WO2021065969A1 - Device and method for preparing cell suspension - Google Patents

Device and method for preparing cell suspension Download PDF

Info

Publication number
WO2021065969A1
WO2021065969A1 PCT/JP2020/037054 JP2020037054W WO2021065969A1 WO 2021065969 A1 WO2021065969 A1 WO 2021065969A1 JP 2020037054 W JP2020037054 W JP 2020037054W WO 2021065969 A1 WO2021065969 A1 WO 2021065969A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell suspension
mounting portion
cells
mixing
container
Prior art date
Application number
PCT/JP2020/037054
Other languages
French (fr)
Japanese (ja)
Inventor
祐香 嶋津
りさ 結城
徹哉 山口
Original Assignee
テルモ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by テルモ株式会社 filed Critical テルモ株式会社
Publication of WO2021065969A1 publication Critical patent/WO2021065969A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • the present invention relates to devices and methods for preparing cell suspensions.
  • Non-Patent Document 1 fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for repair of myocardial tissue damaged by ischemic heart disease such as angina and myocardial infarction.
  • Forming a sheet-like cell culture by this method requires more cells than usual, so when freezing and / or thawing the cells, a device was devised to minimize physicochemical damage to the cells.
  • a device was devised to minimize physicochemical damage to the cells.
  • frozen cells are suspended together with a freezing medium, stored in a frozen cell storage container (cryotube, etc.), frozen, transferred to a hospital, and subjected to a warm bath, so that the cells are contained in the frozen cell storage container. It is necessary to thaw the cryopreserved cells of. (Patent Documents 4 and 5).
  • the above-mentioned system had a complicated structure for realizing an operation unit and a control unit, a high manufacturing cost, and a complicated operation.
  • the present invention has been made in consideration of these points, and it is possible to efficiently prepare a cell suspension while reducing the risk of contamination by having a simple structure and a simple operation. It is to provide the means that can be done.
  • the present invention relates to the following.
  • [1] Includes a first mounting portion to which a storage container for accommodating a cell suspension can be mounted, and a second mounting portion to which a mixing container for mixing the cell suspension and the mixed solution can be mounted.
  • [2] The device according to [1], wherein the fluid connection is performed via a flexible tube.
  • [3] The device according to [1] or [2], wherein the first mounting portion is configured to mount a plurality of storage containers.
  • [4] The device according to any one of [1] to [3], which is provided with an injection port for a mixture used for mixing.
  • the first mounting portion and / or the second mounting portion is configured to be openable and closable.
  • a cell suspension comprising a second mounting portion to which a mixing container for mixing the liquid and the mixed liquid can be mounted, and having a connecting portion for fluidly connecting the first mounting portion and the second mounting portion. Transport device.
  • a method for preparing a cell suspension wherein (a) a first mounting portion to which a storage container for storing the cell suspension can be mounted, and the cell suspension and a mixed solution.
  • the method comprising attaching the containment vessel and the mixing vessel to the device and transferring the cell suspension from the containment vessel to the mixing vessel via the device.
  • the step (b) includes the step of injecting the mixture from the inlet for the mixture used for mixing with the cell suspension provided in the device (c) [14]. The method described in.
  • [16] The method according to [14] or [15], which comprises, after step (c), (d) a step of transferring the mixed solution to a storage container or a mixing container.
  • [17] The method according to any one of [14] to [16], wherein the mixed solution is a diluent.
  • [18] A method for treating a disease that is ameliorated by application of a sheet cell culture, the cells prepared using the device according to any one of [1] to [13], or [14].
  • the manufacturing cost can be kept low, and the cell suspension can be efficiently prepared by a simple operation, so that it depends on the procedure of the operator.
  • the work level can be made uniform.
  • the cell suspension can be prepared aseptically without exposing the cell suspension to the outside air, and the risk of contamination can be reduced.
  • harmful substances can be appropriately removed by a simple operation, and the work can be made uniform, so that the cell viability is improved.
  • cryopreserved cells can be thawed efficiently and quickly by a simple mechanism, and cell suspensions can be collected efficiently and aseptically by a simple mechanism.
  • the collected cell suspension can be rapidly and aseptically transferred to the mixing vessel without contaminating the surroundings. Therefore, the cell viability is improved because the next step, such as dilution of the cell suspension, can be made quickly and the effects of cytotoxic components on the cells can be minimized.
  • the osmotic load when diluting a cell suspension can be controlled by a simple mechanism, the number of dead cells due to a sudden change in osmotic pressure is reduced, and the number of recoverable living cells is increased. .. In particular, even when the cells are used after thawing without undergoing growth culture, it is possible to secure a sufficient number of viable cells.
  • FIG. 1 is a conceptual diagram of a device according to the first embodiment of the present invention.
  • FIG. 2 is a conceptual diagram showing a usage example of the device according to the first embodiment of the present invention.
  • the present invention has a first mounting portion to which a storage container for storing a cell suspension can be mounted and a first mounting portion to which a mixing container for mixing the cell suspension and a mixed solution can be mounted.
  • the present invention relates to a device in which a first mounting portion and a second mounting portion are fluidly connected, including a mounting portion.
  • the present invention is a method for preparing a cell suspension in one aspect, wherein (a) a first mounting portion to which a storage container for storing the cell suspension can be mounted, and a cell suspension.
  • a step of providing a device comprising a second mounting portion to which a mixing container for mixing the liquid and the mixed liquid can be mounted, the first mounting portion and the second mounting portion being fluid connectable, (b) cells.
  • the method relates to the method comprising mounting a containment vessel and a mixing vessel containing the suspension on the device and transferring the cell suspension from the containing vessel to the mixing vessel via the device.
  • the cell suspension is obtained by thawing cryopreserved cells.
  • the "cryopreserved cell” usually means the cryopreserved cell itself, but may also mean one cryopreserved unit of the cryopreserved cell.
  • the cryopreservation unit means a group of cells that are cryopreserved together as a group, for example, one tube. Therefore, in this case, when the "cryopreserved cells" are thawed, the frozen "cell suspension” is obtained.
  • the "osmotic pressure load at the time of dilution” means the rate of change of the osmotic pressure per unit time that changes due to the addition of the diluent.
  • the osmotic load varies depending on factors such as the addition rate of the diluent (addition amount per unit time) and the difference in osmotic pressure between the diluent and the solution to be diluted (for example, a thawed cell suspension).
  • the “maximum osmotic pressure load” means the maximum value among the osmotic pressure loads that change due to the addition of the diluent from the start of dilution to the end of dilution.
  • the present invention is characterized in that a cell suspension obtained by thawing cryopreserved cells is diluted, and the change in osmotic pressure at the time of dilution is sufficiently slowed down.
  • a culture medium or the like is added to the thawed cell suspension for the purpose of reducing the influence of cytotoxic components in the thawed cell suspension. And dilute.
  • the present inventors have found that when the cells are rapidly diluted in such a dilution step, the viability of the cells is reduced due to the damage caused to the cells by the sudden change in the osmotic pressure of the suspension.
  • any method known in the art may be used, for example, slowing down the rate of adding the diluent or using a diluent having a small difference in osmotic pressure from the cell suspension.
  • the diluent that can be used in the present invention is not particularly limited, but one that does not cause physicochemical damage to cells is preferable.
  • the diluent include, but are not limited to, liquid media such as DMEM medium, Hanks balanced salt solution, buffer solution such as PBS, isotonic solution such as physiological saline, and distilled water. .. Further, other components such as albumin may be further added to these.
  • the unit Osm is used as a unit of osmotic pressure
  • 1 Osm means an osmotic pressure equivalent to the osmotic pressure of an ideal solution of 1 mol / L.
  • the osmotic pressure load at the time of dilution is expressed as a change in osmotic pressure per second (unit: Osm / sec) under normal temperature conditions, but any unit can express the magnitude of the change in osmotic pressure. May be used, and examples thereof include, but are not limited to, the rate of addition of the diluent, the rate of increase in volume or weight, and the like.
  • the osmotic pressure load may be obtained by measuring the osmotic pressure change in real time, or by obtaining the osmotic pressure at a certain two time points and obtaining the average change per unit time between them.
  • the osmotic load at the start of addition of the diluent generally permeates in that time width. It is considered that the maximum value of the pressure load is reached and the osmotic pressure load decreases as the amount of the diluent added increases.
  • the osmotic pressure at the start of addition of the diluent and the osmotic pressure after a unit time thereof (for example, 1 second in the above example of the present invention). Is obtained, and the difference in osmotic pressure at both time points is regarded as the maximum osmotic pressure load at the dilution.
  • the dilution may be carried out while the thawed cell suspension is placed in a cryopreservation container or transferred to a mixing container (centrifuge tube or the like).
  • a mixing container centrifuge tube or the like.
  • the cryopreservation container after transferring the cell suspension is rinsed with a diluted solution, and this rinse solution is added to the cell suspension.
  • the addition of such a rinse solution also corresponds to the dilution of the present invention. Therefore, in one embodiment of the present invention, the diluent comprises a rinse solution obtained by transferring the thawed cell suspension to a preparation container and then rinsing the cryopreservation container.
  • Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, heart stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) And so on.
  • adherent somatic cells eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin.
  • stem cells eg, tissue stem cells such as myoblasts, heart stem cells, embryonic stem cells, pluripot
  • Somatic cells may be differentiated from stem cells, especially iPS cells.
  • Non-limiting examples of cells capable of forming a sheet-like cell culture include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots).
  • the cell suspension obtained by thawing may contain a cytotoxic component (for example, DMSO), the influence of the cytotoxic component can be reduced by diluting it.
  • a cytotoxic component for example, DMSO
  • the present invention is characterized in that the osmotic pressure change is sufficiently slowed down at the time of this dilution, that is, the osmotic pressure load is sufficiently reduced, thereby increasing the viability of the thawed cells.
  • the threshold value of "sufficiently slow osmotic pressure change” can change depending on the cells used, melting conditions, temperature, and the like.
  • a normal cryopreservation solution for example, DMEM medium containing about 10% DMSO
  • a normal diluted solution for example, commercially available DMEM medium
  • the maximum osmotic load is about 250 mOsm / sec or less in some embodiments. It is preferably about 220 mOsm / sec. It is more preferably about 100 mOsm / sec or less, and even more preferably about 50 mOsm / sec or less.
  • the lower limit of the maximum osmotic load may not be set in particular, but a normal cryopreservation solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example)
  • a normal cryopreservation solution for example, DMEM medium containing about 10% DMSO
  • a normal diluted solution for example, DMEM medium containing about 10% DMSO
  • a normal diluted solution for example
  • the maximum osmotic load is 2 mOsm / sec to 250 mOsm / sec, more preferably 2 mOsm / sec to 220 mOsm / sec, and even more preferably 20 mOsm / sec to 100 mOsm / sec. From the viewpoint of the amount of viable cells that can be recovered, it is most preferably 40 mOsm / sec to 50 mOsm / sec.
  • the maximum osmotic load is the maximum osmotic load from the start of dilution to 3-fold dilution, and in another embodiment, until 2-fold dilution. Maximum osmotic load between.
  • dilution is performed while measuring the osmotic pressure and adjusting the maximum osmotic load.
  • the osmotic pressure may be measured continuously at all times, or may be performed at specific time intervals such as every 1 second, every 10 seconds, every 30 seconds, every 1 minute.
  • any method known in the art can be used, and the osmotic pressure can be measured by using, for example, an osmotic meter or the like, without limitation.
  • the "containment container” refers to a container that can contain a cell suspension, seal it, and store it aseptically.
  • the storage container is not particularly limited, and examples thereof include a cryotube and a cryovial.
  • the containment vessel is preferably constructed of a freezing material.
  • one configuration and another configuration are "fluid-connected" so that these two configurations are connected by a tube-like tube through which the fluid flows without being exposed to the outside air. It means to be composed.
  • "preparation" of a cell suspension includes thawing, collecting, transferring, diluting, rinsing, etc. of the cell suspension.
  • Thawing a cell suspension means thawing cryopreserved cells to obtain a cell suspension
  • thawing a cell suspension means thawing cryopreserved cells contained in a storage container.
  • the cryopreserved cells may be thawed in a separate container.
  • Collection of cell suspension means collecting cell suspension contained in a storage container, and includes collecting cell suspension contained in a plurality of storage containers.
  • the transfer of the cell suspension means the transfer of the cell suspension contained in the storage container to the mixing container via the flow path, and in one embodiment, the transfer moves the storage container to a position higher than the mixing container. This is done by positioning and using potential energy to move the cell suspension.
  • Diluting the cell suspension means adding (mixing) the diluent to the cell suspension, and in one embodiment, the diluent may be added to the cell suspension transferred to the mixing vessel. ..
  • the diluent may include a rinse solution obtained by rinsing the cryopreservation container after transferring the thawed cell suspension to another container.
  • Another aspect of the invention is a method of treating a disease that is ameliorated by the application of a sheet cell culture, using the devices of the invention or using cells prepared by the methods of the invention.
  • the present invention relates to a method comprising applying the sheet-like cell culture produced in the above-mentioned to a subject in need thereof.
  • the treatment method of the present invention may include applying a sheet cell culture prepared using the cells prepared by the method of the present invention to a subject in need thereof.
  • the treatment method of the present invention may further include collecting cells from a subject or a tissue from which the cells are supplied, prior to the step of producing the sheet-shaped cell culture. ..
  • the subject from which the cell or tissue to be the source of the cell is collected is the same individual as the subject to whom the sheet-like cell culture or composition is administered.
  • the subject from which the cell or tissue from which the cell is source is collected is a separate entity of the same species as the subject receiving the administration, such as a sheet cell culture or composition.
  • the subject from which the cell or the tissue that is the source of the cell is collected is an individual that is different from the subject to which the sheet-like cell culture or composition is administered.
  • the term "subject” means any individual organism, preferably an animal, more preferably a mammal, even more preferably a human.
  • the subject may be healthy or suffer from some disease, but the disease improved by the application of the sheet-like cell culture (for example, a disease related to tissue abnormality).
  • the treatment typically means a subject who has or is at risk of developing the disease.
  • treatment shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at the cure, temporary remission or prevention of the disease.
  • treatment refers to delaying or stopping the progression of a disease that is ameliorated by the application of sheet cell culture (eg, a disease associated with tissue abnormalities), regression or disappearance of a lesion, or the onset of the disease.
  • sheet cell culture eg, a disease associated with tissue abnormalities
  • regression or disappearance of a lesion e.g. a disease associated with tissue abnormalities
  • the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-shaped cell cultures, etc.).
  • the amount is preferably an amount that prevents the onset and recurrence of the disease or cures the disease.
  • an amount that does not cause an adverse effect exceeding the benefit of administration is preferable.
  • Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art.
  • the size of the tissue lesion to be treated can be an important index for determining the effective amount.
  • the application method typically includes direct application to an organization.
  • the application frequency is typically once per treatment, but it is possible to apply a plurality of sheets when the desired effect is not obtained.
  • a method of applying the sheet-shaped cell culture of the present invention or the like so as to be attached to a diseased part of tissues is used.
  • the tissue to be treated is not limited, for example, heart (myocardium), cornea, retina, esophagus, skin, articular cartilage, liver, pancreas, gingiva, kidney, thyroid gland, skeletal muscle, etc.
  • the middle ear and the like can be mentioned.
  • the diseases to be treated include, for example, heart diseases (for example, myocardial injuries (myocardial infarction, cardiac trauma, etc.)) and myocardial diseases (ischemic myocardial disease, dilated myocardial disease, dilated phase fertilizer).
  • the application site moves frequently due to beating or peristaltic movement, such as when the application tissue is an organ such as the heart, it may fall off from the application site simply by attaching the sheet-shaped cell culture of the present invention. is there. Therefore, in a preferred embodiment of the treatment method of the present invention, after applying the sheet-shaped cell culture or the like of the present invention to the diseased site, the sheet-shaped cell culture or the like of the present invention is sutured and fixed through a suture needle or the like through the penetrating site. Is included. As a result, it is possible to prevent the sheet-shaped cell culture or the like of the present invention applied to the diseased part from falling off from the applied part.
  • FIG. 1 is a conceptual diagram of a device according to the first embodiment of the present invention.
  • FIG. 2 is a conceptual diagram showing a usage example of the device according to the first embodiment of the present invention.
  • the device includes a first mounting portion 1 and a second mounting portion 2.
  • the first mounting portion 1 is configured so that the storage container C can be mounted.
  • the second mounting portion 2 is configured so that the mixing container M can be mounted.
  • the first mounting portion 1 may be configured so that a plurality of storage containers C can be mounted.
  • the first mounting portion 1 and the second mounting portion 2 may be directly fluid-connected, or the first mounting portion 1 and the second mounting portion 2 are fluid-connected via a flexible tube 4. You may.
  • the portion where the first mounting portion 1 and the second mounting portion 2 are fluidly connected is referred to as a connecting portion.
  • the first mounting portion 1 and the second mounting portion 2 are configured so that the openings of the storage container C and the mixing container M can be liquid-tightly mounted, respectively.
  • the first mounting portion 1 may be provided with an injection port 3 for a mixed solution used for mixing with the cell suspension contained in the storage container C in the flow path.
  • a control means 5 capable of controlling the flow rate of a liquid (for example, a mixed liquid) flowing through the flow path may be connected to the flow path (for example, a connecting portion or a second mounting portion 2).
  • a sterilization filter 6 for taking in outside air into the flow path may be connected to the flow path.
  • the inlet 3, the control means 5, and the sterilization filter 6 are arbitrary configurations that can be included in the device according to the use of the device.
  • the storage container C containing the cell suspension is mounted on the first mounting portion 1, and the cell suspension and the mixed solution can be sufficiently mixed.
  • a mixing container M having a volume for example, a centrifuge tube
  • the storage container C and the mixing container M are attached to the device in this way, a flow path (closed circuit) in which the storage container C, the device, and the mixing container M are fluidly connected in this order is formed. Therefore, the cell suspension contained in the storage container C can be aseptically transferred to the mixing container M without being exposed to the outside air through the device and without contaminating the surroundings. At this time, the transfer can be made more efficient by taking in the outside air into the closed circuit through the sterilization filter 6.
  • the tube 4 when mounting the storage container C, the tube 4 is bent and the first mounting portion 1 is inverted so that the portion to which the storage container C is mounted faces the opening of the storage container C.
  • the cells can be easily mounted on the first mounting portion 1 with the opening of the storage container C facing upward, so that leakage of the cell suspension in the storage container C can be prevented.
  • the storage container C may be mounted on the first mounting portion 1 in a clean room or the like, or the device itself may be flipped upside down without bending the tube 4.
  • the storage container C When the cryopreserved cells are contained in the storage container C, the storage container C can be subjected to thawing (for example, a hot bath) with the opening of the storage container C mounted upward.
  • thawing for example, a hot bath
  • a plurality of storage containers C can be mounted on the first mounting portion 1, these can be subjected to a hot bath at the same time.
  • a hot bath Normally, when multiple containers for storing frozen cells are subjected to a hot bath, they may float on the water surface due to their buoyancy, so the operator had to grasp them by hand and submerge them in water. By doing so, such work can be made more efficient.
  • the tube 4 is bent to invert the first mounting portion 1 and position it at a position higher than the second mounting portion 2. Then, using potential energy, the cell suspension is transferred from the storage container C to the mixing container M via the device. Inversion may invert the device itself upside down. By using the device in this way, the process from thawing of the cell suspension to transfer can be performed aseptically even outside the clean room.
  • the tube 4 is bent again to invert the first mounting portion 1, and the mixed solution (in this case, the diluted solution) is injected from the injection port 3. Then, the inside of the storage container C can be rinsed. In this way, by reversing the first mounting portion 1 without reversing the mixing container M, the inside of the storage container C is rinsed without causing the cell suspension transferred to the mixing container M to flow back to the storage container C. You can also do it. It is also possible to improve the cell collection rate by applying a cell non-adhesive treatment to the inner surface of the flow path.
  • the side opposite to the mounting portion of the first mounting portion 1, that is, the second mounting portion 2 side is formed in a funnel shape.
  • Cell suspensions contained in a plurality of storage containers C can be efficiently collected.
  • the process from thawing of the cell suspension to collection can be performed aseptically and efficiently even outside the clean room.
  • the cells it is preferable to use cells in the logarithmic growth phase from the viewpoint of the amount of live cells recovered.
  • the first mounting portion 1 is positioned higher than the second mounting portion 2.
  • this rinse solution can be added to the cell suspension.
  • the diluent is further injected from the injection port 3 as the mixture, the diluent can be added to the cell suspension transferred to the mixing vessel M.
  • the control means 5 is connected to the device, the flow rate of the fluid, that is, the addition rate of the diluent (addition amount per unit time) can be controlled by the control means 5.
  • the addition rate of the diluent can be controlled by adjusting the positional relationship between the first mounting portion 1 and the second mounting portion 2. That is, when the position of the first mounting portion 1 is lower than or the same as the position of the second mounting portion 2, the mixed liquid injected from the injection port 3 does not flow to the second mounting portion 2, but moves to a higher position. By doing so, it can be added to the second mounting portion 2, and by making it higher, the addition rate can be increased.
  • the amount of addition may be controlled per unit time by crushing the tube 4 by hand to narrow the flow path.
  • the control means 5 may connect a clip-like object such as a clamp capable of crushing the tube 4 to the flow path to control the cross-sectional area of the flow path, or provide a valve in the flow path to provide a flow path.
  • the cross-sectional area of the valve may be controlled by the degree of opening of the valve, or a valve whose degree of opening changes depending on the weight of the fluid flowing through the flow path may be provided in the flow path so that the flow rate is automatically controlled.
  • a method of slowing down the rate of adding the diluent can be used, but if the rate of addition is slow, it takes time to sufficiently dilute the cell suspension.
  • the proportion of the diluted solution in the cell suspension increases, so it can be said that rapid dilution of the cell suspension is unlikely to occur even if a larger amount of the diluted solution is added. Therefore, in the present invention, by controlling the addition rate of the diluent so as to increase as the dilution progresses, the time required for the dilution step can be significantly shortened.
  • the threshold value of "sufficiently slow osmotic pressure change” can change depending on the cells used, melting conditions, temperature, and the like.
  • a normal cryopreservation solution for example, DMEM medium containing about 10% DMSO
  • a normal diluted solution for example, commercially available DMEM medium
  • the maximum osmotic load is about 250 mOsm / sec or less in some embodiments. It is preferably about 220 mOsm / sec. It is more preferably about 100 mOsm / sec or less, and even more preferably about 50 mOsm / sec or less.
  • the lower limit of the maximum osmotic load may not be set in particular, but a normal cryopreservation solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example, commercially available).
  • DMEM medium or the like it is preferably about 2 mOsm / sec or more, more preferably about 20 mOsm / sec or more, and even more preferably about 40 mOsm / sec or more.
  • the maximum osmotic load is 2 mOsm / sec to 250 mOsm / sec, more preferably 2 mOsm / sec to 220 mOsm / sec, and even more preferably 20 mOsm / sec to 100 mOsm / sec. From the viewpoint of the amount of viable cells that can be recovered, it is most preferably 40 mOsm / sec to 50 mOsm / sec.
  • the maximum osmotic pressure load can be suitably controlled by controlling the injection amount of the diluent to be injected from the injection port 3, controlling the flow rate of the flow path by the control means 5, controlling the flow velocity using potential energy, and the like.
  • the cell suspension since the cell suspension has a simple structure, the production cost can be kept low, and the cell suspension can be efficiently prepared by a simple operation.
  • the work level can be made uniform without depending on the procedure.
  • the cell suspension can be prepared aseptically without exposing the cell suspension to the outside air, and the risk of contamination can be reduced.
  • harmful substances can be appropriately removed by a simple operation, and the work can be made uniform, so that the cell viability is improved.
  • cryopreserved cells can be thawed efficiently and quickly by a simple mechanism, and cell suspensions can be collected efficiently and aseptically by a simple mechanism.
  • the collected cell suspension can be rapidly and aseptically transferred to the mixing vessel without contaminating the surroundings. Therefore, the cell viability is improved because the next step, such as dilution of the cell suspension, can be made quickly and the effects of cytotoxic components on the cells can be minimized.
  • the osmotic pressure load at the time of diluting the cell suspension can be controlled by a simple mechanism, so that the work can be made uniform, and the number of dead cells due to a sudden change in osmotic pressure can be reduced, thereby recovering. Improves the number of living cells. In particular, even when the cells are used after thawing without undergoing growth culture, it is possible to secure a sufficient number of viable cells.
  • the present invention is not limited to this.
  • the case where the diluted solution is used as the mixed solution has been described, but as the mixed solution, any liquid that can be added to the cell suspension can be freely selected and placed in the mixing container M. It can be added to the transferred cell suspension.
  • the mixed solution can be added aseptically.
  • the mixed solution that can be mixed with the cell suspension includes, but is not limited to, a diluted solution, a diluted solution, a waste solution, a medium, a washing solution, a pretreatment solution, and a phosphate buffer solution (PBS).
  • PBS phosphate buffer solution
  • the first mounting portion 1 and / or the second mounting portion 2 may be provided so that the cell suspension can be prepared without using the clean room. It can also be configured to be openable and closable. That is, when the first mounting portion 1 and the second mounting portion 2 are closed, the flow path formed by the first mounting portion 1 and the second mounting portion 2 (which may include the tube 4) is sterile. Can be kept in a state.
  • the risk of contamination at the time of mounting can be reduced.
  • the mounting portion of the first mounting portion 1 and / or the second mounting portion 2 is covered with a film or the like, and an opening / closing means for tearing the film when the storage container C and / or the mixing container M is mounted is applied. Therefore, the risk of contamination at the time of mounting can be reduced.
  • the device may include a first mounting portion 1, a second mounting portion 2, and a mixing container M mounted on the second mounting portion 2. Then, the first mounting portion 1 is configured to be openable and closable, and when the storage container C is not mounted, it is closed to form the first mounting portion 1, the second mounting portion 2, and the mixing container M. The space (flow path) can be aseptically closed. Then, the cell suspension can be transferred by opening the storage container C when it is attached. In this case, at least the first mounting portion 1 may be configured to be openable and closable. In the present invention, each configuration can be replaced with any configuration capable of exerting the same function, or any configuration can be added.
  • the device of the present invention can be used in sequence by, for example, the following steps.
  • (2) A step of thawing the storage container and thawing the cryopreservation fine to obtain a cell suspension.
  • (3) The step of transferring the cell suspension from the storage container to the mixing container via the device.
  • (4) A step of injecting the diluted solution from the injection port provided in the device.
  • the device may be equipped with a storage container containing the cell suspension, in which case step (2) may be omitted.
  • the diluted solution injected in step (4) is transferred to a storage container, the remaining cell suspension is rinsed, and this rinse solution is transferred to a mixing container to increase the recovery rate of living cells. You may.
  • the maximum osmotic load may be adjusted by connecting a control means to the device and controlling the flow rate of the fluid flowing through the flow path.
  • Sheet cell cultures made using the devices of the invention or with cells prepared by the methods of the invention are applied to subjects in need thereof and improved by application of the sheet cell cultures. Diseases can also be treated.

Abstract

The purpose of the present invention is to provide a means with which it is possible to efficiently prepare a cell suspension while reducing the risk of occurrence of contamination using a simple operation and with a simple configuration. The present invention pertains to a device including a first installation part in which an accommodation container for accommodating a cell suspension can be installed, and a second installation part in which a mixture container for mixing the cell suspension and a mixed liquid can be installed, the device being such that the first installation part and the second installation part are fluidly connected.

Description

細胞懸濁液を調製するためのデバイスおよび方法Devices and methods for preparing cell suspensions
　本発明は、細胞懸濁液を調製するためのデバイスおよび方法に関する。 The present invention relates to devices and methods for preparing cell suspensions.
　近年、損傷した組織等の修復のために、種々の細胞を移植する試みが行われている。例えば、狭心症、心筋梗塞などの虚血性心疾患により損傷した心筋組織の修復のために、胎児心筋細胞、骨格筋芽細胞、間葉系幹細胞、心臓幹細胞、ＥＳ細胞、ｉＰＳ細胞等の利用が試みられている（非特許文献１）。 In recent years, attempts have been made to transplant various cells for repairing damaged tissues and the like. For example, use of fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for repair of myocardial tissue damaged by ischemic heart disease such as angina and myocardial infarction. Has been attempted (Non-Patent Document 1).
　このような試みの一環として、スキャフォールドを利用して形成した細胞構造物や、細胞をシート状に形成したシート状細胞培養物が開発されてきた（例えば、特許文献１～３参照） As part of such an attempt, cell structures formed by using scaffolds and sheet-like cell cultures in which cells are formed in sheet form have been developed (see, for example, Patent Documents 1 to 3).
　再生医療に基づく新たな治療方法の確立とともに、自家細胞を凍結させて保存し、人工組織やシート状細胞培養物などの三次元構造体を形成したり、直接細胞を移植したりする際に、かかる凍結保存細胞を融解して自家細胞を回収し、それを用いて治療を行う機会が近年増加してきている。 With the establishment of new therapeutic methods based on regenerative medicine, when autologous cells are frozen and stored to form three-dimensional structures such as artificial tissues and sheet-like cell cultures, or when cells are directly transplanted. In recent years, there have been increasing opportunities to thaw such cryopreserved cells, recover autologous cells, and perform treatment using the cells.
　この方法でシート状細胞培養物を形成する場合、通常よりも多くの細胞を必要とするため、細胞を凍結および／または融解する際に、細胞に物理化学的ダメージを極力与えない工夫がされている。一般的に、凍結された細胞は凍結用培地と共に懸濁して凍結細胞保存用容器（クライオチューブなど）に収容して凍結させ、病院に移送して温浴に供することで、凍結細胞保存用容器内の凍結保存細胞を融解する作業が必要である。（特許文献４、５）。 Forming a sheet-like cell culture by this method requires more cells than usual, so when freezing and / or thawing the cells, a device was devised to minimize physicochemical damage to the cells. There is. Generally, frozen cells are suspended together with a freezing medium, stored in a frozen cell storage container (cryotube, etc.), frozen, transferred to a hospital, and subjected to a warm bath, so that the cells are contained in the frozen cell storage container. It is necessary to thaw the cryopreserved cells of. (Patent Documents 4 and 5).
　そして、凍結保存細胞を融解して生細胞を回収する際、融解した細胞懸濁液中にある細胞毒性成分の影響を低減させるためなどの目的で、融解した細胞懸濁液に希釈液などを加えて希釈する必要がある。この際に、希釈液注入速度を制御することで、急激な浸透圧変化によって死に至る細胞数を減少させ、それにより回収可能な生細胞数を向上させることができるシステムが開示されている（特許文献６）。 Then, when the cryopreserved cells are thawed and the living cells are recovered, a diluted solution or the like is added to the thawed cell suspension for the purpose of reducing the influence of cytotoxic components in the thawed cell suspension. In addition, it needs to be diluted. At this time, a system is disclosed that can reduce the number of cells dying due to a sudden change in osmotic pressure by controlling the dilution injection rate, thereby increasing the number of viable cells that can be recovered (patented). Document 6).
特表２００７－５２８７５５号公報Special Table 2007-528755 特開２０１０－８１８２９号公報Japanese Unexamined Patent Publication No. 2010-81829 特開２０１０－２２６９９１号公報Japanese Unexamined Patent Publication No. 2010-226991 特開２０１８－４２６９２号公報Japanese Unexamined Patent Publication No. 2018-42692 国際公開第２０１４／０７４８５９号International Publication No. 2014/074859 特開２０１５－１８１４３３号公報Japanese Unexamined Patent Publication No. 2015-181433
　上記のようなシステムは、動作部や制御部を実現するための構造が複雑で、製造コストが高く、操作が複雑であった。また、細胞懸濁液を調製して生細胞を確実に回収するためには、コンタミネーション発生のリスクを下げるために、クリーンルームでの作業が必要であった。 The above-mentioned system had a complicated structure for realizing an operation unit and a control unit, a high manufacturing cost, and a complicated operation. In addition, in order to prepare a cell suspension and reliably recover live cells, it was necessary to work in a clean room in order to reduce the risk of contamination.
　本発明はこのような点を考慮してなされたものであり、簡単な構造を有し、かつ簡単な操作で、コンタミネーション発生のリスクを下げながら、効率よく細胞懸濁液を調製することができる手段を提供することにある。 The present invention has been made in consideration of these points, and it is possible to efficiently prepare a cell suspension while reducing the risk of contamination by having a simple structure and a simple operation. It is to provide the means that can be done.
　すなわち本発明は、以下に関する。
［１］細胞懸濁液を収容するための収容容器を装着可能な第一装着部と、細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、第一装着部と第二装着部とが流体連結されている、デバイス。
［２］流体連結が、可撓性のチューブを介して行われる、［１］に記載のデバイス。
［３］第一装着部が、複数の収容容器を装着可能に構成されている、［１］または［２］に記載のデバイス。
［４］混合に使用する混合液のための注入口が設けられている、［１］～［３］のいずれかに記載のデバイス。
［５］混合液が希釈液である、［１］～［４］のいずれかに記載のデバイス。
［６］流量を制御可能な制御手段をさらに含む、［１］～［５］のいずれかに記載のデバイス。
［７］流路に外気を取り込むための滅菌フィルターが接続されている、［１］～［６］のいずれかに記載のデバイス。
［８］第一装着部および／または第二装着部が、開閉可能に構成されている、［１］～［７］のいずれかに記載のデバイス。
［９］細胞懸濁液中の細胞を混合するための容器に移送するためのデバイスで、前記細胞懸濁液を収容する複数の収容容器を装着可能な第一装着部と、前記細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、前記第一装着部と前記第二装着部とを流体連結する連結部を有する、細胞懸濁液の移送デバイス。 That is, the present invention relates to the following.
[1] Includes a first mounting portion to which a storage container for accommodating a cell suspension can be mounted, and a second mounting portion to which a mixing container for mixing the cell suspension and the mixed solution can be mounted. A device in which the first mounting part and the second mounting part are fluidly connected.
[2] The device according to [1], wherein the fluid connection is performed via a flexible tube.
[3] The device according to [1] or [2], wherein the first mounting portion is configured to mount a plurality of storage containers.
[4] The device according to any one of [1] to [3], which is provided with an injection port for a mixture used for mixing.
[5] The device according to any one of [1] to [4], wherein the mixed solution is a diluent.
[6] The device according to any one of [1] to [5], further comprising a control means capable of controlling the flow rate.
[7] The device according to any one of [1] to [6], to which a sterilization filter for taking in outside air is connected to the flow path.
[8] The device according to any one of [1] to [7], wherein the first mounting portion and / or the second mounting portion is configured to be openable and closable.
[9] A device for transferring cells in a cell suspension to a container for mixing, a first mounting portion on which a plurality of storage containers for accommodating the cell suspension can be mounted, and the cell suspension. A cell suspension comprising a second mounting portion to which a mixing container for mixing the liquid and the mixed liquid can be mounted, and having a connecting portion for fluidly connecting the first mounting portion and the second mounting portion. Transport device.
［１０］流体連結部が、可撓性のチューブにより構成され、該チューブを介して流体連結が行われる、［９］に記載のデバイス。
［１１］前記第一装着部に、混合に使用するための混合液を注入する注入口が設けられている、［９］または［１０］に記載のデバイス。
［１２］前記混合液が希釈液である、［９］～［１１］のいずれかに記載のデバイス。
［１３］前記連結部または前記第二装着部のいずれかに、前記混合液の流入量を制御可能な制御手段をさらに含む、［９］～［１２］のいずれかに記載のデバイス。
［１４］細胞懸濁液を調製するための方法であって、（ａ）細胞懸濁液を収容するための収容容器を装着可能な第一装着部と、細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、第一装着部と第二装着部とが流体連結可能である、デバイスを供するステップ、（ｂ）細胞懸濁液を収容した収容容器および混合容器をデバイスに装着し、収容容器から混合容器にデバイスを介して細胞懸濁液を移送するステップを含む、前記方法。
［１５］ステップ（ｂ）の後に、（ｃ）デバイスに設けられている細胞懸濁液との混合に使用する混合液のための注入口から、混合液を注入するステップを含む、［１４］に記載の方法。
［１６］ステップ（ｃ）の後に、（ｄ）混合液を収容容器または混合容器に移送するステップを含む、［１４］または［１５］に記載の方法。
［１７］混合液が希釈液である、［１４］～［１６］のいずれかに記載の方法。
［１８］シート状細胞培養物の適用により改善される疾患を処置する方法であって、［１］～［１３］のいずれかに記載のデバイスを使用して調製された細胞、または［１４］～［１７］のいずれかに記載の方法で調製された細胞を使用して製造されたシート状細胞培養物を、それを必要とする対象に適用することを含む、前記方法。 [10] The device according to [9], wherein the fluid connecting portion is composed of a flexible tube, and the fluid connecting is performed through the tube.
[11] The device according to [9] or [10], wherein the first mounting portion is provided with an injection port for injecting a mixed liquid for use in mixing.
[12] The device according to any one of [9] to [11], wherein the mixed solution is a diluent.
[13] The device according to any one of [9] to [12], wherein either the connecting portion or the second mounting portion further includes a control means capable of controlling the inflow amount of the mixed liquid.
[14] A method for preparing a cell suspension, wherein (a) a first mounting portion to which a storage container for storing the cell suspension can be mounted, and the cell suspension and a mixed solution. A step of providing a device, comprising a second mounting portion to which a mixing vessel for mixing can be mounted, the first mounting portion and the second mounting portion being fluid connectable, (b) containing a cell suspension. The method comprising attaching the containment vessel and the mixing vessel to the device and transferring the cell suspension from the containment vessel to the mixing vessel via the device.
[15] The step (b) includes the step of injecting the mixture from the inlet for the mixture used for mixing with the cell suspension provided in the device (c) [14]. The method described in.
[16] The method according to [14] or [15], which comprises, after step (c), (d) a step of transferring the mixed solution to a storage container or a mixing container.
[17] The method according to any one of [14] to [16], wherein the mixed solution is a diluent.
[18] A method for treating a disease that is ameliorated by application of a sheet cell culture, the cells prepared using the device according to any one of [1] to [13], or [14]. The method described above, comprising applying a sheet cell culture prepared using the cells prepared by the method according to any one of [17] to a subject in need thereof.
　本発明によれば、簡単な構造を有するため、製造コストを低く抑えることができ、かつ簡単な操作で、効率よく細胞懸濁液を調製することができるため、作業者の手技に依存することなく、作業レベルを均一化することができる。
　本発明によれば、細胞懸濁液の調製を細胞懸濁液を外気に曝すことなく無菌的に行うことができ、コンタミネーション発生のリスクを下げることができる。また、簡単な操作で有害物質を適性に除去でき、作業を均一化できるため、細胞の生存率が向上する。 According to the present invention, since it has a simple structure, the manufacturing cost can be kept low, and the cell suspension can be efficiently prepared by a simple operation, so that it depends on the procedure of the operator. The work level can be made uniform.
According to the present invention, the cell suspension can be prepared aseptically without exposing the cell suspension to the outside air, and the risk of contamination can be reduced. In addition, harmful substances can be appropriately removed by a simple operation, and the work can be made uniform, so that the cell viability is improved.
　本発明によれば、凍結保存細胞の融解を簡単な機構で効率良く迅速に行うことができ、また、細胞懸濁液の収集を簡単な機構で効率良く無菌的に行うことができ、さらに、収集された細胞懸濁液を周囲を汚染することなく迅速に無菌的に混合容器に移送することができる。したがって、細胞懸濁液の希釈などの次のステップに、素早く移行することができ、細胞毒性成分の細胞への影響を最小限に抑えることができるため、細胞の生存率が向上する。 According to the present invention, cryopreserved cells can be thawed efficiently and quickly by a simple mechanism, and cell suspensions can be collected efficiently and aseptically by a simple mechanism. The collected cell suspension can be rapidly and aseptically transferred to the mixing vessel without contaminating the surroundings. Therefore, the cell viability is improved because the next step, such as dilution of the cell suspension, can be made quickly and the effects of cytotoxic components on the cells can be minimized.
　本発明によれば、細胞懸濁液の希釈時の浸透圧負荷を簡単な機構で制御でき、急激な浸透圧変化によって死に至る細胞数を減少させ、それにより回収可能な生細胞数を向上させる。とくに融解後に増殖培養を経ずに使用する場合であっても、十分な生細胞数を確保することが可能となる。 According to the present invention, the osmotic load when diluting a cell suspension can be controlled by a simple mechanism, the number of dead cells due to a sudden change in osmotic pressure is reduced, and the number of recoverable living cells is increased. .. In particular, even when the cells are used after thawing without undergoing growth culture, it is possible to secure a sufficient number of viable cells.
図１は、本発明の第１実施形態に係るデバイスの概念図である。FIG. 1 is a conceptual diagram of a device according to the first embodiment of the present invention. 図２は、本発明の第１実施形態に係るデバイスの使用例を示す概念図である。FIG. 2 is a conceptual diagram showing a usage example of the device according to the first embodiment of the present invention.
　本発明は、１つの側面において、細胞懸濁液を収容するための収容容器を装着可能な第一装着部と、細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、第一装着部と第二装着部とが流体連結されている、デバイスに関する。
　本発明は、１つの側面において、細胞懸濁液を調製するための方法であって、（ａ）細胞懸濁液を収容するための収容容器を装着可能な第一装着部と、細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、第一装着部と第二装着部とが流体連結可能である、デバイスを供するステップ、（ｂ）細胞懸濁液を収容した収容容器および混合容器をデバイスに装着し、収容容器から混合容器にデバイスを介して細胞懸濁液を移送するステップを含む、前記方法に関する。本発明の一態様において、細胞懸濁液は、凍結保存細胞を融解して得られたものである。 In one aspect, the present invention has a first mounting portion to which a storage container for storing a cell suspension can be mounted and a first mounting portion to which a mixing container for mixing the cell suspension and a mixed solution can be mounted. (Ii) The present invention relates to a device in which a first mounting portion and a second mounting portion are fluidly connected, including a mounting portion.
The present invention is a method for preparing a cell suspension in one aspect, wherein (a) a first mounting portion to which a storage container for storing the cell suspension can be mounted, and a cell suspension. A step of providing a device, comprising a second mounting portion to which a mixing container for mixing the liquid and the mixed liquid can be mounted, the first mounting portion and the second mounting portion being fluid connectable, (b) cells. The method relates to the method comprising mounting a containment vessel and a mixing vessel containing the suspension on the device and transferring the cell suspension from the containing vessel to the mixing vessel via the device. In one aspect of the invention, the cell suspension is obtained by thawing cryopreserved cells.
　本発明において、「凍結保存細胞」とは、通常は凍結保存された細胞そのものを意味するが、凍結保存された細胞の１つの凍結保存単位を意味することもある。この場合、凍結保存単位とは、例えば１つのチューブなど、１群として一緒に凍結保存される細胞群を意味する。したがってこの場合、「凍結保存細胞」を融解した場合、凍結されていた「細胞懸濁液」が得られることとなる。 In the present invention, the "cryopreserved cell" usually means the cryopreserved cell itself, but may also mean one cryopreserved unit of the cryopreserved cell. In this case, the cryopreservation unit means a group of cells that are cryopreserved together as a group, for example, one tube. Therefore, in this case, when the "cryopreserved cells" are thawed, the frozen "cell suspension" is obtained.
　本発明において、「希釈時の浸透圧負荷」とは、希釈液を加えたことによって変化する浸透圧の単位時間当たりの変化率を意味する。浸透圧負荷は、希釈液の添加速度（単位時間当たりの添加量）や、希釈液と希釈対象の液（例えば融解した細胞懸濁液など）との浸透圧の差などの因子により変化する。「最大浸透圧負荷」とは、希釈開始から希釈終了までの間に、希釈液の添加により変化する浸透圧負荷のうち、最大の数値を意味する。 In the present invention, the "osmotic pressure load at the time of dilution" means the rate of change of the osmotic pressure per unit time that changes due to the addition of the diluent. The osmotic load varies depending on factors such as the addition rate of the diluent (addition amount per unit time) and the difference in osmotic pressure between the diluent and the solution to be diluted (for example, a thawed cell suspension). The “maximum osmotic pressure load” means the maximum value among the osmotic pressure loads that change due to the addition of the diluent from the start of dilution to the end of dilution.
　本発明は、凍結保存細胞を融解して得られた細胞懸濁液を希釈し、その希釈時の浸透圧変化を十分緩慢にすることを特徴とするものである。通常凍結保存細胞を融解して生細胞を回収する際、融解した細胞懸濁液中にある細胞毒性成分の影響を低減させるためなどの目的で、融解した細胞懸濁液に培養培地などを加えて希釈する。本発明者らは、かかる希釈の工程において急激に希釈すると、懸濁液の浸透圧の急激な変化により細胞に与えられるダメージにより、細胞の生存率が低下してしまうことを見出した。 The present invention is characterized in that a cell suspension obtained by thawing cryopreserved cells is diluted, and the change in osmotic pressure at the time of dilution is sufficiently slowed down. Normally, when cryopreserved cells are thawed and live cells are collected, a culture medium or the like is added to the thawed cell suspension for the purpose of reducing the influence of cytotoxic components in the thawed cell suspension. And dilute. The present inventors have found that when the cells are rapidly diluted in such a dilution step, the viability of the cells is reduced due to the damage caused to the cells by the sudden change in the osmotic pressure of the suspension.
　希釈時の浸透圧変化が十分緩慢であれば、細胞に与えられるダメージが低減され、生細胞の回収量が増大する。浸透圧変化を緩慢にする方法は、例えば希釈液を添加する速度を遅くする、細胞懸濁液と浸透圧差の少ない希釈液を用いるなど、当該技術分野において知られたあらゆる方法を用いてよいが、例えば細胞毒性を有する希釈液を用いないなど、浸透圧負荷以外のダメージを与えないことが好ましい。 If the change in osmotic pressure during dilution is slow enough, the damage given to the cells will be reduced and the amount of live cells recovered will increase. As a method for slowing down the change in osmotic pressure, any method known in the art may be used, for example, slowing down the rate of adding the diluent or using a diluent having a small difference in osmotic pressure from the cell suspension. For example, it is preferable not to cause damage other than osmotic load, such as not using a diluent having cytotoxicity.
　本発明において用い得る希釈液は特に限定されないが、細胞に物理化学的ダメージを与えないものが好ましい。希釈液の例としては、これに限定するものではないが、例えばＤＭＥＭ培地などの液体培地、ハンクス平衡塩溶液、ＰＢＳなどの緩衝液、生理食塩水などの等張液、蒸留水などが挙げられる。また、これらにさらにアルブミンなど他の成分が添加されていてもよい。 The diluent that can be used in the present invention is not particularly limited, but one that does not cause physicochemical damage to cells is preferable. Examples of the diluent include, but are not limited to, liquid media such as DMEM medium, Hanks balanced salt solution, buffer solution such as PBS, isotonic solution such as physiological saline, and distilled water. .. Further, other components such as albumin may be further added to these.
　本明細書においては、単位Ｏｓｍは浸透圧の単位として用いており、１Ｏｓｍは１ｍｏｌ／Ｌの理想溶液の有する浸透圧と等価な浸透圧を意味するものである。また、希釈時の浸透圧負荷は、室温条件下における１秒あたりの浸透圧の変化（単位：Ｏｓｍ／秒）として表しているが、浸透圧変化の大きさを表現できる単位であればどんな単位を用いてもよく、これに限定するものではないが、例えば希釈液の添加速度、体積または重量の増加速度などが挙げられる。 In this specification, the unit Osm is used as a unit of osmotic pressure, and 1 Osm means an osmotic pressure equivalent to the osmotic pressure of an ideal solution of 1 mol / L. The osmotic pressure load at the time of dilution is expressed as a change in osmotic pressure per second (unit: Osm / sec) under normal temperature conditions, but any unit can express the magnitude of the change in osmotic pressure. May be used, and examples thereof include, but are not limited to, the rate of addition of the diluent, the rate of increase in volume or weight, and the like.
　浸透圧負荷は、浸透圧変化をリアルタイムで計測してもよいし、ある２つの時点での浸透圧を求め、その間の単位時間当たりの平均変化として求めてもよい。ある時間幅において一定の速度で希釈液を添加している場合、同一の希釈液を添加している限りにおいては、一般的に、希釈液添加開始時点での浸透圧負荷がその時間幅における浸透圧負荷の最大値となり、希釈液の添加量が増大するにつれて浸透圧負荷は減少していくと考えられる。したがって、ある態様において、一定の速度で同一の希釈液を添加している場合、希釈液添加開始時の浸透圧およびその単位時間後（例えば本発明の上記例においては１秒後）の浸透圧を求め、両時点での浸透圧の差分をその希釈における最大浸透圧負荷とみなす。 The osmotic pressure load may be obtained by measuring the osmotic pressure change in real time, or by obtaining the osmotic pressure at a certain two time points and obtaining the average change per unit time between them. When the diluent is added at a constant rate in a certain time width, as long as the same diluent is added, the osmotic load at the start of addition of the diluent generally permeates in that time width. It is considered that the maximum value of the pressure load is reached and the osmotic pressure load decreases as the amount of the diluent added increases. Therefore, in a certain embodiment, when the same diluent is added at a constant rate, the osmotic pressure at the start of addition of the diluent and the osmotic pressure after a unit time thereof (for example, 1 second in the above example of the present invention). Is obtained, and the difference in osmotic pressure at both time points is regarded as the maximum osmotic pressure load at the dilution.
　本発明においては、希釈は、融解した細胞懸濁液を凍結保存容器に入れたまま行ってもよいし、混合容器（遠沈管など）に移して行ってもよい。混合容器に移して行う場合、融解細胞の生存率を上げるため、細胞懸濁液を移した後の凍結保存容器を希釈液でリンスし、このリンス液を細胞懸濁液に添加する。かかるリンス液の添加もまた本発明の希釈に該当する。したがって本発明の一態様において、希釈液は融解した細胞懸濁液を調製容器に移した後の凍結保存容器をリンスしたリンス液を含む。 In the present invention, the dilution may be carried out while the thawed cell suspension is placed in a cryopreservation container or transferred to a mixing container (centrifuge tube or the like). When transferring to a mixing container, in order to increase the viability of thawed cells, the cryopreservation container after transferring the cell suspension is rinsed with a diluted solution, and this rinse solution is added to the cell suspension. The addition of such a rinse solution also corresponds to the dilution of the present invention. Therefore, in one embodiment of the present invention, the diluent comprises a rinse solution obtained by transferring the thawed cell suspension to a preparation container and then rinsing the cryopreservation container.
　本方法に用いることができる細胞の例としては、限定されずに、接着細胞（付着性細胞）を含む。接着細胞は、例えば、接着性の体細胞（例えば、心筋細胞、線維芽細胞、上皮細胞、内皮細胞、肝細胞、膵細胞、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞、滑膜細胞、軟骨細胞など）および幹細胞（例えば、筋芽細胞、心臓幹細胞などの組織幹細胞、胚性幹細胞、ｉＰＳ（induced pluripotent stem）細胞などの多能性幹細胞、間葉系幹細胞等）などを含む。体細胞は、幹細胞、特にｉＰＳ細胞から分化させたものであってもよい。シート状細胞培養物を形成し得る細胞の非限定例としては、例えば、筋芽細胞（例えば、骨格筋芽細胞など）、間葉系幹細胞（例えば、骨髄、脂肪組織、末梢血、皮膚、毛根、筋組織、子宮内膜、胎盤、臍帯血由来のものなど）、心筋細胞、線維芽細胞、心臓幹細胞、胚性幹細胞、ｉＰＳ細胞、滑膜細胞、軟骨細胞、上皮細胞（例えば、口腔粘膜上皮細胞、網膜色素上皮細胞、鼻粘膜上皮細胞など）、内皮細胞（例えば、血管内皮細胞など）、肝細胞（例えば、肝実質細胞など）、膵細胞（例えば、膵島細胞など）、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞等が挙げられる。本明細書においては、単層の細胞培養物を形成するもの、例えば、筋芽細胞または心筋細胞などが好ましく、とくに好ましくは骨格筋芽細胞またはｉＰＳ細胞由来の心筋細胞である。 Examples of cells that can be used in this method include, but are not limited to, adherent cells (adhesive cells). Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, heart stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) And so on. Somatic cells may be differentiated from stem cells, especially iPS cells. Non-limiting examples of cells capable of forming a sheet-like cell culture include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots). , Muscle tissue, endometrial membrane, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, heart stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelium) Cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), renal cells, adrenal Examples thereof include cells, root membrane cells, gingival cells, bone membrane cells, skin cells and the like. In the present specification, those forming a monolayer cell culture, for example, myoblasts or cardiomyocytes are preferable, and skeletal myoblasts or cardiomyocytes derived from iPS cells are particularly preferable.
　上述のとおり、融解により得られた細胞懸濁液は細胞毒性成分（例えばＤＭＳＯなど）を含み得るため、希釈することで該細胞毒性成分の影響を低減することができる。本発明は、この希釈の際に浸透圧変化を十分緩慢にする、すなわち浸透圧負荷を十分小さくすることにより、融解細胞の生存率を増大させることに特徴を有するものである。 As described above, since the cell suspension obtained by thawing may contain a cytotoxic component (for example, DMSO), the influence of the cytotoxic component can be reduced by diluting it. The present invention is characterized in that the osmotic pressure change is sufficiently slowed down at the time of this dilution, that is, the osmotic pressure load is sufficiently reduced, thereby increasing the viability of the thawed cells.
　本発明において、「十分緩慢な浸透圧変化」の閾値は、用いる細胞、融解の条件、温度などにより変化し得る。例えば通常の凍結保存液（例えば１０％程度のＤＭＳＯを含有するＤＭＥＭ培地など）および通常の希釈液（例えば市販のＤＭＥＭ培地など）を用いる場合、ある態様において最大浸透圧負荷は約２５０ｍＯｓｍ／秒以下であり、約２２０ｍＯｓｍ／秒であることが好ましい。より好ましくは約１００ｍＯｓｍ／秒以下であり、さらに好ましくは約５０ｍＯｓｍ／秒以下である。 In the present invention, the threshold value of "sufficiently slow osmotic pressure change" can change depending on the cells used, melting conditions, temperature, and the like. For example, when a normal cryopreservation solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example, commercially available DMEM medium) are used, the maximum osmotic load is about 250 mOsm / sec or less in some embodiments. It is preferably about 220 mOsm / sec. It is more preferably about 100 mOsm / sec or less, and even more preferably about 50 mOsm / sec or less.
　また、希釈に時間をかけすぎると、例えば細胞懸濁液中の細胞毒性成分の影響で、浸透圧負荷以外の理由によるダメージが細胞に与えられてしまう場合があるため、この観点からは速やかに希釈されることが好ましい。本発明の方法においては、最大浸透圧負荷の下限値は特に設定されなくてもよいが、通常の凍結保存液（例えば１０％程度のＤＭＳＯを含有するＤＭＥＭ培地など）および通常の希釈液（例えば市販のＤＭＥＭ培地など）を用いる場合であれば、約２ｍＯｓｍ／秒以上が好ましく、約２０ｍＯｓｍ／秒以上がより好ましく、約４０ｍＯｓｍ／秒以上がさらに好ましい。 In addition, if it takes too much time to dilute, for example, due to the influence of cytotoxic components in the cell suspension, damage may be given to the cells for reasons other than osmotic load, so from this point of view, promptly. It is preferably diluted. In the method of the present invention, the lower limit of the maximum osmotic load may not be set in particular, but a normal cryopreservation solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example) When using (commercially available DMEM medium or the like), about 2 mOsm / sec or more is preferable, about 20 mOsm / sec or more is more preferable, and about 40 mOsm / sec or more is further preferable.
　したがって本発明の方法の好ましい一態様において、最大浸透圧負荷は、２ｍＯｓｍ／秒～２５０ｍＯｓｍ／秒であり、より好ましくは２ｍＯｓｍ／秒～２２０ｍＯｓｍ／秒であり、さらに好ましくは２０ｍＯｓｍ／秒～１００ｍＯｓｍ／秒であり、回収可能な生細胞量の観点から、最も好ましくは４０ｍＯｓｍ／秒～５０ｍＯｓｍ／秒である。 Therefore, in a preferred embodiment of the method of the present invention, the maximum osmotic load is 2 mOsm / sec to 250 mOsm / sec, more preferably 2 mOsm / sec to 220 mOsm / sec, and even more preferably 20 mOsm / sec to 100 mOsm / sec. From the viewpoint of the amount of viable cells that can be recovered, it is most preferably 40 mOsm / sec to 50 mOsm / sec.
　上述のとおり、本発明の方法において、融解した細胞懸濁液に最初に添加される希釈液が最大浸透圧負荷に最も影響しやすい。したがって、本発明の一態様において、最大浸透圧負荷は、希釈開始から３倍に希釈されるまでの間の最大浸透圧負荷であり、別の一態様においては、２倍に希釈されるまでの間の最大浸透圧負荷である。 As described above, in the method of the present invention, the diluent first added to the thawed cell suspension is most likely to affect the maximum osmotic load. Therefore, in one embodiment of the present invention, the maximum osmotic load is the maximum osmotic load from the start of dilution to 3-fold dilution, and in another embodiment, until 2-fold dilution. Maximum osmotic load between.
　本発明の好ましい一態様において、希釈は浸透圧を計測して最大浸透圧負荷を調節しながら行われる。浸透圧の計測は常時継続的に行われてもよいし、例えば１秒ごと、１０秒ごと、３０秒ごと、１分ごとなど、特定の時間間隔で行われてもよい。浸透圧の計測方法は、当該技術分野において知られたあらゆる方法を用いることができ、これに限定するものではないが、例えばオスモメーターなどを用いて計測することができる。 In a preferred embodiment of the present invention, dilution is performed while measuring the osmotic pressure and adjusting the maximum osmotic load. The osmotic pressure may be measured continuously at all times, or may be performed at specific time intervals such as every 1 second, every 10 seconds, every 30 seconds, every 1 minute. As the method for measuring the osmotic pressure, any method known in the art can be used, and the osmotic pressure can be measured by using, for example, an osmotic meter or the like, without limitation.
　本発明において、「収容容器」とは、細胞懸濁液を収容して密閉し、無菌的に保存できる容器をいう。収容容器としては、特に限定されないが、クライオチューブ、クライオバイアルなどが挙げられる。収容容器は、好ましくは、凍結可能な材料で構成される。
　本発明において、１つの構成と別の構成とが「流体連結される」とは、これら２つの構成がチューブのような管で接続され、これを介して流体が外気に触れることなく流れるように構成されることをいう。 In the present invention, the "containment container" refers to a container that can contain a cell suspension, seal it, and store it aseptically. The storage container is not particularly limited, and examples thereof include a cryotube and a cryovial. The containment vessel is preferably constructed of a freezing material.
In the present invention, one configuration and another configuration are "fluid-connected" so that these two configurations are connected by a tube-like tube through which the fluid flows without being exposed to the outside air. It means to be composed.
　本発明において、細胞懸濁液の「調製」は、細胞懸濁液の融解、収集、移送、希釈、リンスなどを含む。細胞懸濁液の融解とは、凍結保存細胞を融解して、細胞懸濁液を得ることをいい、細胞懸濁液の融解は、収容容器内に収容された凍結保存細胞を融解してもよいし、別の容器で凍結保存細胞を融解してもよい。細胞懸濁液の収集とは、収容容器内に収容された細胞懸濁液を収集することをいい、複数の収容容器内に収容された細胞懸濁液を収集することを含む。 In the present invention, "preparation" of a cell suspension includes thawing, collecting, transferring, diluting, rinsing, etc. of the cell suspension. Thawing a cell suspension means thawing cryopreserved cells to obtain a cell suspension, and thawing a cell suspension means thawing cryopreserved cells contained in a storage container. Alternatively, the cryopreserved cells may be thawed in a separate container. Collection of cell suspension means collecting cell suspension contained in a storage container, and includes collecting cell suspension contained in a plurality of storage containers.
　細胞懸濁液の移送とは、収容容器内に収容された細胞懸濁液を流路を介して混合容器に移送することをいい、一態様において、移送は収容容器を混合容器より高い位置に位置付けて、位置エネルギーを利用して細胞懸濁液を移動させることにより行われる。細胞懸濁液の希釈とは、細胞懸濁液に希釈液を添加（混合）することをいい、一態様において、希釈液は、混合容器に移送された細胞懸濁液に添加してもよい。希釈液は、融解した細胞懸濁液を別の容器に移した後の凍結保存容器をリンスしたリンス液を含んでもよい。 The transfer of the cell suspension means the transfer of the cell suspension contained in the storage container to the mixing container via the flow path, and in one embodiment, the transfer moves the storage container to a position higher than the mixing container. This is done by positioning and using potential energy to move the cell suspension. Diluting the cell suspension means adding (mixing) the diluent to the cell suspension, and in one embodiment, the diluent may be added to the cell suspension transferred to the mixing vessel. .. The diluent may include a rinse solution obtained by rinsing the cryopreservation container after transferring the thawed cell suspension to another container.
　本発明の別の側面は、シート状細胞培養物の適用により改善される疾患を処置する方法であって、本発明のデバイスを使用して、または本発明の方法で調製された細胞を使用して製造されたシート状細胞培養物を、それを必要とする対象に適用することを含む方法に関する。 Another aspect of the invention is a method of treating a disease that is ameliorated by the application of a sheet cell culture, using the devices of the invention or using cells prepared by the methods of the invention. The present invention relates to a method comprising applying the sheet-like cell culture produced in the above-mentioned to a subject in need thereof.
　本発明の処置方法は、本発明の方法で調製された細胞を使用して製造されたシート状細胞培養物をそれを必要とする対象に適用するステップを含んでもよい。本発明の処置方法は、シート状細胞培養物を製造するステップの前に、対象からシート状細胞培養物を製造するための細胞または細胞の供給源となる組織を採取するステップをさらに含んでもよい。一態様において、細胞または細胞の給源となる組織を採取する対象は、シート状細胞培養物または組成物等の投与を受ける対象と同一の個体である。別の態様において、細胞または細胞の供給源となる組織を採取する対象は、シート状細胞培養物または組成物等の投与を受ける対象とは同種の別個体である。別の態様において、細胞または細胞の給源となる組織を採取する対象は、シート状細胞培養物または組成物等の投与を受ける対象とは異種の個体である。 The treatment method of the present invention may include applying a sheet cell culture prepared using the cells prepared by the method of the present invention to a subject in need thereof. The treatment method of the present invention may further include collecting cells from a subject or a tissue from which the cells are supplied, prior to the step of producing the sheet-shaped cell culture. .. In one embodiment, the subject from which the cell or tissue to be the source of the cell is collected is the same individual as the subject to whom the sheet-like cell culture or composition is administered. In another embodiment, the subject from which the cell or tissue from which the cell is source is collected is a separate entity of the same species as the subject receiving the administration, such as a sheet cell culture or composition. In another embodiment, the subject from which the cell or the tissue that is the source of the cell is collected is an individual that is different from the subject to which the sheet-like cell culture or composition is administered.
　本発明において、用語「対象」は、任意の生物個体、好ましくは動物、さらに好ましくは哺乳動物、さらに好ましくはヒトの個体を意味する。本発明において、対象は健常であっても、何らかの疾患に罹患していてもよいものとするが、シート状細胞培養物の適用により改善される疾患（例えば、組織の異常に関連する疾患など）の処置が企図される場合には、典型的には当該疾患に罹患しているか、罹患するリスクを有する対象を意味する。 In the present invention, the term "subject" means any individual organism, preferably an animal, more preferably a mammal, even more preferably a human. In the present invention, the subject may be healthy or suffer from some disease, but the disease improved by the application of the sheet-like cell culture (for example, a disease related to tissue abnormality). When the treatment is intended, it typically means a subject who has or is at risk of developing the disease.
　また、用語「処置」は、疾患の治癒、一時的寛解または予防などを目的とする医学的に許容される全ての種類の予防的および／または治療的介入を包含するものとする。例えば、「処置」の用語は、シート状細胞培養物の適用により改善される疾患（例えば、組織の異常に関連する疾患など）の進行の遅延または停止、病変の退縮または消失、当該疾患発症の予防または再発の防止などを含む、種々の目的の医学的に許容される介入を包含する。 Also, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at the cure, temporary remission or prevention of the disease. For example, the term "treatment" refers to delaying or stopping the progression of a disease that is ameliorated by the application of sheet cell culture (eg, a disease associated with tissue abnormalities), regression or disappearance of a lesion, or the onset of the disease. Includes medically acceptable interventions for a variety of purposes, including prevention or prevention of recurrence.
　本発明において、有効量とは、例えば、疾患の発症や再発を抑制し、症状を軽減し、または進行を遅延もしくは停止し得る量（例えば、シート状細胞培養物のサイズや重量、枚数など）であり、好ましくは、当該疾患の発症および再発を予防し、または当該疾患を治癒する量である。また、投与による利益を超える悪影響が生じない量が好ましい。かかる量は、例えば、マウス、ラット、イヌまたはブタなどの実験動物や疾患モデル動物における試験などにより適宜決定することができ、このような試験法は当業者によく知られている。また、処置の対象となる組織病変の大きさは、有効量決定のための重要な指標となり得る。 In the present invention, the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-shaped cell cultures, etc.). The amount is preferably an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause an adverse effect exceeding the benefit of administration is preferable. Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.
　適用方法としては、典型的には組織への直接的な適用が挙げられる。適用頻度は、典型的には１回の処置につき１回であるが、所望の効果が得られない場合などには、複数のシートを適用することも可能である。組織への直接的な適用は、例えば本発明のシート状細胞培養物等を組織の疾患部位に貼付するように適用する方法などが用いられる。 The application method typically includes direct application to an organization. The application frequency is typically once per treatment, but it is possible to apply a plurality of sheets when the desired effect is not obtained. For direct application to tissues, for example, a method of applying the sheet-shaped cell culture of the present invention or the like so as to be attached to a diseased part of tissues is used.
　処置の対象となる組織（適用組織）としては、限定されずに、例えば、心臓（心筋）、角膜、網膜、食道、皮膚、関節、軟骨、肝臓、膵臓、歯肉、腎臓、甲状腺、骨格筋、中耳などが挙げられる。また、処置の対象となる疾患としては、限定されずに、例えば、心疾患（例えば、心筋傷害（心筋梗塞、心外傷等）、心筋症（虚血性心筋症、拡張型心筋症、拡張相肥大型心筋症等）など）、角膜疾患（例えば、角膜上皮幹細胞疲弊症、角膜損傷（熱・化学腐食）、角膜潰瘍、角膜混濁、角膜穿孔、角膜瘢痕、スティーブンス・ジョンソン症候群、眼類天疱瘡など）、網膜疾患（例えば、網膜色素変性症、加齢黄斑変性症など）、食道疾患（例えば、食道手術（食道ガン除去）後の食道の炎症・狭窄の予防など）、皮膚疾患（例えば、皮膚損傷（外傷、熱傷）など）、関節疾患（例えば、変形性関節炎など）、軟骨疾患（例えば、軟骨の損傷など）、肝疾患（例えば、慢性肝疾患など）、膵臓疾患（例えば、糖尿病など）、歯科疾患（例えば、歯周病など）、腎臓疾患（例えば、腎不全、腎性貧血、腎性骨異栄養症など）、甲状腺疾患（例えば、甲状腺機能低下症など）、筋疾患（例えば、筋損傷、筋炎など）、中耳疾患（例えば、中耳炎など）が挙げられる。 The tissue to be treated (applicable tissue) is not limited, for example, heart (myocardium), cornea, retina, esophagus, skin, articular cartilage, liver, pancreas, gingiva, kidney, thyroid gland, skeletal muscle, etc. The middle ear and the like can be mentioned. The diseases to be treated include, for example, heart diseases (for example, myocardial injuries (myocardial infarction, cardiac trauma, etc.)) and myocardial diseases (ischemic myocardial disease, dilated myocardial disease, dilated phase fertilizer). (Major myocardial disease, etc.), Corneal disease (eg, corneal epithelial stem cell exhaustion, corneal injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens Johnson syndrome, ocular herbitis ), Retinal diseases (eg, retinal pigment degeneration, age-related yellow spot degeneration, etc.), esophageal diseases (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)), skin diseases (eg, prevention Skin damage (trauma, burns, etc.), joint disease (eg, osteoarthritis), cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes, etc.) ), Dental disease (eg periodontal disease), kidney disease (eg renal failure, renal anemia, renal osteodystrophy, etc.), thyroid disease (eg, hypothyroidism), muscle disease (eg, hypothyroidism) , Muscle injury, myitis, etc.), middle ear diseases (eg, middle ear inflammation, etc.).
　適用組織が心臓などの臓器である場合など、適用箇所が拍動や蠕動運動などにより頻繁に動く場合、本発明のシート状細胞培養物等を貼付するだけでは適用箇所から脱落してしまう場合がある。したがって本発明の処置方法の好ましい一態様において、本発明のシート状細胞培養物等を疾患部位に適用した後、貫通部位に縫合針などを通して本発明のシート状細胞培養物等を縫合固定することが含まれる。これにより疾患部位に適用された本発明のシート状細胞培養物等が、適用箇所から脱落することを防止できる。 When the application site moves frequently due to beating or peristaltic movement, such as when the application tissue is an organ such as the heart, it may fall off from the application site simply by attaching the sheet-shaped cell culture of the present invention. is there. Therefore, in a preferred embodiment of the treatment method of the present invention, after applying the sheet-shaped cell culture or the like of the present invention to the diseased site, the sheet-shaped cell culture or the like of the present invention is sutured and fixed through a suture needle or the like through the penetrating site. Is included. As a result, it is possible to prevent the sheet-shaped cell culture or the like of the present invention applied to the diseased part from falling off from the applied part.
　以下、本発明を、図面を参照しつつ、好適な実施形態に基づき、詳細に説明する。なお、図中の各部材の大きさは説明のために適宜強調されており、実際の比率、大きさを示すものではない。 Hereinafter, the present invention will be described in detail based on a preferred embodiment with reference to the drawings. The size of each member in the figure is emphasized as appropriate for the sake of explanation, and does not indicate the actual ratio or size.
＜第１実施形態＞
　まず、本発明の第１実施形態に係るデバイスについて説明する。図１は、本発明の第１実施形態に係るデバイスの概念図である。図２は、本発明の第１実施形態に係るデバイスの使用例を示す概念図である。 <First Embodiment>
First, the device according to the first embodiment of the present invention will be described. FIG. 1 is a conceptual diagram of a device according to the first embodiment of the present invention. FIG. 2 is a conceptual diagram showing a usage example of the device according to the first embodiment of the present invention.
　図１に示されるように、デバイスは、第一装着部１および第二装着部２を含む。第一装着部１は、収容容器Ｃを装着可能に構成されている。第二装着部２は、混合容器Ｍを装着可能に構成されている。第一装着部１は、複数の収容容器Ｃを装着可能に構成されていてもよい。第一装着部１と第二装着部２とは、直接的に流体連結してもよいし、第一装着部１と第二装着部２とを可撓性のチューブ４を介して流体連通してもよい。本発明において、第一装着部１と第二装着部２とが流体連結される部分を連結部という。第一装着部１および第二装着部２はそれぞれ、収容容器Ｃおよび混合容器Ｍの開口部を液密に装着できるように構成されている。 As shown in FIG. 1, the device includes a first mounting portion 1 and a second mounting portion 2. The first mounting portion 1 is configured so that the storage container C can be mounted. The second mounting portion 2 is configured so that the mixing container M can be mounted. The first mounting portion 1 may be configured so that a plurality of storage containers C can be mounted. The first mounting portion 1 and the second mounting portion 2 may be directly fluid-connected, or the first mounting portion 1 and the second mounting portion 2 are fluid-connected via a flexible tube 4. You may. In the present invention, the portion where the first mounting portion 1 and the second mounting portion 2 are fluidly connected is referred to as a connecting portion. The first mounting portion 1 and the second mounting portion 2 are configured so that the openings of the storage container C and the mixing container M can be liquid-tightly mounted, respectively.
　第一装着部１には、収容容器Ｃに収容される細胞懸濁液との混合に使用する混合液のための注入口３を流路に設けてもよい。流路を流れる液体（例えば、混合液）の流量を制御可能な制御手段５を流路（例えば、連結部または第二装着部２）に接続してもよい。流路に外気を取り込むための滅菌フィルター６を流路に接続してもよい。これら注入口３、制御手段５および滅菌フィルター６は、デバイスの使用に合わせて、デバイスに含むことができる任意の構成である。 The first mounting portion 1 may be provided with an injection port 3 for a mixed solution used for mixing with the cell suspension contained in the storage container C in the flow path. A control means 5 capable of controlling the flow rate of a liquid (for example, a mixed liquid) flowing through the flow path may be connected to the flow path (for example, a connecting portion or a second mounting portion 2). A sterilization filter 6 for taking in outside air into the flow path may be connected to the flow path. The inlet 3, the control means 5, and the sterilization filter 6 are arbitrary configurations that can be included in the device according to the use of the device.
　図２に示されるように、デバイスを使用する際は、細胞懸濁液が収容された収容容器Ｃを第一装着部１に装着し、細胞懸濁液と混合液との混合が十分可能な容積を有する混合容器Ｍ（例えば、遠沈管）を第二装着部２に装着する。このように収容容器Ｃおよび混合容器Ｍをデバイスに装着すると、収容容器Ｃ、デバイス、混合容器Ｍの順に流体連結された流路（閉回路）が形成される。したがって、収容容器Ｃに収容された細胞懸濁液を、デバイスを介して外気に触れさせることなく、かつ周囲を汚染することなく混合容器Ｍに無菌的に移送することができる。この際に、滅菌フィルター６を介して外気を閉回路に取り込むことで、移送を効率化することもできる。 As shown in FIG. 2, when using the device, the storage container C containing the cell suspension is mounted on the first mounting portion 1, and the cell suspension and the mixed solution can be sufficiently mixed. A mixing container M having a volume (for example, a centrifuge tube) is mounted on the second mounting portion 2. When the storage container C and the mixing container M are attached to the device in this way, a flow path (closed circuit) in which the storage container C, the device, and the mixing container M are fluidly connected in this order is formed. Therefore, the cell suspension contained in the storage container C can be aseptically transferred to the mixing container M without being exposed to the outside air through the device and without contaminating the surroundings. At this time, the transfer can be made more efficient by taking in the outside air into the closed circuit through the sterilization filter 6.
　図２Ａに示されるように、収容容器Ｃを装着する際は、チューブ４を屈曲させて第一装着部１を反転させて、収容容器Ｃを装着する部分が収容容器Ｃの開口と対向するようにする。これにより、収容容器Ｃの開口を上向きにした状態で簡単に第一装着部１に装着することができるため、収容容器Ｃ内の細胞懸濁液の漏出を防ぐことができる。収容容器Ｃの第一装着部１への装着は、クリーンルームなどで行ってもよいし、反転は、チューブ４を屈曲させずに、デバイス自体の上下を反転させてもよい。 As shown in FIG. 2A, when mounting the storage container C, the tube 4 is bent and the first mounting portion 1 is inverted so that the portion to which the storage container C is mounted faces the opening of the storage container C. To. As a result, the cells can be easily mounted on the first mounting portion 1 with the opening of the storage container C facing upward, so that leakage of the cell suspension in the storage container C can be prevented. The storage container C may be mounted on the first mounting portion 1 in a clean room or the like, or the device itself may be flipped upside down without bending the tube 4.
　収容容器Ｃに凍結保存細胞が収容されている場合は、収容容器Ｃの開口部を上向きに装着した状態で、収容容器Ｃを融解（例えば、温浴）に供することができる。第一装着部１に複数の収容容器Ｃを装着できる場合は、これらを同時に温浴に供することができる。通常、複数の凍結細胞保存用容器を温浴に供する場合、その浮力で水面に浮遊する事があるため、作業者はこれらを手で把持して水中に沈める必要があったが、本デバイスを使用することで、かかる作業を効率化することができる。 When the cryopreserved cells are contained in the storage container C, the storage container C can be subjected to thawing (for example, a hot bath) with the opening of the storage container C mounted upward. When a plurality of storage containers C can be mounted on the first mounting portion 1, these can be subjected to a hot bath at the same time. Normally, when multiple containers for storing frozen cells are subjected to a hot bath, they may float on the water surface due to their buoyancy, so the operator had to grasp them by hand and submerge them in water. By doing so, such work can be made more efficient.
　凍結保存細胞を融解して細胞懸濁液が得られると、図２Ｂに示されるように、チューブ４を屈曲させて第一装着部１を反転し、第二装着部２より高い位置に位置付ける。そして、位置エネルギーを利用して、細胞懸濁液をデバイスを介して収容容器Ｃから混合容器Ｍに移送する。反転は、デバイス自体の上下を反転させてもよい。このようにデバイスを使用することで、細胞懸濁液の融解から移送まで、クリーンルームの外であっても無菌的に行うことができる。 When the cryopreserved cells are thawed to obtain a cell suspension, as shown in FIG. 2B, the tube 4 is bent to invert the first mounting portion 1 and position it at a position higher than the second mounting portion 2. Then, using potential energy, the cell suspension is transferred from the storage container C to the mixing container M via the device. Inversion may invert the device itself upside down. By using the device in this way, the process from thawing of the cell suspension to transfer can be performed aseptically even outside the clean room.
　また、融解細胞の生存率を上げるため、図２Ａに示されるように、再度チューブ４を屈曲させて第一装着部１を反転させ、混合液（この場合、希釈液）を注入口３から注入し、収容容器Ｃ内をリンスすることができる。このように、混合容器Ｍを反転することなく、第一装着部１を反転させることで、混合容器Ｍに移送された細胞懸濁液を収容容器Ｃ逆流させることなく、収容容器Ｃ内をリンスすることもできる。流路の内表面に、細胞非接着処理を施すことで、細胞の収集率を向上させることもできる。 Further, in order to increase the survival rate of the thawed cells, as shown in FIG. 2A, the tube 4 is bent again to invert the first mounting portion 1, and the mixed solution (in this case, the diluted solution) is injected from the injection port 3. Then, the inside of the storage container C can be rinsed. In this way, by reversing the first mounting portion 1 without reversing the mixing container M, the inside of the storage container C is rinsed without causing the cell suspension transferred to the mixing container M to flow back to the storage container C. You can also do it. It is also possible to improve the cell collection rate by applying a cell non-adhesive treatment to the inner surface of the flow path.
　第一装着部１を複数の収容容器Ｃを装着できるように構成する場合は、第一装着部１の装着部とは反対側、すなわち第二装着部２側を漏斗状に形成することで、複数の収容容器Ｃに収容されている細胞懸濁液を効率良く収集することができる。このようにデバイスを使用することで、細胞懸濁液の融解から収集まで、クリーンルームの外であっても無菌的に効率よく行うことができる。細胞は、対数増殖期の細胞を用いるのが、生細胞回収量の観点から好ましい。 When the first mounting portion 1 is configured so that a plurality of storage containers C can be mounted, the side opposite to the mounting portion of the first mounting portion 1, that is, the second mounting portion 2 side is formed in a funnel shape. Cell suspensions contained in a plurality of storage containers C can be efficiently collected. By using the device in this way, the process from thawing of the cell suspension to collection can be performed aseptically and efficiently even outside the clean room. As the cells, it is preferable to use cells in the logarithmic growth phase from the viewpoint of the amount of live cells recovered.
　次に、図２Ｂに示されるように、第一装着部１を第二装着部２より高い位置に位置付ける。この際に、収容容器Ｃ内をリンスした場合は、このリンス液を細胞懸濁液に添加することができる。そして、混合液としてさらに希釈液を注入口３から注入した場合、混合容器Ｍに移送されている細胞懸濁液に希釈液を添加することができる。デバイスに制御手段５が接続されている場合は、流体の流量、すなわち希釈液の添加速度（単位時間当たりの添加量）を制御手段５で制御することができる。 Next, as shown in FIG. 2B, the first mounting portion 1 is positioned higher than the second mounting portion 2. At this time, when the inside of the storage container C is rinsed, this rinse solution can be added to the cell suspension. Then, when the diluent is further injected from the injection port 3 as the mixture, the diluent can be added to the cell suspension transferred to the mixing vessel M. When the control means 5 is connected to the device, the flow rate of the fluid, that is, the addition rate of the diluent (addition amount per unit time) can be controlled by the control means 5.
　また、希釈液の添加速度は、第一装着部１と第二装着部２との位置関係を調節することで、制御することもできる。すなわち、第一装着部１の位置が第二装着部２の位置より低いか同じである場合は、注入口３から注入した混合液は、第二装着部２へ流れないが、高い位置に移動することで第二装着部２へ添加することができ、より高くすることで、添加速度を速くすることもできる。チューブ４を手で圧し潰して流路を狭くすることで、単位時間当たりの添加量を制御してもよい。 Further, the addition rate of the diluent can be controlled by adjusting the positional relationship between the first mounting portion 1 and the second mounting portion 2. That is, when the position of the first mounting portion 1 is lower than or the same as the position of the second mounting portion 2, the mixed liquid injected from the injection port 3 does not flow to the second mounting portion 2, but moves to a higher position. By doing so, it can be added to the second mounting portion 2, and by making it higher, the addition rate can be increased. The amount of addition may be controlled per unit time by crushing the tube 4 by hand to narrow the flow path.
　このように、流路の断面積や、位置エネルギーを適宜調節することで、十分緩慢な浸透圧変化が起こるように制御することができる。制御手段５は、クレンメのようなチューブ４を圧し潰すことができるクリップ状のものを流路に接続し、流路の断面積を制御してもよいし、流路に弁を設け、流路の断面積を弁の開放度合いで制御してもよいし、流路を流れる流体の重さによって開放度合いが変わる弁を流路に設け、流量が自動的に制御されるようにしてもよい。 In this way, by appropriately adjusting the cross-sectional area of the flow path and the potential energy, it is possible to control so that a sufficiently slow osmotic pressure change occurs. The control means 5 may connect a clip-like object such as a clamp capable of crushing the tube 4 to the flow path to control the cross-sectional area of the flow path, or provide a valve in the flow path to provide a flow path. The cross-sectional area of the valve may be controlled by the degree of opening of the valve, or a valve whose degree of opening changes depending on the weight of the fluid flowing through the flow path may be provided in the flow path so that the flow rate is automatically controlled.
　浸透圧変化を緩慢にするためには、希釈液を添加する速度を遅くする方法を用いることができるが、添加速度が遅い場合は、細胞懸濁液を十分に希釈するのに時間がかかる。しかしながら、希釈が進むにつれて、希釈液の細胞懸濁液に占める割合は増大するため、希釈液をより多く添加しても細胞懸濁液の急激な希釈は起こりにくいと言える。したがって、本発明においては、希釈液の添加速度を、希釈が進むにつれて大きくなるように制御することで、希釈工程にかかる時間を大幅に短縮することができる。 In order to slow down the change in osmotic pressure, a method of slowing down the rate of adding the diluent can be used, but if the rate of addition is slow, it takes time to sufficiently dilute the cell suspension. However, as the dilution progresses, the proportion of the diluted solution in the cell suspension increases, so it can be said that rapid dilution of the cell suspension is unlikely to occur even if a larger amount of the diluted solution is added. Therefore, in the present invention, by controlling the addition rate of the diluent so as to increase as the dilution progresses, the time required for the dilution step can be significantly shortened.
　本発明において、「十分緩慢な浸透圧変化」の閾値は、用いる細胞、融解の条件、温度などにより変化し得る。例えば通常の凍結保存液（例えば１０％程度のＤＭＳＯを含有するＤＭＥＭ培地など）および通常の希釈液（例えば市販のＤＭＥＭ培地など）を用いる場合、ある態様において最大浸透圧負荷は約２５０ｍＯｓｍ／秒以下であり、約２２０ｍＯｓｍ／秒であることが好ましい。より好ましくは約１００ｍＯｓｍ／秒以下であり、さらに好ましくは約５０ｍＯｓｍ／秒以下である。 In the present invention, the threshold value of "sufficiently slow osmotic pressure change" can change depending on the cells used, melting conditions, temperature, and the like. For example, when a normal cryopreservation solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example, commercially available DMEM medium) are used, the maximum osmotic load is about 250 mOsm / sec or less in some embodiments. It is preferably about 220 mOsm / sec. It is more preferably about 100 mOsm / sec or less, and even more preferably about 50 mOsm / sec or less.
　また、希釈に時間をかけすぎると、例えば細胞懸濁液中の細胞毒性成分の影響で、浸透圧負荷以外の理由によるダメージが細胞に与えられてしまう場合があるため、この観点からは速やかに希釈されることが好ましい。本発明においては、最大浸透圧負荷の下限値は特に設定されなくてもよいが、通常の凍結保存液（例えば１０％程度のＤＭＳＯを含有するＤＭＥＭ培地など）および通常の希釈液（例えば市販のＤＭＥＭ培地など）を用いる場合であれば、約２ｍＯｓｍ／秒以上が好ましく、約２０ｍＯｓｍ／秒以上がより好ましく、約４０ｍＯｓｍ／秒以上がさらに好ましい。 In addition, if it takes too much time to dilute, for example, due to the influence of cytotoxic components in the cell suspension, damage may be given to the cells for reasons other than osmotic load, so from this point of view, promptly. It is preferably diluted. In the present invention, the lower limit of the maximum osmotic load may not be set in particular, but a normal cryopreservation solution (for example, DMEM medium containing about 10% DMSO) and a normal diluted solution (for example, commercially available). When DMEM medium or the like is used, it is preferably about 2 mOsm / sec or more, more preferably about 20 mOsm / sec or more, and even more preferably about 40 mOsm / sec or more.
　したがって本発明の好ましい一態様において、最大浸透圧負荷は、２ｍＯｓｍ／秒～２５０ｍＯｓｍ／秒であり、より好ましくは２ｍＯｓｍ／秒～２２０ｍＯｓｍ／秒であり、さらに好ましくは２０ｍＯｓｍ／秒～１００ｍＯｓｍ／秒であり、回収可能な生細胞量の観点から、最も好ましくは４０ｍＯｓｍ／秒～５０ｍＯｓｍ／秒である。最大浸透圧負荷は、注入口３から注入する希釈液の注入量の制御、制御手段５による流路の流量の制御、位置エネルギーを利用した流速の制御などにより好適にコントロールすることができる。 Therefore, in a preferred embodiment of the present invention, the maximum osmotic load is 2 mOsm / sec to 250 mOsm / sec, more preferably 2 mOsm / sec to 220 mOsm / sec, and even more preferably 20 mOsm / sec to 100 mOsm / sec. From the viewpoint of the amount of viable cells that can be recovered, it is most preferably 40 mOsm / sec to 50 mOsm / sec. The maximum osmotic pressure load can be suitably controlled by controlling the injection amount of the diluent to be injected from the injection port 3, controlling the flow rate of the flow path by the control means 5, controlling the flow velocity using potential energy, and the like.
　以上のように、本発明によれば、簡単な構造を有するため、製造コストを低く抑えることができ、かつ簡単な操作で、効率よく細胞懸濁液を調製することができるため、作業者の手技に依存することなく、作業レベルを均一化することができる。
　本発明によれば、細胞懸濁液の調製を細胞懸濁液を外気に曝すことなく無菌的に行うことができ、コンタミネーション発生のリスクを下げることができる。また、簡単な操作で有害物質を適性に除去でき、作業を均一化できるため、細胞の生存率が向上する。 As described above, according to the present invention, since the cell suspension has a simple structure, the production cost can be kept low, and the cell suspension can be efficiently prepared by a simple operation. The work level can be made uniform without depending on the procedure.
According to the present invention, the cell suspension can be prepared aseptically without exposing the cell suspension to the outside air, and the risk of contamination can be reduced. In addition, harmful substances can be appropriately removed by a simple operation, and the work can be made uniform, so that the cell viability is improved.
　本発明によれば、凍結保存細胞の融解を簡単な機構で効率良く迅速に行うことができ、また、細胞懸濁液の収集を簡単な機構で効率良く無菌的に行うことができ、さらに、収集された細胞懸濁液を周囲を汚染することなく迅速に無菌的に混合容器に移送することができる。したがって、細胞懸濁液の希釈などの次のステップに、素早く移行することができ、細胞毒性成分の細胞への影響を最小限に抑えることができるため、細胞の生存率が向上する。 According to the present invention, cryopreserved cells can be thawed efficiently and quickly by a simple mechanism, and cell suspensions can be collected efficiently and aseptically by a simple mechanism. The collected cell suspension can be rapidly and aseptically transferred to the mixing vessel without contaminating the surroundings. Therefore, the cell viability is improved because the next step, such as dilution of the cell suspension, can be made quickly and the effects of cytotoxic components on the cells can be minimized.
　本発明によれば、細胞懸濁液の希釈時の浸透圧負荷を簡単な機構で制御できるため作業を均一化でき、急激な浸透圧変化によって死に至る細胞数を減少させ、それにより回収可能な生細胞数を向上させる。とくに融解後に増殖培養を経ずに使用する場合であっても、十分な生細胞数を確保することが可能となる。 According to the present invention, the osmotic pressure load at the time of diluting the cell suspension can be controlled by a simple mechanism, so that the work can be made uniform, and the number of dead cells due to a sudden change in osmotic pressure can be reduced, thereby recovering. Improves the number of living cells. In particular, even when the cells are used after thawing without undergoing growth culture, it is possible to secure a sufficient number of viable cells.
　以上、本発明を第１実施形態に係るデバイスを使用した例について説明したが、本発明はこれに限定されるものではない。例えば、上記の実施形態では、混合液として希釈液を使用する場合を説明したが、混合液は、細胞懸濁液に添加することができる任意の液体を自由に選択して、混合容器Ｍに移送された細胞懸濁液に添加することができる。本発明のデバイスを使用することで、混合液を無菌的に添加することができる。細胞懸濁液と混合できる混合液としては、限定されずに、希釈液、希釈液、廃液、培地、洗浄液、前処理液、リン酸緩衝液（PBS）が挙げられる。 Although the example in which the device according to the first embodiment is used in the present invention has been described above, the present invention is not limited to this. For example, in the above embodiment, the case where the diluted solution is used as the mixed solution has been described, but as the mixed solution, any liquid that can be added to the cell suspension can be freely selected and placed in the mixing container M. It can be added to the transferred cell suspension. By using the device of the present invention, the mixed solution can be added aseptically. The mixed solution that can be mixed with the cell suspension includes, but is not limited to, a diluted solution, a diluted solution, a waste solution, a medium, a washing solution, a pretreatment solution, and a phosphate buffer solution (PBS).
　また、上記では、デバイスを用いてクリーンルームの内外で細胞懸濁液を調製する場合を説明したが、クリーンルームを使用せずに行えるように、第一装着部１および／または第二装着部２を開閉可能に構成することもできる。すなわち、第一装着部１と第二装着部２とが閉鎖されている場合、第一装着部１と第二装着部２（チューブ４を含む場合もある）とで形成される流路を無菌状態に保つことができる。 Further, in the above description, the case where the cell suspension is prepared inside and outside the clean room using the device has been described, but the first mounting portion 1 and / or the second mounting portion 2 may be provided so that the cell suspension can be prepared without using the clean room. It can also be configured to be openable and closable. That is, when the first mounting portion 1 and the second mounting portion 2 are closed, the flow path formed by the first mounting portion 1 and the second mounting portion 2 (which may include the tube 4) is sterile. Can be kept in a state.
　そして、収容容器Ｃおよび／または混合容器Ｍを装着した後に、第一装着部１および／または第二装着部２を開放することにより、装着時におけるコンタミネーション発生のリスクを下げることができる。また、第一装着部１および／または第二装着部２の装着部分をフィルムなどで覆い、収容容器Ｃおよび／または混合容器Ｍを装着した際にフィルムが破けるような開閉手段を適用することで、装着時のコンタミネーション発生のリスクを下げることができる。 Then, by opening the first mounting portion 1 and / or the second mounting portion 2 after mounting the storage container C and / or the mixing container M, the risk of contamination at the time of mounting can be reduced. Further, the mounting portion of the first mounting portion 1 and / or the second mounting portion 2 is covered with a film or the like, and an opening / closing means for tearing the film when the storage container C and / or the mixing container M is mounted is applied. Therefore, the risk of contamination at the time of mounting can be reduced.
　デバイスは、第一装着部１と、第二装着部２と、第二装着部２に装着されている混合容器Ｍとを含んでもよい。そして、第一装着部１を開閉可能に構成して、収容容器Ｃを装着していないときはこれを閉鎖することにより、第一装着部１、第二装着部２および混合容器Ｍで構成される空間（流路）を無菌的に閉回路にすることができる。そして、収容容器Ｃを装着した際に開放することにより、細胞懸濁液の移送を行うこともできる。この場合は、少なくとも第一装着部１が開閉可能に構成されていればよい。
　本発明においては、各構成は、同様の機能を発揮し得る任意のものと置換することができ、あるいは、任意の構成を付加することもできる。 The device may include a first mounting portion 1, a second mounting portion 2, and a mixing container M mounted on the second mounting portion 2. Then, the first mounting portion 1 is configured to be openable and closable, and when the storage container C is not mounted, it is closed to form the first mounting portion 1, the second mounting portion 2, and the mixing container M. The space (flow path) can be aseptically closed. Then, the cell suspension can be transferred by opening the storage container C when it is attached. In this case, at least the first mounting portion 1 may be configured to be openable and closable.
In the present invention, each configuration can be replaced with any configuration capable of exerting the same function, or any configuration can be added.
　本発明のデバイスの使用は、例えば以下の工程によって順次行うことができる。
　（１）凍結保存細を収容した収容容器と混合容器とをデバイスに装着するステップ。
　（２）収容容器を融解に供して凍結保存細を融解して細胞懸濁液を得るステップ。
　（３）収容容器から混合容器にデバイスを介して細胞懸濁液を移送するステップ。
　（４）デバイスに設けられた注入口から希釈液を注入するステップ。
　（５）希釈液を混合容器に移送して、細胞懸濁液に添加するステップ。 The device of the present invention can be used in sequence by, for example, the following steps.
(1) A step of attaching a storage container containing a cryopreservation fine and a mixing container to a device.
(2) A step of thawing the storage container and thawing the cryopreservation fine to obtain a cell suspension.
(3) The step of transferring the cell suspension from the storage container to the mixing container via the device.
(4) A step of injecting the diluted solution from the injection port provided in the device.
(5) A step of transferring the diluent to a mixing vessel and adding it to the cell suspension.
　デバイスには、細胞懸濁液を収容した収容容器を装着してもよく、この場合は、ステップ（２）を省略してもよい。ステップ（４）において注入した希釈液を収容容器に移送し、残留している細胞懸濁液をリンスして、このリンス液を混合容器に移送することで、生細胞の回収率を高めるようにしてもよい。ステップ（５）において、デバイスに制御手段を接続し、流路を流れる流体の流量を制御することで、最大浸透圧負荷を調製してもよい。 The device may be equipped with a storage container containing the cell suspension, in which case step (2) may be omitted. The diluted solution injected in step (4) is transferred to a storage container, the remaining cell suspension is rinsed, and this rinse solution is transferred to a mixing container to increase the recovery rate of living cells. You may. In step (5), the maximum osmotic load may be adjusted by connecting a control means to the device and controlling the flow rate of the fluid flowing through the flow path.
　本発明のデバイスを使用して、または本発明の方法で調製された細胞で製造されたシート状細胞培養物を、それを必要とする対象に適用し、シート状細胞培養物の適用により改善される疾患を処置することもできる。 Sheet cell cultures made using the devices of the invention or with cells prepared by the methods of the invention are applied to subjects in need thereof and improved by application of the sheet cell cultures. Diseases can also be treated.
Ｃ　　　　　　　　　　　　収容容器
Ｍ　　　　　　　　　　　　混合容器
１　　　　　　　　　　　　第一装着部
２　　　　　　　　　　　　第二装着部
３　　　　　　　　　　　　注入口
４　　　　　　　　　　　　連結部（チューブ）
５　　　　　　　　　　　　制御手段
６　　　　　　　　　　　　滅菌フィルター C Storage container M Mixing container 1 First mounting part 2 Second mounting part 3 Injection port 4 Connecting part (tube)
5 Control means 6 Sterilization filter

Claims (18)

  1. 　細胞懸濁液を収容するための収容容器を装着可能な第一装着部と、細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、第一装着部と第二装着部とが流体連結されている、デバイス。 The first mounting portion includes a first mounting portion to which a storage container for accommodating the cell suspension can be mounted, and a second mounting portion to which a mixing container for mixing the cell suspension and the mixed solution can be mounted. A device in which the mounting part and the second mounting part are fluidly connected.
  2. 　流体連結が、可撓性のチューブを介して行われる、請求項１に記載のデバイス。 The device according to claim 1, wherein the fluid connection is performed via a flexible tube.
  3. 　第一装着部が、複数の収容容器を装着可能に構成されている、請求項１または２に記載のデバイス。 The device according to claim 1 or 2, wherein the first mounting portion is configured so that a plurality of storage containers can be mounted.
  4. 　混合に使用する混合液のための注入口が設けられている、請求項１～３のいずれか一項に記載のデバイス。 The device according to any one of claims 1 to 3, which is provided with an injection port for a mixture used for mixing.
  5. 　混合液が希釈液である、請求項１～４のいずれか一項に記載のデバイス。 The device according to any one of claims 1 to 4, wherein the mixed solution is a diluent.
  6. 　流量を制御可能な制御手段をさらに含む、請求項１～５のいずれか一項に記載のデバイス。 The device according to any one of claims 1 to 5, further comprising a control means capable of controlling the flow rate.
  7. 　流路に外気を取り込むための滅菌フィルターが接続されている、請求項１～６のいずれか一項に記載のデバイス。 The device according to any one of claims 1 to 6, wherein a sterilization filter for taking in outside air is connected to the flow path.
  8. 　第一装着部および／または第二装着部が、開閉可能に構成されている、請求項１～７のいずれか一項に記載のデバイス。 The device according to any one of claims 1 to 7, wherein the first mounting portion and / or the second mounting portion is configured to be openable and closable.
  9. 　細胞懸濁液中の細胞を混合するための容器に移送するためのデバイスで、前記細胞懸濁液を収容する複数の収容容器を装着可能な第一装着部と、前記細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、前記第一装着部と前記第二装着部とを流体連結する連結部を有する、細胞懸濁液の移送デバイス。 A device for transferring cells in a cell suspension to a container for mixing, a first mounting portion on which a plurality of storage containers for accommodating the cell suspension can be mounted, and mixing with the cell suspension. A cell suspension transfer device comprising a second mounting portion to which a mixing container for mixing with a liquid can be mounted, and having a connecting portion for fluidly connecting the first mounting portion and the second mounting portion.
  10. 　流体連結部が、可撓性のチューブにより構成され、該チューブを介して流体連結が行われる、請求項９に記載のデバイス。 The device according to claim 9, wherein the fluid connecting portion is composed of a flexible tube, and the fluid connecting is performed through the tube.
  11. 　前記第一装着部に、混合に使用するための混合液を注入する注入口が設けられている、請求項９または１０に記載のデバイス。 The device according to claim 9 or 10, wherein the first mounting portion is provided with an injection port for injecting a mixed liquid for use in mixing.
  12. 　前記混合液が希釈液である、請求項９～１１のいずれか一項に記載のデバイス。 The device according to any one of claims 9 to 11, wherein the mixed solution is a diluent.
  13. 　前記連結部または前記第二装着部のいずれかに、前記混合液の流入量を制御可能な制御手段をさらに含む、請求項９～１２のいずれか一項に記載のデバイス。 The device according to any one of claims 9 to 12, further comprising a control means capable of controlling the inflow amount of the mixed liquid in either the connecting portion or the second mounting portion.
  14. 　細胞懸濁液を調製するための方法であって、
    （ａ）細胞懸濁液を収容するための収容容器を装着可能な第一装着部と、細胞懸濁液と混合液との混合のための混合容器を装着可能な第二装着部とを含み、第一装着部と第二装着部とが流体連結可能である、デバイスを供するステップ、
    （ｂ）細胞懸濁液を収容した収容容器および混合容器をデバイスに装着し、収容容器から混合容器にデバイスを介して細胞懸濁液を移送するステップを含む、前記方法。     A method for preparing cell suspensions,
    (A) Includes a first mounting portion to which a storage container for storing the cell suspension can be mounted and a second mounting portion to which a mixing container for mixing the cell suspension and the mixed solution can be mounted. , The step of providing the device, where the first mounting part and the second mounting part can be fluidly connected,
    (B) The method comprising attaching a storage container and a mixing container containing a cell suspension to a device and transferring the cell suspension from the storage container to the mixing container via the device.
  15. 　ステップ（ｂ）の後に、（ｃ）デバイスに設けられている細胞懸濁液との混合に使用する混合液のための注入口から、混合液を注入するステップを含む、請求項１４に記載の方法。 14. The 14th aspect of claim 14, comprising injecting the mixture after step (b) through an inlet for the mixture used for mixing with the cell suspension provided in the device (c). Method.
  16. 　ステップ（ｃ）の後に、（ｄ）混合液を収容容器または混合容器に移送するステップを含む、請求項１４または１５に記載の方法。 The method according to claim 14 or 15, which comprises, after step (c), (d) a step of transferring the mixed solution to a storage container or a mixing container.
  17. 　混合液が希釈液である、請求項１４～１６のいずれか一項に記載の方法。 The method according to any one of claims 14 to 16, wherein the mixed solution is a diluent.
  18. 　シート状細胞培養物の適用により改善される疾患を処置する方法であって、請求項１～１３のいずれか一項に記載のデバイスを使用して調製された細胞、または請求項１４～１７のいずれか一項に記載の方法で調製された細胞を使用して製造されたシート状細胞培養物を、それを必要とする対象に適用することを含む、前記方法。 A method of treating a disease that is ameliorated by application of a sheet cell culture, wherein the cells are prepared using the device according to any one of claims 1 to 13, or claims 14 to 17. The method described above, comprising applying a sheet cell culture prepared using the cells prepared by the method according to any one of the above to a subject in need thereof.
PCT/JP2020/037054 2019-09-30 2020-09-30 Device and method for preparing cell suspension WO2021065969A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019-179005 2019-09-30
JP2019179005 2019-09-30

Publications (1)

Publication Number Publication Date
WO2021065969A1 true WO2021065969A1 (en) 2021-04-08

Family

ID=75336986

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/037054 WO2021065969A1 (en) 2019-09-30 2020-09-30 Device and method for preparing cell suspension

Country Status (1)

Country Link
WO (1) WO2021065969A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01158957A (en) * 1987-12-11 1989-06-22 Komatsu Ltd Sterilizing apparatus
JPH1084942A (en) * 1996-09-19 1998-04-07 Terumo Corp Connected container and culturing method of bacteria
JP2010226991A (en) * 2009-03-26 2010-10-14 Terumo Corp Method for producing sheet-shaped cell culture
WO2013114845A1 (en) * 2012-02-01 2013-08-08 東洋製罐グループホールディングス株式会社 Cell culture kit, and method of using cell culture kit

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01158957A (en) * 1987-12-11 1989-06-22 Komatsu Ltd Sterilizing apparatus
JPH1084942A (en) * 1996-09-19 1998-04-07 Terumo Corp Connected container and culturing method of bacteria
JP2010226991A (en) * 2009-03-26 2010-10-14 Terumo Corp Method for producing sheet-shaped cell culture
WO2013114845A1 (en) * 2012-02-01 2013-08-08 東洋製罐グループホールディングス株式会社 Cell culture kit, and method of using cell culture kit

Similar Documents

Publication Publication Date Title
JP6495603B2 (en) Method for producing laminate of sheet-shaped cell culture and fibrin gel
JP2022107023A (en) Decellularization and recellularization of organs and tissues
Ko et al. Bioengineered transplantable porcine livers with re-endothelialized vasculature
Arora et al. Banking stem cells from human exfoliated deciduous teeth (SHED): saving for the future
JP4943844B2 (en) 3D tissue structure
JP6463275B2 (en) Human and large mammalian lung bioreactors
JP6599089B2 (en) Laminated body of sheet-shaped cell culture having reinforcing portion and fibrin gel
CN104922059A (en) Umbilical cord mesenchymal stem cell injection and preparation method and application thereof
US20020197240A1 (en) Marrow stem cell (MSC) transplantation for use in tissue and/or organ repair
CN108743620A (en) Bioactive materials promote source of human stem cell excretion body to treat corneal injury
JP2012516853A (en) Therapeutic use of differentiated endothelial progenitor cells
WO2021065969A1 (en) Device and method for preparing cell suspension
JP6846402B2 (en) Method for producing a laminate of sheet-shaped cell culture and fibrin gel
KR102431887B1 (en) media for implantation
JP2021052660A (en) Container and method for preparing cell suspension
CN111867644A (en) Implantable cell dressing for treating disease
WO2021065991A1 (en) Sheet-shaped cell culture preparation device
JP7262443B2 (en) Sheet-like cell culture with penetrating structures
Gaillard et al. Minimally invasive liver preconditioning for hepatocyte transplantation in rats
WO2020067434A1 (en) Method for cryopreserving cells
WO2020067442A1 (en) Method for cryopreserving cells
JP2023125045A (en) Medical equipment for administering liquid including liquid factor produced by cell, to target region
Sreekanth et al. PERIODONTAL REGENERATION THROUGH CELL SHEET ENGINEERING-A NOVEL APPROACH
Kalter andTheoMMHdeBy Tissue banking programmes in Europe

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20872667

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20872667

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP