, Advanced Medical Promotion Foundation
  • SNL cells manufactured by DS Pharma Biomedical, product number: EC07032801
  • the medium is a medium for human ES cell culture (Dulbecco's modified Eagle medium / nutrient mixture F-12 Ham (manufactured by Sigma Aldrich, product number: D6421), 500 mL, knockout serum substitute (manufactured by Invitrogen, product number) : 10828-028) 125 mL, non-essential amino acid solution (manufactured by Sigma-Aldrich, product number: M7145) 5 mL, 200 mM L-glutamine (manufactured by Invitrogen, product number: 25030-081) 6.25 mL, 0.1 M 2-mercapto PBS (Invitrogen, product number: 21985) 500 ⁇ L with ethanol added, bFGF (manufactured by Wako Pure Chemical Industries, product number: 064-04541) final concentration of 5 ng / mL, and in the feeder-less condition, ReproFF2 medium (Repr A medium in which b
  • Cell seeding was performed by suspending iPS cells in 10 mL of medium and introducing the cells into OptiCell using OptiCell Tip (trademark).
  • peeling method in addition to the usual peeling method by enzyme treatment, a peeling method using an ultrasonic cleaner and a peeling method using an ultrasonic crusher were tested.
  • trypsin-EDTA 0.25% trypsin, 1 mM EDTA ⁇ 4Na
  • US CLEANER manufactured by ASONE, product number: US-2A
  • the ultrasonic generation conditions of the ultrasonic cleaner were an output of 80 W and a frequency of 40 kHz.
  • peeling using an ultrasonic crusher is performed by using an ultrasonic probe (UR-21P) manufactured by TOMY equipped with an ultrasonic wave generator having a tip having a diameter of 2 mm. This was done by contacting from the outside.
  • the ultrasonic probe was oscillated with an output of 32 W and a frequency of 28 kHz.
  • Example 2 Subculture of iPS cells detached by applying high-frequency vibrations
  • iPS cell colonies that started differentiation that adversely affect the culture were removed and detached using an ultrasonic disrupter. Whether undifferentiated iPS cell colonies can be recovered using an ultrasonic disrupter and then used for subsequent subculture, whether or not the cells can be suitably used while maintaining a good undifferentiated state It was confirmed.
  • the culture vessel, iPS cells, medium and ultrasonic crusher were the same as in Example 1. After seeding the cells in OptiCell, the iPS cell colonies that started to differentiate were quickly and appropriately selectively removed by an ultrasonic crusher as soon as they were discovered. Thereafter, when iPS cells became confluent in the culture vessel, undifferentiated iPS cells were detached by an ultrasonic disrupter and subcultured. iPS cells could be detached easily and quickly by moving up, down, left and right with the probe of the ultrasonic crusher in contact with the gas permeable membrane. Further, the iPS cells detached in this way were in the form of a sheet (FIG. 2A). Sheet-shaped iPS cells could be easily made into cell clusters of a size suitable for passage by pipetting. Thereafter, the iPS cell mass was suspended in a fresh medium and seeded in OptiCell.
  • iPS cells could be suitably cultured even after being detached and subcultured using an ultrasonic disrupter (FIG. 2B).
  • a long-term passage test was conducted to confirm the safety and stability of the peeling method using high-frequency vibration. Specifically, in order to confirm the differentiation state of iPS cells after 10 passages, alkaline phosphatase staining was performed using iPS cells passaged 10 times under feederless conditions (FIG. 2C) or iPS cells. Expression of Oct3 / 4 (FIG. 2D), Nanog (FIG. 2E), SSEA3 (FIG. 2F), and TRA-1-60 (FIG. 2G), known as undifferentiated markers of cells, was confirmed by immunostaining.
  • Anti-Oct3 / 4 antibody (Santa Cruz Biotechnology, product number: sc-5279), anti-Nanog antibody (ReproCell, product number: RAB0003P), anti-SSEA3 antibody (Chemicon, product number: MAB4303)
  • immunostaining was performed according to a conventional method using an anti-TRA-1-60 antibody (manufactured by Chemicon, product number: MAB4306).
  • the cells were well stained with alkaline phosphatase staining, as shown in FIG. 2C, and all undifferentiated markers were found as shown in FIGS. It was expressed well.
  • Example 2 From the results of Example 2, even when the iPS cell colonies that started differentiation were removed by applying high-frequency vibration, other cells left on the adhesion surface were not adversely affected, and high-frequency vibration was applied. It was revealed that the detached cells themselves can be successfully used for subsequent subculture while maintaining the undifferentiated state. In particular, when an ultrasonic crusher is used, the cells can be detached and collected in a sheet form without destroying the cell-cell adhesion, and the collected cells can be collected by pipetting or the like. It became clear that it can be used suitably for subsequent subculture.
  • pluripotent stem cells can be well cultured in an undifferentiated state even in closed culture by applying high-frequency vibration to remove and / or collect cells. It is considered that high-frequency vibration can be used for detachment of cells other than pluripotent stem cells, and it can be suitably used for recovery of necessary cells and removal of unnecessary cells in all adherent cells. It became.

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Abstract

L'invention a pour objectif de fournir un procédé pour détacher par choix de région des cellules d'une surface d'adhésion d'un réceptacle de culture, et à l'aide d'un moyen sans contact. L'invention a également pour objectif de fournir un procédé de culture ainsi qu'un procédé de repiquage de cellules mettant en œuvre ce procédé de détachement. Le procédé de l'invention permet de détacher des cellules par choix de région à partir d'une surface d'adhésion, à l'aide de vibrations de haute fréquence.
PCT/JP2013/069906 2012-07-23 2013-07-23 PROCÉDÉ DE DÉTACHEMENT DE CELLULES PAR CHOIX DE RÉGION ET PROCÉDÉ DE CULTURE AINSI QUE PROCÉDÉ DE REPIQUAGE DE CELLULES METTANT EN œUVRE CE PROCÉDÉ DE DÉTACHEMENT WO2014017481A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016052269A (ja) * 2014-09-03 2016-04-14 テルモ株式会社 シート状細胞培養物回収システムおよび方法
JP2016116460A (ja) * 2014-12-19 2016-06-30 パナソニック株式会社 細胞培養装置
WO2019044804A1 (fr) * 2017-08-28 2019-03-07 学校法人慶應義塾 Méthode de production de cellule et dispositif de production de cellule
CN109706078A (zh) * 2019-02-25 2019-05-03 常州市第一人民医院 一种细胞分选系统及不良细胞分选方法
US10538731B2 (en) * 2015-02-19 2020-01-21 Commissariat A L'energie Atomique Et Aux Energies Alternatives Device for manipulating biological cells using a vibrating support
WO2023079905A1 (fr) * 2021-11-04 2023-05-11 株式会社日立製作所 Dispositif de décollement de cellules et procédé de décollement de cellules

Families Citing this family (8)

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Publication number Priority date Publication date Assignee Title
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JP6004149B1 (ja) * 2015-03-16 2016-10-05 パナソニック株式会社 ピペットチップ及びピペッティング方法
WO2017006942A1 (fr) 2015-07-06 2017-01-12 株式会社朝日ラバー Récipient renfermant des cellules et procédé de culture cellulaire à l'aide d'un tel récipient
JP6593589B2 (ja) * 2015-09-30 2019-10-23 澁谷工業株式会社 細胞塊シートの作製方法および細胞塊シート
JP6956956B2 (ja) * 2017-08-08 2021-11-02 学校法人慶應義塾 細胞生産方法
JP2023067822A (ja) * 2021-10-29 2023-05-16 キヤノン株式会社 細胞剥離液及び細胞剥離方法、細胞保存方法
JP2024000178A (ja) * 2022-06-20 2024-01-05 株式会社日立製作所 細胞剥離装置および細胞剥離方法
JP2024077845A (ja) * 2022-11-29 2024-06-10 キヤノン株式会社 細胞剥離方法、細胞剥離システム及び情報処理装置

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006314204A (ja) * 2005-05-10 2006-11-24 Chiyoda Corp 培養容器からの動物細胞の剥離方法および剥離回収方法
JP2008000008A (ja) * 2006-06-20 2008-01-10 Seiko Instruments Inc 細胞剥離方法
JP2008079554A (ja) * 2006-09-28 2008-04-10 Chiyoda Corp 閉鎖系の細胞回収装置及び細胞培養装置並びに細胞の回収方法及び培養方法
WO2011028524A1 (fr) * 2009-08-24 2011-03-10 Wisconsin Alumni Research Foundation Progéniteur rétinien humain sensiblement pur, progéniteur de cerveau antérieur, et cultures de cellules d'épithélium pigmentaire rétinien et leurs procédés de fabrication
JP2011115080A (ja) * 2009-12-02 2011-06-16 Terumo Corp シート状細胞培養物剥離システム
WO2011108503A1 (fr) * 2010-03-01 2011-09-09 独立行政法人科学技術振興機構 Procédé de détachement de cellules mises en culture, dispositif de détachement de cellules utilisé dans ledit procédé pour détacher des cellules mises en culture et incubateur
JP2012039947A (ja) * 2010-08-19 2012-03-01 Osaka Prefecture Univ 細胞培養用基材

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006314204A (ja) * 2005-05-10 2006-11-24 Chiyoda Corp 培養容器からの動物細胞の剥離方法および剥離回収方法
JP2008000008A (ja) * 2006-06-20 2008-01-10 Seiko Instruments Inc 細胞剥離方法
JP2008079554A (ja) * 2006-09-28 2008-04-10 Chiyoda Corp 閉鎖系の細胞回収装置及び細胞培養装置並びに細胞の回収方法及び培養方法
WO2011028524A1 (fr) * 2009-08-24 2011-03-10 Wisconsin Alumni Research Foundation Progéniteur rétinien humain sensiblement pur, progéniteur de cerveau antérieur, et cultures de cellules d'épithélium pigmentaire rétinien et leurs procédés de fabrication
JP2011115080A (ja) * 2009-12-02 2011-06-16 Terumo Corp シート状細胞培養物剥離システム
WO2011108503A1 (fr) * 2010-03-01 2011-09-09 独立行政法人科学技術振興機構 Procédé de détachement de cellules mises en culture, dispositif de détachement de cellules utilisé dans ledit procédé pour détacher des cellules mises en culture et incubateur
JP2012039947A (ja) * 2010-08-19 2012-03-01 Osaka Prefecture Univ 細胞培養用基材

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016052269A (ja) * 2014-09-03 2016-04-14 テルモ株式会社 シート状細胞培養物回収システムおよび方法
JP2016116460A (ja) * 2014-12-19 2016-06-30 パナソニック株式会社 細胞培養装置
US10538731B2 (en) * 2015-02-19 2020-01-21 Commissariat A L'energie Atomique Et Aux Energies Alternatives Device for manipulating biological cells using a vibrating support
WO2019044804A1 (fr) * 2017-08-28 2019-03-07 学校法人慶應義塾 Méthode de production de cellule et dispositif de production de cellule
CN109706078A (zh) * 2019-02-25 2019-05-03 常州市第一人民医院 一种细胞分选系统及不良细胞分选方法
WO2023079905A1 (fr) * 2021-11-04 2023-05-11 株式会社日立製作所 Dispositif de décollement de cellules et procédé de décollement de cellules

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