WO2017187680A1 - Système de culture de cellules à grande échelle et dispositif de transfert de liquide de cellules de récipient à récipient destiné à y recevoir le système de culture de cellules, et dispositif rotatif de culture de cellules - Google Patents

Système de culture de cellules à grande échelle et dispositif de transfert de liquide de cellules de récipient à récipient destiné à y recevoir le système de culture de cellules, et dispositif rotatif de culture de cellules Download PDF

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Publication number
WO2017187680A1
WO2017187680A1 PCT/JP2017/002558 JP2017002558W WO2017187680A1 WO 2017187680 A1 WO2017187680 A1 WO 2017187680A1 JP 2017002558 W JP2017002558 W JP 2017002558W WO 2017187680 A1 WO2017187680 A1 WO 2017187680A1
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WIPO (PCT)
Prior art keywords
vessel
culture
cell
spheroids
plunger
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PCT/JP2017/002558
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English (en)
Japanese (ja)
Inventor
尚史 津村
寿公 植村
雄大 内田
Original Assignee
株式会社ジェイテックコーポレーション
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.)
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Priority claimed from JP2017007993A external-priority patent/JP6268342B2/ja
Application filed by 株式会社ジェイテックコーポレーション filed Critical 株式会社ジェイテックコーポレーション
Priority to EP20190032.1A priority Critical patent/EP3770245A1/fr
Priority to CN201780025397.1A priority patent/CN109072157A/zh
Priority to US16/094,346 priority patent/US20190119623A1/en
Priority to EP17788970.6A priority patent/EP3450535A4/fr
Publication of WO2017187680A1 publication Critical patent/WO2017187680A1/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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/10—Apparatus for enzymology or microbiology rotatably mounted

Definitions

  • the present invention relates to a mass cell culture system suitable for culturing a large number of pluripotent stem cells and adherent cells used in regenerative medicine and the like, an inter-cell cell fluid transfer device and a rotating cell culture device used therefor.
  • Non-Patent Documents 1 to 3 The discovery of artificial pluripotent stem cells (often referred to herein as “iPS cells”) (Non-Patent Documents 1 to 3) has increased the momentum for practical application of regenerative medicine using them.
  • iPS cells artificial pluripotent stem cells
  • the amount of about 10 6 cells used in the laboratory is far from sufficient, and for clinical application, the number of cells on the order of 10 9 to 10 10 is necessary.
  • the mass culture technology has not been well established.
  • feeder cells such as mouse embryo-derived primary cultured fibroblasts (MEF) and STO cells in order to maintain and maintain an undifferentiated state.
  • contamination of feeder cells is a major obstacle to use in regenerative medicine.
  • Feeders can be developed by culturing iPS cells on the surface of a matrix coated with Matrigel, or by using a culture method using laminin or laminin partial peptide coating. Methods have been developed that can be cultured without the presence of cells. Further, instead of ordinary dish culture, culture using a bag is also performed. However, even in a feeder-free culture system, it is necessary to repeat the culture on the coated substrate, so that the culture process is complicated, and the cost of the culture is significantly increased, so that one patient can be treated. There is a serious problem that the cost of the system becomes enormous. On the other hand, in the case of adherent cells, it is necessary to add the drug after culturing and peel off the adherent cells, and the use of the drug for regenerative medicine also causes a problem.
  • Patent Documents 4 to 6 a flat cylindrical culture vessel equipped with a gas permeable membrane on the back side (rear side) is attached to the horizontal rotation shaft of the rotation control device, and is rotated while being cantilevered on the back side. This balances the gravity, buoyancy, and the force received from the flow of the culture fluid due to the rotation of the spheroids in the culture vessel, creating a pseudo-microgravity environment that is 1 / 100th of the ground gravity on a time average, and the spheroids do not settle An apparatus that realizes a state of floating softly in an area has been proposed.
  • an observation window is provided on the front side of the culture vessel, and the rotation speed is controlled according to the growth and floating state of the cells by imaging the spheroids with the camera through the window, and the spheroids are always floated in the certain area. Maintained in a state.
  • Patent Document 7 a method for efficiently mass-culturing stable iPS cells that retain undifferentiated properties. That is, the invention described in Patent Document 7 maintains undifferentiated properties even in the absence of feeder cells and coating agents by culturing pluripotent stem cells, particularly iPS cells, in a pseudo microgravity environment. It is possible to proliferate pluripotent stem cells and form spheroids, and to proliferate pluripotent stem cells in a closed system with low risk of contamination, which has the advantage of improving safety .
  • Patent Document 8 (i) a step of suspension culture of a pluripotent stem cell until the average diameter of the cell mass becomes about 200 to about 300 ⁇ m, and (ii) a cell mass obtained by the step (i).
  • a method for maintaining and amplifying pluripotent stem cells is disclosed, which comprises repeating the step of dividing cells into uniform cell masses having an average diameter of about 80 to about 120 ⁇ m. This method is performed in a stationary state using a culture vessel such as a dish in a medium containing a water-soluble polymer component having a viscosity that does not cause adhesion between cell masses.
  • this method uses a special medium to increase the specific gravity of the medium and cultivate the cell mass in a state where it has floated inside without being settled, so that some molecules secreted by the cell mass flow. Since it remains around the cell mass, it has the problem of being affected by it. In fact, when the diameter of the cell mass exceeds 300 ⁇ m, a microenvironment is formed due to the influence of cytokinin etc. secreted by the cell, differentiation is induced, and necrosis occurs in the central part of the cell mass. It has been pointed out that the recovery rate deteriorates. Furthermore, when filtering the cell mass, it is necessary to pass the entire amount of the cell mass suspension medium through the filter using a pipetman, which is a laborious and labor-intensive operation. In addition, it is difficult to separate the medium and the cell mass, and it takes time to exchange the medium.
  • spheroids of pluripotent stem cells formed by suspension culture are separated into small spheroids and dispersed in a culture solution, and in a pseudo microgravity environment such as rotational culture. Can be cultured in large quantities while maintaining an undifferentiated state.
  • spheroids are typically mechanically (mechanically) passed through a filter in order to break them into small spheroids (30 to 200 cells). Specifically, pressure is applied to the spheroids using a pipette or the like to pass through the filter.
  • the present invention intends to solve pluripotent stem cells used in regenerative medicine and the like, particularly iPS cells, even in the absence of feeder cells and coating agents. It can be maintained and cultured in large quantities, and it can also be subcultured by eliminating variations due to operator skills. Furthermore, adherent cells can also be cultured in large quantities in a floating state without being attached.
  • An object of the present invention is to provide a suitable mass cell culture system, an inter-cell cell fluid transfer device and a rotating cell culture device used therefor.
  • the present invention has constituted the following large-scale cell culture system, an inter-cell cell fluid transfer device and a rotating cell culture device used therefor.
  • a mass cell culture system for culturing a large number of pluripotent stem cells and adherent cells by performing subculture using a syringe-structured vessel and transferring the spheroid and culture solution in a closed system,
  • a front flange and a back flange having the same outer shape are integrally provided at both ends of the outer cylinder portion, the head serving as the solution inlet / outlet is closed with a removable cap, and the space surrounded by the plunger gasket is provided.
  • a mass cell culture system characterized in that it can be rotated and cultured using the front flange and the back flange in a state filled with a cell solution in which cells are suspended in a culture solution.
  • the inner surface on the tip side of the outer cylinder portion is formed as a conical recess, and the inclination angle ⁇ of the inner surface is set in a range of 80 ° to 160 ° in the central angle, according to (1) or (2) Mass cell culture system.
  • a treatment between subcultures comprising a cell concentration step of transferring the cultured spheroids from the culture vessel to the concentration vessel, wherein the cell concentration step includes connecting the heads of the culture vessel and the concentration vessel to each other with a connection tool.
  • the intercellular fluid transfer device capable of driving the plunger by pushing and pulling it, while holding the outer tube portion of each vessel with the head of the culture vessel in a downward posture.
  • a treatment between subcultures comprising a dispensing / refining step for transferring a predetermined amount of the cell solution in which the spheroids in the concentrated vessel are concentrated to a plurality of new culture vessels;
  • the head of the concentrated vessel containing the water and the head of the new culture vessel are connected to the intercellular fluid transfer device while the head of the new culture vessel is connected with the connection device with a built-in filter, and the plunger of the concentrated vessel is pushed in The plunger of the culture vessel is pulled out, and a predetermined amount of the cell solution in which the spheroids in the concentrated vessel are concentrated is transferred to the culture vessel through the filter having a function of crushing the spheroids into small spheroids.
  • the large-scale cell culture system comprising a dispensing / refining step for transferring a predetermined amount of the cell solution in which the spheroids in the concentrated vessel are concentrated to a plurality of new culture vessels.
  • New culture connected to the head of the culture vessel, including a dilution step for adding a new culture solution to the culture vessel in which a predetermined amount of the cell solution enriched with spheroids is transferred, which is treatment between subcultures.
  • the mass cell culture system according to (6) wherein a culture solution is supplied into the vessel from a solution supply source.
  • An inter-cell cell fluid transfer device capable of transferring a solution from one vessel to the other vessel, wherein the vessel has a front flange and a back flange having the same outer shape at both ends of the outer cylinder portion.
  • the head serving as the solution inlet / outlet is closed with a detachable cap, and the space surrounded by the gasket of the plunger is filled with the cell solution in which the cells are suspended in the culture solution.
  • the fixed portion includes an outer tube portion holding portion for fixing the outer tube portion of each vessel in a state where the heads of the two vessels are connected to each other by a connecting tool, and a plunger.
  • a plunger holding portion for holding a plunger button provided at the tip of the movable portion and a drive mechanism portion for driving the movable portion.
  • a rotating cell culture apparatus capable of rotating a culture vessel filled with a cell solution in which cells are suspended in a culture solution at a predetermined speed, and capable of culturing cells in a floating state in a pseudo microgravity environment,
  • the vessel is integrally provided with a front flange and a back flange having the same outer shape at both ends of the outer cylinder portion, the head serving as the solution inlet / outlet is closed with a detachable cap, and the vessel is enclosed in a space surrounded by the plunger gasket.
  • the lower portions of the front flange and the back flange are rotatably supported by a pair of rollers, and at least one roller in contact with the front flange or the back flange is a drive roller that is rotated by a drive motor.
  • the other is driven rollers, and the posture is maintained by abutting the radially outer surfaces of the front flange and the back flange.
  • Rotating cell culture apparatus characterized by comprising a control unit.
  • pluripotent stem cells and adhesive cells used in regenerative medicine and the like can be cultured in large quantities and efficiently regardless of operator skills.
  • the culture vessel has a syringe structure, cell concentration, filtering, dilution, and rotation culture in subculture can all be performed in a closed system, and there is little risk of contamination.
  • Filtering can be performed by transferring the cultured cells to another vessel only through a filter built-in connector, and the spheroids can be mechanically refined into small spheroids without using any chemicals.
  • iPS cells can be cultured in large quantities while maintaining undifferentiation.
  • the vessel used for the mass cell culture system of the present invention is shown, (a) is a partial sectional view of a culture vessel having a capacity of 50 ml, and (b) is a partial sectional view of a concentrated vessel having a capacity of 10 ml. It is sectional drawing of the decomposition
  • the cell concentration step for transferring the cultured spheroids from the culture vessel to the concentration vessel is shown, (a) is a sectional view of the state where the culture vessel and the head of the concentration vessel are connected via a connector, and (b) is the lower part of the culture vessel. It is sectional drawing of the state which transferred the spheroid settled in to the concentration vessel.
  • the concentration vessel and the head of a new culture vessel are connected via a connector with a built-in filter, and the dispensing and refining step is shown, in which the spheroid is finely divided into small spheroids, and (a) shows the concentration vessel and the first culture.
  • a cross-sectional view with the head of the vessel connected (b) a cross-sectional view with the 1 / n spheroid transferred to the first culture vessel, and (c) a 1 / n spheroid with the second culture vessel (D) is a cross-sectional view of a state in which 1 / n spheroids have been transferred to the nth culture vessel.
  • the dilution step which adds a new culture solution to the culture vessel which transferred 1 / n spheroid is shown, (a) is sectional drawing of the state which connected the culture solution supply pipe to the head of the culture vessel, (b) is a culture vessel Sectional drawing of the state which inject
  • (c) is a sectional view of the rotation culture vessel form which attached the cap to the head and isolate
  • FIG. 18 is a sectional view taken along line XX of FIG. It is a disassembled perspective view of the rotating cell culture apparatus of the modification of 2nd Embodiment.
  • FIG. 25 is a sectional view taken along line YY in FIG. 24.
  • FIG. 25 is a sectional view taken along line ZZ in FIG. 24. It is a fragmentary perspective view which shows a drive mechanism.
  • phase contrast image of a small spheroid after passing through a 70 ⁇ m filter (a) shows a phase contrast image with a low magnification, and shows a phase contrast image with a high magnification.
  • 15 is a phase difference image of a spheroid after culturing the small spheroids of FIG. 15, (a) shows a low magnification phase difference image, and (b) shows a high magnification phase difference image.
  • phase contrast image of spheroids after 3 days of rotation culture of iPS cells (253G1 cells) using 10 ml vessel (a) is a phase contrast image of spheroids after rotation culture in mTeSR1 medium, and (b) is AK02N. It is a phase contrast image of the spheroid after rotation culture in a culture medium. It is a phase contrast image of spheroids after 3 days of rotation culture of iPS cells (409B2 cells) using 10 ml vessel, (a) is a phase contrast image of spheroids after rotation culture in mTeSR1 medium, and (b) is AK02N. It is a phase contrast image of the spheroid after rotation culture in a culture medium. It is a phase contrast image of a small spheroid immediately after crushing at each passage (P1 to P10) in a continuous passage culture experiment.
  • the “cell” is a cultured cell, which is an ES cell or iPS cell pluripotent stem cell or adhesive cell.
  • the cell in the present invention may be derived from any mammal (for example, mouse, rat, guinea pig, hamster, rabbit, dog, cat, monkey, cow, etc.), but it is more preferably a human-derived iPS cell. .
  • the medium (culture solution) for suspending cells is not particularly limited as long as it is a medium suitable for culturing those cells.
  • Antibiotics such as FBS (fetal calf serum) and Antibiotic-Antimycotic may be added to the medium.
  • the culture solution used in the mass cell culture system of the present invention has such a property that, when left standing, spheroids mixed in a dispersed state settle and accumulate in the lower part.
  • spheroid refers to a cell mass in which a large number of cells aggregate, and typically refers to a cell mass having a diameter of 300 ⁇ m or more, for example, 300 to 2000 ⁇ m.
  • small spheroid means a relatively small cell mass composed of a relatively small number of cells, and typically about 3 to 1,000 cell masses, for example, 5 to 600 cells. , 10 to 200 cells, 30 to 100 cells or 20 to 40 cell masses are included.
  • 1 to 7 show a vessel used for the culture of the present invention and a procedure for culturing a large amount of cells using the same, in which 1 is a syringe, 2 is a culture vessel, 3 is a concentrated vessel, and 4 is a connector. . Further, a cell or spheroid is indicated by a symbol S, a culture fluid is indicated by a symbol M, and a cell fluid in a state where the cells or spheroids are suspended in the culture fluid is indicated by a symbol SM.
  • the syringe 1 is integrally provided with a front flange 6 and a back flange 7 having the same outer shape at both ends of a cylindrical outer cylinder portion 5, and provided at the center of the front flange 6.
  • a head 8 serving as a discharge port has a standard luer lock structure, and a gasket 11 that is slidably elastically fitted into the outer cylinder portion 5 is provided at the distal end of the shaft portion 10 of the plunger 9, and a plunger is provided at the proximal end.
  • the button 12 is provided.
  • the shaft portion 10 of the plunger 9 is structured to be separable into a short shaft portion 10A on the distal end side and a long shaft portion 10B on the proximal end side.
  • a female screw 10C is provided at the end of the shaft portion 10A
  • a male screw 10D that is screwed into the female screw 10C is formed at the end of the shaft portion 10B, and a detachable structure is adopted.
  • notches 10E are formed on the outer surfaces.
  • the shape of the tip 11A of the gasket 11 is a conical convex portion, and the inner surface of the front end side of the outer cylinder portion 5, that is, the shape of the inner surface 6A of the front flange 6 is a conical recess that receives the tip 11A.
  • the liquid inside the outer cylinder portion 5 can be discharged without leaving.
  • a stainless steel head 8 having a luer lock structure is integrally formed on the outer surface of the front flange 6.
  • the inclination angle of the inner surface 6A having a tapered shape is important for efficiently discharging the spheroids settled as described later from the head 8 together with a small amount of culture medium.
  • the inclination angle ⁇ of the inner surface 6A is set to a central angle in the range of 80 ° to 160 °, more preferably in the range of 90 ° to 150 °. If the inclination angle of the inner surface 6A is too large, it is difficult to collect the spheroids in the vicinity of the ejection holes of the head 8 when the spheroids are sedimented with the axis directed in the vertical direction, while the inclination angle of the inner surface 6A is too small.
  • the shape of the tip 11A is a conical convex portion in the present embodiment, it may have the same inclination angle as the inner surface 6A or a larger inclination angle, or may be a flat surface (180 °). .
  • the inclination angle ⁇ of the inner surface 6A is 150 ° as a central angle.
  • the syringe 1 is made of a material that can be sterilized such as autoclave sterilization.
  • the outer cylinder portion 5 is made of transparent pyrex glass
  • the front flange 6, the back flange 7, and the shaft portion 10 of the plunger 9 are made of stainless steel, in order to observe the floating state and the settling state of the internal spheroids.
  • the gasket 11 of the plunger 9 is made of PTFE.
  • the material of the syringe 1 is not limited to that described above, and the front flange 6 and the back flange 7 may be made of Pyrex glass or formed of a transparent synthetic resin material, like the outer cylinder portion 5. Good.
  • the whole may be integrally molded, or divided into a plurality of parts and bonded to each other. Fusion or ultrasonic fusion may be performed.
  • the culture vessel 2 uses the syringe 1 as it is. However, when the culture vessel 2 is mounted on a rotating cell culture apparatus to be described later and rotationally cultured, a cap C is attached to the head 8 as shown in FIG.
  • the shaft portion 10B of the plunger 9 is used in a separated state.
  • the syringe 1 and the culture vessel 2 are synonymous and used without distinction.
  • the culture vessel 2 for rotating culture refers to the form shown in FIG.
  • the culture vessel 2 shown in FIG. 1A has a capacity of 50 ml, the front flange 6 and the back flange 7 have a diameter of 50 mm ⁇ , and the outer dimension between the front flange 6 and the back flange 7 is 91 mm.
  • the stroke of the plunger 9 is about 60 mm.
  • the culture vessel 2 is configured to cover the head 8 with a cap C, fill a space surrounded by the gasket 11 of the plunger 9 with a cell solution in which cells are suspended in a culture solution, and perform rotary culture.
  • the syringe 1 shown in FIG.1 (b) is the completely same structure except the diameter of the outer cylinder part 5 and the gasket 11 compared with the said syringe 1 of Fig.1 (a), and a capacity
  • capacitance is 10 ml. , External dimensions.
  • the syringe 1 is used as a concentrated vessel 3 in the present invention, and is used to transfer the cultured spheroids from the culture vessel 2 and dispense them into other new culture vessels 2, but of course a culture with a small volume. It can also be used as a vessel. Since the structure of the concentrated vessel 3 is the same as that of the culture vessel 2, the same components are denoted by the same reference numerals and the description thereof is omitted.
  • the connector 4 is used when transferring the culture solution between the culture vessel 2 and the concentrated vessel 3, and each of the vessels 2, 3
  • the heads 8 and 8 can be connected to each other.
  • the connector 4 includes a first member 13 and a second member 14.
  • the first member 13 is provided with a flow path 15 at the center, a housing portion 16 having a hollow inside at one end in the axial direction, a female screw portion 17 at the inner periphery of the housing portion 16, and at the other end in the axial direction.
  • a connection 18 with the head 8 is provided.
  • the second member 14 has a flow path 19 at the center, a male screw portion 20 that is screwed into the female screw portion 17 of the first member 13 on one axial end side, and the head 8 on the other axial end side.
  • a connecting portion 21 is provided.
  • the recessed part 22 is formed in the front-end
  • the filter 23, packing 24, and the spacer 25 are laminated
  • the packing 24 and the spacer 25 have a center hole communicating with the flow paths 15 and 19.
  • the filter 23 may be sandwiched between two packings 24 and 24.
  • the filter 23 may be a filter having a filtration particle size capable of crushing the produced spheroid into smaller spheroids having a smaller size, and has a filtration particle size of 40 to 100 ⁇ m, preferably 60 to 80 ⁇ m, for example 70 ⁇ m. Those are preferred. Note that small spheroids obtained by crushing through a filter generally have an elongated shape. Spheroids may be crushed once or twice or more through the filter 23. When crushing by filter passage is performed twice or more, a filter 23 having the same filtration particle size may be used or a filter 23 having a different filtration particle size may be used for each filter passage.
  • the crushed spheroids may be further crushed through the filter 23 having a larger filtration particle size, thereby obtaining the size of the obtained small spheroids.
  • the thickness (especially the major axis) can be made narrower.
  • a 200-mesh stainless steel filter is used as the filter 23, and the filtration particle size is 70 ⁇ m.
  • the connector 4 incorporating the filter 23 By using the connector 4 incorporating the filter 23, the cell fluid containing spheroids is transferred from one vessel to another vessel, and at the same time, the size of the spheroids according to the filtration particle size passes through the filter 23. It is crushed into small spheroids. If the cell fluid is simply transferred from one vessel to another without disrupting the spheroids, the connector 4 from which the filter 23 has been removed may be used.
  • a predetermined number of cells and a culture solution are injected into the culture vessel 2 to prepare a cell solution of about 1 ⁇ 10 4 to 1 ⁇ 10 5 cells / cm 3 .
  • the treatment after the number of cells grows about 5 times by culture will be described.
  • FIG. 5 a cell concentration step for transferring cultured spheroids from a culture vessel to a concentration vessel will be described.
  • the shaft portion 10B of the plunger 9 of the culture vessel 2 is connected to the shaft portion 10B which has been separated during the culture to form a syringe having a normal structure, and the head 8 of the culture vessel 2
  • the cap C is removed from the connection vessel 4 and connected to one connection portion 21 of the connection device 4 without a filter, and the other connection portion 18 of the connection device 4 is connected to the head 8 of the concentration vessel 3 so that the culture vessel 2
  • the head 8 is placed so as to face downward and is left to stand. Since the connector 4 has a structure that can be used in both directions, the connection portion 18 may be connected to the head 8 of the culture vessel 2 and the connection portion 21 may be connected to the head 8 of the concentration vessel 3.
  • the concentration vessel 3 and the heads 8 and 8 of the new culture vessel 2 are connected via the connecting device 4 with a built-in filter, and the spheroid is finely divided into small spheroids for dispensing.
  • the steps will be described.
  • the head 8 of the concentrated vessel 3 containing the concentrated cell fluid SM and the head 8 of the new first culture vessel 2 are connected by the connecting device 4 with a built-in filter.
  • the plunger 9 of the concentrated vessel 3 is pushed in, and at the same time, the plunger 9 of the culture vessel 2 is pulled out, and the cell solution SM in which the spheroid S in the concentrated vessel 3 is concentrated is extracted.
  • 1/5 is transferred to the culture vessel 2.
  • the second new culture vessel 2 is connected, and 1/5 of the cell fluid SM in the concentrated vessel 3 is transferred to the culture vessel 2.
  • a fifth new culture vessel 2 is connected, and 1/5 of the cell fluid SM in the concentrated vessel 3 is transferred to the culture vessel 2.
  • the spheroids are broken into small spheroids by the filter 23.
  • the spheroid is broken into small spheroids by the filter 23 in this step by using the filter built-in connector 4. That is, after the second generation culture, the filter processing can be performed twice by the next n + 1 generation culture, and the small spheroid can be made more spherical.
  • the culture solution supply pipe 26 is connected to the head 8 of the culture vessel 2 in which the cell solution SM containing 1/5 of the concentrated spheroids is transferred by the dispensing / miniaturization step.
  • the culture medium M is sucked while the plunger 9 is pulled out, and a new culture medium M is added and diluted.
  • a syringe (not shown) containing the new culture solution is prepared, and the syringe is connected to the culture vessel 2 via the connector 4.
  • the culture solution may be injected from the syringe.
  • all of the above-described cell concentration step, dispensing / miniaturization step, and dilution step can be performed in a closed system using the inter-cell cell fluid transfer device A described later.
  • the cap 8 is attached to the head 8 of the culture vessel 2 and closed, and the shaft portion 10B of the plunger 9 is pivoted. Prepared so that it can be separated from the part 10A and mounted on the rotating cell culture apparatus B.
  • the inter-cell cell fluid transfer device A fixes the outer cylinder portion 5 in a state where the heads 8 of the two vessels are connected to each other by the connector 4, and specifically holds the back flange 7 so as not to move in the axial direction. Then, it is an automation device that can hold the plunger button 12 and drive the plunger 9 in the axial direction to transfer a predetermined amount of liquid from one vessel to the other at a constant speed.
  • the first mechanism 27 and the second mechanism 28 facing each other in the same vertical direction are provided with a common base portion 29, and a vessel is attached to each to fix the outer cylinder portion 5.
  • the plunger 9 is driven forward and backward.
  • “advance” means to advance in the direction of discharging liquid from the head 8 of the vessel
  • “retreat” means to advance in the direction of sucking liquid from the outside, which is opposite to advance. Means to go on.
  • the first mechanism 27 includes a fixed portion 30 for fixing the outer cylinder portion 5 of the culture vessel 2, a movable portion 31 for driving the plunger 9 forward and backward, and a drive mechanism portion for driving the movable portion 31. 32.
  • the second mechanism 28 also includes a fixed portion 33 that fixes the outer cylinder portion 5 of the concentration vessel 3, a movable portion 34 that drives the plunger 9 forward and backward, and a drive mechanism portion 35 that drives the movable portion 34. I have.
  • the movable portion 31 and the drive mechanism portion 32 of the first mechanism 27 have the same structure as the movable portion 34 and the drive mechanism portion 35 of the second mechanism 28, and are vertically inverted from each other.
  • the movable portion 31 of the first mechanism 27 and the movable portion 34 of the second mechanism 28 are driven forward and backward in synchronization with each other in accordance with the cross-sectional area of each vessel so that the flow rates flowing through the connector 4 coincide.
  • the base portion 29 fixes a vertical support plate 38 to the central portion of the upper surface of the horizontal base plate 37 in an upright state, and extends along both side edges on the back side of the support plate 38.
  • the reinforcing plates 39, 39 are erected and fixed to the base plate 37 and the support plate 38, and have a U-shaped structure with high rigidity in plan view.
  • a linear guide 40 that extends in the vertical direction constituting the drive mechanism portion 32 and a linear actuator 41 provided along the linear guide 40 are disposed on the upper front surface of the support plate 38.
  • the movable portion 31 is held by the linear guide 40 so as to be movable up and down, and the movable portion 31 is driven by the linear actuator 41.
  • the linear guide 40 is composed of two parallel guide rails 42, 42, and a movable block 43, 43 on which the movable portion 31 moves along the guide rails 42, 42 is provided on a stage 44 having a rear surface. It is fixed.
  • the linear actuator 41 is composed of a feed screw mechanism 46 that is rotationally driven by a drive motor 45 that can control the rotational speed, such as a stepping motor or a servo motor.
  • a screw shaft 46A of the feed screw mechanism 46 is disposed in parallel between the guide rails 42, 42, and a nut member 46B linked to the screw shaft 46A so as to be able to advance and retract is fixed to the back surface of the stage 44.
  • a ball screw with higher accuracy may be used.
  • a linear guide 47 extending in the vertical direction constituting the drive mechanism portion 35 and a linear actuator 48 provided along the linear guide 47 are disposed at the lower front portion of the support plate 38.
  • the movable portion 34 is held by the linear guide 47 so as to be movable up and down, and the movable portion 34 is driven by the linear actuator 48.
  • the linear guide 47 is composed of two parallel guide rails 49, 49, and the movable part 34 moves on the guide rails 49, 49 on a stage 51 provided on the back with movable blocks 50, 50. It is fixed.
  • the linear actuator 48 is constituted by a feed screw mechanism 53 that is rotationally driven by a drive motor 52 such as a stepping motor or a servo motor that can control the rotational speed.
  • a screw shaft 53A of the feed screw mechanism 53 is arranged in parallel between the guide rails 49, 49, and a nut member 53B linked to the screw shaft 53A so as to be able to advance and retract is fixed to the back surface of the stage 51.
  • a ball screw with higher accuracy may be used instead of the feed screw mechanism 53.
  • the fixing portion 30 of the first mechanism 27 and the fixing portion 33 of the second mechanism 28 are directly or indirectly attached to a common fixing stage 54 that is fixed in parallel with a space on the surface side of the support plate 38.
  • the fixed portion 33 is directly attached to the lower portion of the fixed stage 54, and the fixed portion 30 is attached to a movable stage 55 provided on the upper portion of the fixed stage 54 and movable in the vertical direction.
  • the fixing portion 33 is connected to the fixing portion 30 via a distance adjusting tool 56.
  • the spacing adjustment tool 56 is a mechanism that expands and contracts by a screw mechanism, and the fixing portion 30 is fixed to the fixing stage 54 via the spacing adjustment tool 56 and the fixing portion 33.
  • the reason for providing the distance adjusting tool 56 is to absorb errors in the dimensions of the connecting tool 4 and the connection depth of the connecting tool 4 with respect to the head 8 of the vessel.
  • the movable portion 31 of the first mechanism 27 is provided on the stage 44, and the plunger holding portion 57 that holds the plunger button 12 of the plunger 9 with respect to the stage 44 is finely adjusted in the vertical direction. It can be fixed. Specifically, with the adjustment plate 58 joined to the surface of the stage 44, the pin 59 and the slit groove 60 are moved and guided in the vertical direction, and a long hole extending in the vertical direction opened at the center of the adjustment plate 58. A tightening screw 62 is screwed to the stage 44 through 61. The handle 63 provided on the tightening screw 62 can be easily tightened and loosened manually.
  • the plunger holding portion 57 is configured by an engagement recess 64 that engages the plunger button 12 from the front side.
  • the movable portion 34 of the second mechanism 28 is provided on the stage 51, and a plunger holding portion 65 that holds the plunger button 12 of the plunger 9 with respect to the stage 51 is positioned in the vertical direction.
  • the adjustment plate 66 is joined to the surface of the stage 51, and is moved and guided in the vertical direction by the pin 67 and the slit groove 68, and a long hole extending in the vertical direction opened at the center of the adjustment plate 66.
  • the fastening screw 70 is screwed to the stage 51 through 69.
  • the handle 71 provided on the tightening screw 70 can be easily manually tightened and loosened.
  • the plunger holding portion 65 is configured by an engagement recess 72 that engages the plunger button 12 from the front side.
  • the fixed portion 30 of the first mechanism 27 is provided with an outer tube portion holding portion 73 that holds the outer tube portion 5 of the culture vessel 2.
  • the outer tube portion holding portion 73 includes a U-shaped recess 74 that receives the outer tube portion 5, a flange fitting groove 75 that fits the front flange 6, It comprises a holding member 76 that holds and holds the front flange 6 from the outside.
  • the holding member 76 is configured so that one end can be horizontally rotated and the other end can be elastically engaged and disengaged by a hook 77.
  • the outer cylinder holding part 73 can hold the back flange 7 at the same time.
  • the fixing part 33 of the second mechanism 28 is provided with an outer cylinder part holding part 78 that holds the outer cylinder part 5 of the concentrated vessel 3.
  • the outer tube portion holding portion 78 includes a U-shaped recess 79 that receives the outer tube portion 5, a flange fitting groove 80 that fits the front flange 6,
  • the holding member 81 holds the front flange 6 from outside.
  • the holding member 81 is configured so that one end thereof can be horizontally rotated and the other end can be elastically engaged and disengaged by a hook 82.
  • the outer cylinder part holding part 78 can hold the back flange 7 at the same time.
  • a limit mechanism for limiting the movable range of each movable part is provided for safety and to determine the home position.
  • the limit mechanism is composed of a proximity sensor provided in the fixed part and a restriction piece provided in the movable part. When the restriction sensor detects the restriction piece, the drive motors 45 and 52 are forcibly stopped. Further, a rubber grounding pad 83,... Is provided on the lower surface of the base plate 37, and rubber grounding pads 84,. It can be used even when A is tilted horizontally.
  • the heads 8 of the culture vessel 2 and the concentrated vessel 3 is flange-fitted in a state in which the holding members 76 and 81 are released and the fastening screws 62 and 70 are loosened.
  • the plunger button 12 is engaged with the engagement recess 64, the front flange 6 of the concentration vessel 3 is engaged with the flange engagement groove 80, and the plunger button 12 is engaged with the engagement groove 75. Engage with the recess 72 and then close the holding members 76 and 81 to hold the front flange 6 respectively. Then, the tightening screws 62 and 70 are tightened and set.
  • the new culture vessel 2 is incorporated into the concentrated vessel 3 as shown in FIG. In the state where it is connected via the connector 4, it is attached to the inter-cell cell fluid transfer device A in the same manner as described above.
  • the state shown in FIG. 12 corresponds to the state shown in FIG. Also in this case, the drive motor 45 of the first mechanism 27 and the drive motor 52 of the second mechanism 28 are rotationally driven to advance the movable portion 31 and push the plunger 9 of the concentration vessel 3 at the same time.
  • the movable part 34 is retracted, the plunger 9 of the culture vessel 2 is pulled out, and the cell solution in which the spheroids in the concentrated vessel 3 are concentrated is transferred to the culture vessel 2 by a predetermined amount.
  • the lower concentrated vessel 3 can be a simple container, and the culture vessel 2 can be filtered and transferred to the container while crushing the spheroids into small spheroids. That is, the filtration operation is possible even with an apparatus including only the first mechanism 27 of the inter-cell cell fluid transfer apparatus A.
  • This rotating cell culture apparatus B is capable of simultaneously rotating a plurality of culture vessels 2 in the state shown in FIG.
  • the rotating cell culture apparatus B of this embodiment simultaneously rotates four culture vessels 2,... At a predetermined speed so that cells can be cultured in a floating state in a pseudo microgravity environment.
  • the rotating cell culture apparatus B of the present invention is designed to be compact so that it can be installed and used in an incubator having a temperature control function.
  • the “pseudo microgravity environment” means a simulated microgravity environment artificially created by simulating a microgravity environment in outer space or the like. Such a pseudo microgravity environment is realized, for example, by offsetting the earth's gravity by the stress generated by the rotation.
  • the rotating cell culture apparatus B of the present invention controls the rotation speed of the culture vessel, balances the gravity of the spheroids in the culture vessel, the buoyancy, and the force received from the flow of the culture solution due to the rotation. It is a device that creates a pseudo microgravity environment of about 1/10 to 1/100 and realizes a state where spheroids do not settle and float in a certain area.
  • the rotating cell culture apparatus B rotatably supports the lower portion of the front flange 6 and the back flange 7 of the culture vessel 2 with a pair of driving rollers 85 and 85 and driven rollers 86 and 86, respectively.
  • the outer surface in the radial direction of the flange 7 is stopped by restriction rollers 87 and 87 so as not to be displaced in the axial direction.
  • the restriction roller 87 is a restriction portion in the present invention.
  • a central block 90 having a drive mechanism 89 built in the central portion of the upper surface of a base 88 incorporating a control circuit therein, and the front flange 6 and back of the culture vessel 2 with respect to the central block 90
  • Side blocks 91, 91 are provided with a minimum interval to receive the flange 7, and four pairs of drive rollers 85, 85 are provided on both sides of the central block 90.
  • Two pairs of driven rollers 86, 86 are provided on the side blocks 91, 91 at positions facing 85, respectively. Both the driving roller 85 and the driven roller 86 are provided with rotating shafts 92 and 93 which are horizontal and face the same direction.
  • the regulating roller 87 includes horizontal rotation shafts 95 that are orthogonal to the rotation shafts 92 and 93 at both ends of a projecting portion 94 that projects from the center of the upper surface of the side block 91.
  • the driven rollers 86, 86 stably support one of the front flange 6 and the back flange 7 of the culture vessel 2, and freely rotate as the culture vessel 2 rotates.
  • the restricting roller 87 is driven to rotate only when the outer surfaces in the radial direction of the front flange 6 and the back flange 7 are in contact with each other.
  • the front flange 6 and the back flange 7 are driven by the driving rollers 85 and 85 and the driven rollers 86 and 86. The posture is maintained so as not to fall off.
  • the drive mechanism 89 has pulleys 96,... Fixed at the center of four rotating shafts 92,.
  • a belt preferably a timing belt 97, is wound around the pulleys 96, 96, and further, pulleys 98, 98 fixed to two rotating shafts 92, 92 in a central portion which are different pairs, and a driving shaft 100 of one driving motor 99.
  • a belt preferably a timing belt 102, is wound around the pulley 101 fixed to the pulley 101, and all the driving rollers 85 are driven so as to rotate in the same direction. Since the driving rollers 85 are fixed to both ends of the four rotating shafts 92,..., The four culture vessels 2,. Can be suspended culture.
  • a rotation speed adjustment knob 103 that also functions as a power switch is provided on the side surface of the base 88 to adjust the rotation speed of the drive motor 99.
  • the drive mechanism 89 described above has expandability, and can be configured so that a large number of culture vessels 2 can be rotated and cultured at the same time.
  • by making the dimensions of the front flange 6 and the back flange 7 common it is possible to rotate and culture in the same rotating cell culture apparatus B even if the diameter of the outer cylinder part 5 is different, that is, the capacity is different. is there.
  • the number of culture vessels 2 is increased by subculture, so the number of rotating cell culture apparatuses B to be used increases accordingly. It is necessary to adopt.
  • FIG. 15 shows a syringe 1A of another embodiment used as the culture vessel 2 or the concentrated vessel 3.
  • a front flange 6 and a back flange 7 having the same outer shape are integrally provided at both ends of the cylindrical outer cylinder portion 5 in the same manner as described above, and at the end portion of the outer cylinder portion 5 on the front flange 6 side.
  • the head 8 serving as a discharge port provided in the center portion has a standard luer lock structure, and a gasket that is slidably elastically fitted into the outer cylinder portion 5 at the tip of the short shaft portion 10A of the plunger 9. 11 and a female screw 10C is provided at the other end of the short shaft portion 10A.
  • the inclination angle ⁇ of the inner surface 6A is 90 ° as a central angle.
  • the outer cylinder part 5, the back flange 7 and the luer lock structure head 8 are integrally formed of synthetic resin, and the front flange 6 is screwed to the end of the outer cylinder part 5 from the radial direction.
  • the front flange 6 may be a synthetic resin molded product, and may be bonded, heat-sealed, or ultrasonically fused to the end of the outer cylinder portion 5.
  • the syringe 1A shown in FIG. 15 has a capacity for 10 ml, but basically has the same structure for 50 ml.
  • FIG. 16 shows the connector 4A according to another embodiment.
  • the connecting tool 4 according to the present embodiment includes a first member 105 and a second member 106.
  • the first member 105 is provided with a flow path 15 at the center, a support surface 107 for holding the filter 23 on one end side in the axial direction, and a connecting portion 18 with the head 8 on the other end side in the axial direction. is there.
  • the second member 106 is provided with a flow path 19 at the center, a support surface 108 that is joined to the support surface 107 of the first member 105 on one end side in the axial direction, and a connection with the head 8 on the other end side in the axial direction.
  • a portion 21 is provided.
  • the flange portion 109 formed around the support surface 107 of the first member 105 is fitted into the recess 111 of the flange portion 110 provided around the support surface 108 of the second member 106.
  • the filter 23 is sandwiched between the support surface 107 of the first member 105 and the support surface 108 of the second member 106, and the flange portion 109 of the first member 105 and the flange portion 110 of the second member 106 Are integrated by ultrasonic welding.
  • the flow channels 15 and 19 have a diameter of 2 mm, and the filter 23 also filters spheroids in a region having a diameter of 2 mm. That is, the passage of the culture solution can be suppressed to a minimum by applying a pressure difference to the flow path 15 and the flow path 19 in a state where the entire effective surface of the filter 23 is covered with the spheroid.
  • FIG. 17 shows the rotating cell culture apparatus B1.
  • the rotating cell culture apparatus B1 of this embodiment is an apparatus that can rotate one culture vessel 2 at a predetermined rotational speed with a horizontal axis.
  • the lower portions of the front flange 6 and the back flange 7 of the culture vessel 2 are rotatably supported by a pair of rollers.
  • one side is a pair of a driving roller 112 and a driven roller 113, and the other side is a driven roller 114. , 114 pairs.
  • the central block 116 is fixed to the central portion of the upper surface of the base 115
  • the side block 117 is fixed to the side portion
  • the culture vessel 2 is placed in the concave portion 118 provided on the opposite surface side of each block.
  • the driving roller 120 having a main body fixed to the central block 116 and the driving roller 112 directly connected to the driving shaft 121 and a freely rotating driven roller 113 are arranged in parallel at the bottom of the concave portion 118. Yes.
  • driven rollers 114 and 114 that freely rotate are arranged in parallel at the bottom of the recess 118.
  • ball plungers 122 and 122 for restricting the displacement of the culture vessel 2 in the axial direction are embedded in the standing wall portions of the concave portions 118 of the central block 116 and the side block 117. Normally, a minute gap is provided between the ball plunger 122 and the outer surface of the front flange 6 or the back flange 7 so that the culture vessel 2 can rotate without load.
  • the ball plunger 122 is a restricting portion in the present invention.
  • a fixed leg 123 is detachably provided on one side of the lower surface of the base 115, and an adjuster 124 having an adjustable vertical height is detachably provided on the other side. Further, the base 115 has a level 125 built therein.
  • FIG. 21 is an improved version of the rotating cell culture apparatus B1, in which the central block 116 extends to the other end of the base 115, a space for storing the drive motor 120 is provided therein, and a drive unit is provided for the base 115. And the central block 116 are housed in a sealed state.
  • FIG. 22 and 23 show a rotating cell culture apparatus B2 having a structure in which three of the rotating cell culture apparatuses B1 are arranged on a common base 126 and are detachably attached.
  • the fixing leg 123 and the adjuster 124 of the rotating cell culture apparatus B1 are removed and screwed to the upper surface of the base 126 using the screw holes to which they are attached.
  • adjusters 127,... are attached to the four corners of the base 126 so that the water level can be adjusted.
  • the adjuster 127 the adjuster 124 removed from the rotating cell culture apparatus B1 may be used, and the fixed leg 123 may be used instead of a part of the adjuster 127.
  • FIGS. 24 to 27 are modifications of the rotating cell culture apparatus B, and show a rotating cell culture apparatus B3 that can simultaneously rotate and culture three culture vessels 2 under the same culture conditions. Basically, the same usage as the above-described rotating cell culture apparatus B2 is possible.
  • a driving block 129 incorporating a driving mechanism and an opposing block 130 are fixed on the upper surface of a base 128 so as to face each other.
  • two drive rollers 131, 131 are arranged side by side, and further driven rollers 132, 132 are arranged side by side, and the two drive rollers 131, 131 in the center are cultured in the center.
  • the back flange 7 of the vessel 2 is mounted, and the back flange 7 of the outer culture vessel 2 is mounted on the outer driven roller 132 and one drive roller 131.
  • the two drive rollers 131 and 131 are rotated synchronously in the same direction.
  • the opposing block 130 is provided with driven rollers 133 and 133 having the same size at positions facing the driving rollers 131 and 131, and driven rollers having the same size at positions facing the driving rollers 131 and 131.
  • 134 and 134 are provided.
  • a recess 135 similar to the recess 118 is provided in the drive side block 129 and the opposing block 130, and a ball plunger 136 is provided on each of the standing walls to limit the axial displacement of the culture vessel 2. It is provided corresponding to the flanges 6 and 7.
  • the drive mechanism of the drive rollers 131, 131 is a pulley in which a drive motor 138 is disposed in a box 137 provided below the base 128 and fixed to a drive shaft 139 of the drive motor 138.
  • a timing belt 143 is wound around 140 and a pulley 142 fixed to each rotary shaft 141 of the drive rollers 131 and 131 so that both the drive rollers 131 and 131 are rotationally driven in the same direction.
  • the box 137 incorporates a controller for the drive motor 138 to control the rotation speed of the culture vessel 2 and adjusters 144 and 144 at the lower end to adjust the level. .
  • iPS cells (253G1) having a predetermined number of cells were placed in a culture vessel having a capacity of 50 ml together with a culture solution (mTeSR1), and rotated and cultured in the rotating cell culture apparatus B for 3 days. Then, using the inter-cell cell fluid transfer device A, all spherical spheroids were collected and transferred to a concentrated vessel having a capacity of 10 ml. Then, using the inter-cell cell fluid transfer device A, the small spheroids crushed by passing the spherical spheroids from the concentrated vessel through a 70 ⁇ m filter were transferred to a new 50 ml culture vessel.
  • FIG. 28 is a phase difference image of this small spheroid
  • FIG. 28 (a) is a low magnification phase difference image (bar in the figure is 1000 ⁇ m)
  • FIG. 28 (b) is a high magnification phase difference image (in the figure).
  • the bar in FIG. 5 indicates 500 ⁇ m). It can be seen that the small spheroid after filtration has a slightly long and narrow shape.
  • FIG. 29 is a phase contrast image of a spheroid after culturing small spheroids
  • FIG. 29 (a) is a low-magnification phase contrast image (the bar in the figure is 1000 ⁇ m)
  • FIG. 29 (b) is a high-magnification phase contrast image. (The bar in the figure is 500 ⁇ m).
  • the mass cell culture system of the present invention stably maintained an undifferentiated state until passage 8 and continued to grow at a growth rate of 3 to 5 times. Although it depends on the initial number of cells input, it can be seen that mass culture up to the number of cells on the order of 10 9 to 10 10 can be achieved in about 6 passages with a growth rate of 3 to 5 times. It has been confirmed that it can be performed up to 10 passages using the mass cell culture system according to the present invention.
  • 253G1 cells and 409B2 cells were used as human induced pluripotent stem cells (hiPSCs).
  • 253G1 cells (introduced Oct3 / 4, Sox2, and Klf4; see Non-patent Document 2) are 409B2 cells (introduced Oct3 / 4, Sox2, Klf4, L-Myc, Lin28, and p53 shRNA; non-patent document 2);
  • Patent Document 4 was purchased from RIKEN Bio-Resource Center Cell Materials Development Office (RIKEN BRC CELL BANK) (Japan) under cell number HPS0076.
  • Construction of spherical spheroids 253G1 cells were cultured in human ES / iPS cell maintenance medium mTeSR1 (STEM CELL TECHNOLOGIES) or StemFit AK02N (Ajinomoto) using a 6 cm or 10 cm culture dish coated with Matrigel (BD Biosciences). ), The culture medium was changed every other day, and subcultured using 5 mM EDTA.
  • 253G1 cells used for seeding were detached into small spheroids (loose cell mass having a diameter of about 50 ⁇ m to 200 ⁇ m) composed of about 20 to 40 cells using 5 mM EDTA.
  • FIG. 30 is a phase contrast image of spheroids after 3 days of rotation culture of iPS cells (253G1 cells) using 10 ml vessels
  • FIG. 30 (a) is a phase difference image of spheroids after rotation culture in mTeSR1 medium.
  • FIG. 30 (b) is a phase contrast image of spheroids after rotational culture in AK02N medium.
  • the white bar in the figure indicates 200 ⁇ m.
  • the obtained spherical spheroids were about 50 and the main size was 200 to 400 ⁇ m in diameter.
  • 409B2 cells ⁇ Spheroid production from human induced pluripotent stem cells (hiPSC; 409B2 cells) by three-dimensional culture using syringe-type culture vessels> 409B2 cells are cultured in human ES / iPS cell maintenance medium mTeSR1 (STEM CELL TECHNOLOGIES) or StemFit AK02N (Ajinomoto) using a 6 cm or 10 cm culture dish coated with Matrigel (BD Matrigel TM, BD Biosciences), every other day. The culture medium was exchanged and subcultured using 5 mM EDTA.
  • 409B2 cells used for seeding were detached into small spheroids (loose cell mass having a diameter of about 50 ⁇ m to 200 ⁇ m) consisting of about 20 to 40 cells using 5 mM EDTA.
  • 5 ⁇ 10 5 exfoliated 409B2 cells (small spheroids) are seeded in a 10 ml vessel of a syringe type in mTeSR1 medium (10 ml) containing ROCK inhibitor Y27632 or StemFit AK02N medium (10 ml) containing ROCK inhibitor Y27632, and rotating cell culture Rotating culture was performed at 37 ° C. and 6 rpm for 3 days using the apparatus.
  • FIG. 31 is a phase contrast image of spheroids after 3 days of rotational culture of iPS cells (409B2 cells) using 10 ml vessels
  • FIG. 31 (a) is a phase difference image of spheroids after rotational culture in mTeSR1 medium
  • FIG. 31 (b) is a phase contrast image of spheroids after rotational culture in AK02N medium.
  • the white bar in the figure indicates 200 ⁇ m.
  • the obtained spherical spheroids were about 50 and the main size was 200 to 400 ⁇ m in diameter.
  • ⁇ Continuous subculture test> a continuous subculture test was performed.
  • the procedure is schematically as shown in FIGS.
  • the culture vessel was used for rotational culture, and the small spheroids were transferred to a new culture vessel at the same time as filtration using the inter-cell cell fluid transfer device A during passage.
  • 253G1 cells (5 ⁇ 10 5 cells) exfoliated into small spheroids using 5 mM EDTA were seeded in a syringe-type 10 ml vessel, and using a rotary culture apparatus in a StemFit AK02N medium containing 10 ⁇ M ROCK inhibitor Y27632.
  • Rotating culture was performed for 3 days to produce spherical spheroids.
  • the syringe-type 10 ml vessel used in the rotary culture was connected to the filter built-in connection portion and the collection syringe-type 10 ml vessel, and an inter-cell cell fluid transfer device was used.
  • the spherical spheroids were broken into small spheroids by passing through a filter.
  • the obtained small spheroids were filled with a new medium, and rotated and cultured at 37 ° C. and 6 rpm for 4 days using a rotating cell culture apparatus to produce spherical spheroids.
  • FIG. 32 shows a phase difference image of a small spheroid immediately after crushing at each passage.
  • Spherical spheroids could be obtained each time during each culture period. Their main diameter was 200 to 400 ⁇ m, and the maximum diameter was over 700 ⁇ m.
  • the small spheroids at the time of each crush had a similar rod-like form. Contamination did not occur in the total culture period of 40 days.
  • the present invention can be used to efficiently cultivate pluripotent stem cells, particularly iPS cells and adherent cells at a low cost for use in regenerative medicine and the like.
  • a Vessel cell fluid transfer device B, B1, B2, B3 rotating cell culture device, C cap, M medium, S spheroids, SM cell fluid, 1 syringe, 2 culture vessels, 3 Concentrated vessel, 4 Connector, 5 outer cylinder part, 6 front flange, 7 Back flange, 8 head, 9 Plunger, 10 Shaft, 10A shaft, 10B shaft, 10C female screw, 10D male screw, 10E notch, 11 gasket, 12 plunger button, 13 first member, 14 second member, 15 flow path, 16 Housing part, 17 Female thread part, 18 connections, 19 flow paths, 20 male screw part, 21 connection part, 22 recesses, 23 filters, 24 packing, 25 spacer, 26 culture solution supply pipe, 27 first mechanism, 28 second mechanism, 29 base part, 30 fixed parts, 31 movable parts, 32 drive mechanism part, 33 fixing part, 34 movable part, 35 drive mechanism part, 36 protective covers, 37 base plate, 38 support plate, 39 reinforcement plate, 40 linear guide, 41 linear actuator, 42 guide rails, 43 movable blocks

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Abstract

Le problème décrit par la présente invention consiste : à préparer un système de culture de cellules à grande échelle, moyennant quoi il devient possible de cultiver une cellule souche pluripotente, particulièrement une cellule iPS, qui a pour objet d'être utilisée en médecine régénérative ou similaire, sur une grande échelle en l'absence d'une cellule nourricière ou d'un agent d'enduction tout en conservant son état non différencié, il devient également possible de sous-cultiver la cellule tout en éliminant les fluctuations qui résultaient des différences de compétence entre les opérateurs, et il devient également possible de cultiver une cellule adhérente sous un état de flottement ; en un dispositif de transfert de liquide de cellules de récipient à récipient qui peut être utilisé dans le système ; et en un dispositif rotatif de culture de cellules. La solution selon l'invention consiste en un système de culture de cellules à grande échelle destiné à la sous-culture et destiné à la translocation entre des sphéroïdes et un milieu de culture liquide dans un système fermé à l'aide d'un récipient présentant une structure type seringue en vue de produire des cellules sur une grande échelle, où, dans le récipient, une collerette avant et une collerette arrière, qui présentent la même forme externe circulaire l'une que l'autre, sont prévues d'une seule pièce aux deux extrémités d'une partie externe de cylindre et une partie tête est coiffée d'un capuchon détachable, de sorte que le récipient peut exécuter une culture rotative en utilisant la collerette avant et la collerette arrière d'une manière telle qu'un liquide de cellules, qui comprend des cellules en suspension dans un milieu de culture liquide, soit rempli dans un espace fermé comportant un joint d'étanchéité d'un piston.
PCT/JP2017/002558 2016-04-27 2017-01-25 Système de culture de cellules à grande échelle et dispositif de transfert de liquide de cellules de récipient à récipient destiné à y recevoir le système de culture de cellules, et dispositif rotatif de culture de cellules WO2017187680A1 (fr)

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EP20190032.1A EP3770245A1 (fr) 2016-04-27 2017-01-25 Système de culture de cellules à grande échelle
CN201780025397.1A CN109072157A (zh) 2016-04-27 2017-01-25 大量细胞培养系统、该系统所使用的容器间细胞液移送装置以及旋转细胞培养装置
US16/094,346 US20190119623A1 (en) 2016-04-27 2017-01-25 Large-scale cell culture system and inter-vessel cell liquid transfer device to be used therein, and rotary cell culture device
EP17788970.6A EP3450535A4 (fr) 2016-04-27 2017-01-25 Système de culture de cellules à grande échelle et dispositif de transfert de liquide de cellules de récipient à récipient destiné à y recevoir le système de culture de cellules, et dispositif rotatif de culture de cellules

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JP2016-089837 2016-04-27
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JP2017007993A JP6268342B2 (ja) 2016-04-27 2017-01-19 大量細胞培養システム及びそれに用いる回転細胞培養装置

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JP2021525106A (ja) * 2018-06-01 2021-09-24 ヘルプ・ステム・セル・イノベイションズ・カンパニー・リミテッドＨｅｌｐ Ｓｔｅｍ Ｃｅｌｌ Ｉｎｎｏｖａｔｉｏｎｓ Ｃｏ．， Ｌｔｄ． 分化用培地及びオリゴデンドロサイト前駆細胞の製造方法
CN113624576A (zh) * 2021-08-03 2021-11-09 深路医学科技(武汉)有限公司 一种半自动液基细胞制片机
CN114621872A (zh) * 2022-02-24 2022-06-14 河南茵特赛尔生物技术有限公司 一种免疫细胞培养液自动补液装置
CN117774432A (zh) * 2024-02-27 2024-03-29 南昌大学第一附属医院 一种细菌培养基挤出装置

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JP7114134B2 (ja) 2018-06-01 2022-08-08 ヘルプ・ステム・セル・イノベイションズ・カンパニー・リミテッド 分化用培地及びオリゴデンドロサイト前駆細胞の製造方法
CN113624576A (zh) * 2021-08-03 2021-11-09 深路医学科技(武汉)有限公司 一种半自动液基细胞制片机
CN114621872A (zh) * 2022-02-24 2022-06-14 河南茵特赛尔生物技术有限公司 一种免疫细胞培养液自动补液装置
CN114621872B (zh) * 2022-02-24 2022-09-16 河南茵特赛尔生物技术有限公司 一种免疫细胞培养液自动补液装置
CN117774432A (zh) * 2024-02-27 2024-03-29 南昌大学第一附属医院 一种细菌培养基挤出装置

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