WO2019059278A1 - Method for separating and collecting cells from biological tissue - Google Patents

Method for separating and collecting cells from biological tissue Download PDF

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Publication number
WO2019059278A1
WO2019059278A1 PCT/JP2018/034815 JP2018034815W WO2019059278A1 WO 2019059278 A1 WO2019059278 A1 WO 2019059278A1 JP 2018034815 W JP2018034815 W JP 2018034815W WO 2019059278 A1 WO2019059278 A1 WO 2019059278A1
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Prior art keywords
container
solution
volume
enzyme
aqueous layer
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PCT/JP2018/034815
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French (fr)
Japanese (ja)
Inventor
林　真司
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/04—Phase separators; Separation of non fermentable material; Fractionation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/09—Means for pre-treatment of biological substances by enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes

Definitions

  • the present invention relates to a method of enzymatically treating a living tissue such as adipose tissue to separate and recover cells.
  • At least a part of nucleated cells contained in adipose tissue are living tissue stem cells, which can be differentiated into various cells such as mature adipocytes, bone cells, chondrocytes, myoblasts, vascular endothelial cells, etc. It has been known. A method for efficiently separating and collecting such pluripotent adipose-derived living tissue stem cells is extremely important from the viewpoint of regenerative medicine development.
  • the living tissue As a method of obtaining useful cells from living tissue including useful cells such as adipose tissue, the living tissue is degraded with digestive enzymes to release useful cells, and then the cells are subjected to separation steps such as centrifugation, filtration and the like to obtain useful cells. Methods for recovering are known.
  • Example 1 of Patent Document 1 an adipose tissue and a collagenase solution are accommodated in a 50 mL centrifugal tube, shaken at 37 ° C., 120 times / min for 1 hour to perform an enzyme reaction, and then a filter It is described that filtration and centrifugation are performed to obtain a precipitated cell population (SVF fraction) as sediment.
  • SVF fraction precipitated cell population
  • Patent Document 2 as a device for separating non-fat cells from a fat tissue sample, a substance of a first sheet, a substance of a second sheet bonded to the substance of the first sheet, and a substance of the first sheet An apparatus is disclosed comprising a plurality of chambers defined between a substance and the substance of the second sheet. Then, it is disclosed that the adipose tissue sample is treated with a dissociation solution containing an enzyme such as collagenase to dissociate the adipose tissue sample in a first chamber which is one of a plurality of chambers.
  • a dissociation solution containing an enzyme such as collagenase
  • centrifugation is performed after enzymatic treatment of a living tissue to separate it into an aqueous layer containing useful cells and an oil layer containing oil-soluble components such as lipids, and the aqueous layer is recovered.
  • Methods are known.
  • centrifugation has a large load on cells, so the cells are easily damaged and is not preferable for the purpose of recovering useful cells.
  • a method for separating and recovering cells from a living tissue which comprises: Carrying out the enzyme treatment of the living tissue in a mixture of the living tissue and the enzyme solution of volume V 1 in a container to form an enzyme-treated solution containing free cells; And 2.
  • the container is a container in which a liquid outlet is formed
  • the recovery of the aqueous layer in steps 3 and 4 includes disposing the container so that the outlet is positioned vertically below the aqueous layer, and recovering the aqueous layer through the outlet, (1) to (6) The method described in either.
  • the present specification includes the disclosure content of Japanese Patent Application No. 2017-180178 based on which the priority of the present application is based.
  • living tissue can be subjected to enzyme treatment to separate and recover cells under low load conditions for cells.
  • FIG. 1 illustrates one embodiment of a container for enzymatic treatment of biological tissue. It is a schematic diagram for demonstrating the procedure of collection
  • FIG. 7 illustrates one embodiment of a filtration and storage device for filtering and storing the recovered cell-containing aqueous layer.
  • the biological tissue is typically a biological tissue collected from an animal, such as fat, skin, blood vessels, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine, amniotic membrane, placenta, umbilical cord, etc. And biological tissues derived from The methods disclosed herein are particularly useful for removing interstitial vascular fraction (SVF) cells from adipose tissue.
  • SVF interstitial vascular fraction
  • the adipose tissue is typically mammalian adipose tissue, such as subcutaneous fat of human origin, visceral fat, white fat and brown fat.
  • Adipose tissue may have any shape, for example, one obtained by crushing adipose tissue using a sharp instrument such as scissors, one obtained by mincing using a filter, etc., or one obtained by decomposition using liposuction.
  • the liposuction method is not particularly limited as long as it is a suction method performed in general cosmetic surgery, and for example, a method by ultrasonic liposuction, powered liposome suction using a cannula or the like, syringe suction, etc. It is.
  • cells to be released in the present invention include stem cells, white blood cells, monocytes, granulocytes, lymphocytes, vascular endothelial cells, vascular endothelial precursor cells, pericytes, etc., which need to be collected for cell therapy and experiments.
  • Nucleated cells can be exemplified.
  • the living tissue stem cells are preferably fat-derived mesenchymal stem cells, fat-derived stromal stem cells, and more preferably express at least one selected from CD34, 73, 90, 105, 106, 133, 166 on the cell surface Fat-derived mesenchymal cells, fat-derived stromal stem cells.
  • At least one degradation enzyme selected from collagenase, metalloprotease, dispase, trypsin, hyaluronidase, chymotrypsin, pepsin, aminopeptidase, lipase, amylase and their recombinants can be used.
  • at least one degradable enzyme selected from collagenase, metalloprotease, dispase, trypsin and hyaluronidase from the viewpoint of degradation in a short time and with low invasiveness as an enzyme preferable.
  • aqueous medium such as a culture medium or an isotonic solution can be used.
  • processing container The container used for the enzyme treatment and the layer separation in the method of the present invention (hereinafter sometimes referred to as "processing container") is not particularly limited as long as it is a container capable of containing a liquid.
  • the method of the present invention does not require centrifugation, it is possible to use even containers that are generally difficult to centrifuge, such as bag-like containers.
  • the processing container is preferably a container in which a discharge port for discharging a liquid is formed.
  • a discharge port for discharging a liquid is formed.
  • it is easy to recover the water layer through the outlet.
  • the discharge port is positioned vertically below the water layer to be collected and the discharge port is opened, the water layer is discharged by gravity, and this is compared to a method of collecting the water layer from the top using a pipette etc.
  • it is easy to recover the aqueous layer without disturbing the interface between the oil layer and the aqueous layer and without applying a high load to the cells.
  • At least a part of the processing container is transparent or translucent, and that the layer separation state of the enzyme processing solution can be confirmed.
  • the processing container is more preferably a bag-like container having a wall surface formed of a flexible (flexible) resin sheet, and particularly preferably two opposing resin sheets having flexibility are disposed in an opposing manner.
  • the container is formed in a bag-like shape by bonding the peripheral portion by heat fusion or an adhesive.
  • a processing container 100 which is a specific example of a processing container which is a bag-like container will be described with reference to FIG.
  • the processing container 100 includes a container main body 110 including the first space 114, and an input portion 120A, an input portion 120B, and an input portion 120C in which material inlets 121A, 121B, and 121C to the first space 114 are formed.
  • the discharge unit 130 includes a discharge unit 130A and a discharge unit 130B.
  • the discharge unit 130A includes a discharge unit 130 and discharge openings 131A and 131B of the liquid from the first space 114.
  • the input portion 120A includes a pipe portion 122A that forms an input port 121A of the material into the first space 114, a female luer lock 123A that seals the inlet of the pipe portion 122A in a manner that allows communication with each other, and a removable type And a filter portion 125A for filtering the liquid passing through the pipe portion 122A.
  • the input portion 120B includes a pipe portion 122B that forms an input port 121B of the material into the first space 114, a needleless port 123B that blocks the inlet of the pipe portion 122B in communication, and a removable type that protects the needleless port 123B. And the lid portion 124B of the
  • the input portion 120C includes a pipe portion 122C that forms an input port 121C of the material into the first space 114, a needleless port 123C that blocks the inlet of the pipe portion 122C in communication with each other, and a removable port that protects the needleless port 123C. And a cover 124C of
  • the discharge part 130A includes a pipe part 132A which forms a discharge port 131A of the liquid from the first space 114, a needleless port 133A which closes the outlet of the pipe part 132A in a communicating manner, and a removable part which protects the needleless port 133A. And a lid 134A.
  • the discharge unit 130B includes a pipe portion 132B that forms a discharge port 131B of the liquid from the first space 114, a needleless port 133B that seals the outlet of the pipe portion 132B in a communicating manner, and a removable port that protects the needleless port 133B. And a lid 134B.
  • the processing container 100 is provided with three input units 120, but may be provided with only one, only two, or four or more input units.
  • the input unit 120 is used to input, to the first space 114, a material (for example, a living tissue, an enzyme solution, etc.) used for enzyme treatment of a living tissue in the first space 114 of the processing container 100 or a cleaning solution.
  • the input unit and the discharge unit do not need to be separately provided.
  • the input unit may not be provided, and a discharge unit described later may be used as the input unit.
  • the processing container 100 includes two discharge units 130, but may include only one or three or more discharge units.
  • the discharge unit 130 is used to discharge, from the first space 114, at least a part of an enzyme treatment solution containing free cells formed by enzyme treatment of a living tissue.
  • the container body 110 has the first resin sheet 111 and the second resin sheet 112 disposed opposite to each other, and peripheral portions thereof are joined by heat fusion to form a bag. A portion where the first resin sheet 111 and the second resin sheet 112 are joined by heat fusion is referred to as a fusion part 113.
  • a first space 114 is formed between the first resin sheet 111 and the second resin sheet 112.
  • FIG. 1 is a schematic plan view of the processing container 100 in which the first space 114 is empty, and although not shown, when the reaction mixture containing biological tissue and enzymes is accommodated in the first space 114, the container is not shown.
  • the main body 110 expands in a bag shape. In the first space 114, enzyme treatment of a living tissue is performed.
  • the first resin sheet 111 and the second resin sheet 112 of the pipe sections 122A, 122B, 122C of the input sections 120A, 120B, 120C have one end thereof on the side of one end of the container body 110 in the longitudinal direction.
  • the first resin sheet 111 and the second resin sheet 112 are joined by heat fusion to be integrated with the container body 110.
  • the first resin sheet 111 and the second resin sheet 112 are disposed at one end of the pipe sections 132A and 132B on the other end of the container body 110 in the longitudinal direction.
  • the first resin sheet 111 and the second resin sheet 112 are joined by heat fusion to be integrated with the container body 110.
  • through holes 115, 115 are formed in portions outside the fusion bonded portion 113 on both sides of the side of the container main body 110 where the input portion 120 is disposed.
  • the through hole 115 can be used to hook on a hook or the like.
  • first resin sheet 111 and the second resin sheet 112 and the respective tube portions 122A, 122B, 122C, 132A, and 132B be formed of a material having flexibility.
  • the material having flexibility include soft vinyl chloride, vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin such as polyethylene and polypropylene, styrene-butadiene-styrene copolymer or hydrogenated product thereof
  • flexible resins such as thermoplastic elastomers such as styrene-isoprene-styrene copolymer or a hydrogenated product thereof and mixtures of thermoplastic elastomers and softeners such as polyolefin and ethylene-ethyl acrylate.
  • first resin sheet 111 and the second resin sheet 112 be made of a transparent or translucent material. It is preferable that the inner side surfaces of the first resin sheet 111 and the second resin sheet 112 be subjected to a sashi process because residual liquid can be reduced when the water layer is discharged.
  • FIG. 2 exemplifies the processing container 100 shown in FIG. 1 as a processing container, as described above, other containers can also be used.
  • Step 1 in a vessel, subjected to the enzymatic treatment of the living tissue in a mixture of an enzyme solution of the biological tissue and the volume V 1, a step of forming an enzyme treatment solution containing free cells.
  • an “enzyme solution” refers to a solution in which an enzyme is dissolved or suspended in an aqueous medium.
  • the enzyme solution does not have to be mixed in advance with the whole amount of the enzyme and the aqueous medium to form an enzyme solution, and then the enzyme solution and the aqueous medium may be separately charged into the container to form the enzyme solution in the container. . Therefore the volume V 1 of the enzyme solution can be calculated by summing enzyme loaded into the container in order to carry out the enzyme treatment, the enzyme solution, the volume of the aqueous medium.
  • the volume ratio of the living tissue to the enzyme solution is not particularly limited, but for layer separation in Step 2 and Step 4, the volume V 1 of the enzyme solution is preferably 30% with respect to the volume of the living tissue. It is about -500%, more preferably 30% to 300%, more preferably 50% to 200%, more preferably 70% to 130%.
  • the enzyme treatment of the living tissue can be promoted by shaking the container containing the mixture containing the living tissue and the enzyme solution or stirring the mixture in the container.
  • the temperature and time of the enzyme treatment can be adjusted appropriately.
  • step 1 an enzyme treatment solution A containing free cells is formed in the container (see FIG. 2 (A)).
  • Step 2 is to stand a container containing the enzyme treatment solution after the step 1, the enzyme treatment solution into an oil layer and an aqueous layer, the volume of the aqueous layer is a step of separating to the point not exceeding 0.93V 1.
  • the enzyme-treated solution of the living tissue formed in step 1 is time-lapsed to an oil layer B containing components such as lipids and an aqueous layer C containing free cells in an aqueous medium. Separate with the When the living tissue is fat tissue, fat cells are contained in oil layer B, and fat-derived stem cells are contained in water layer C.
  • step 2 After the completion of step 1, although the whole of the enzyme treatment liquid A is in a turbid state, the oil layer B and the water layer C become clear as time passes, and finally they are separated into two layers with clear boundaries. Volume of water layer when fully separated two layers, the volume close to the enzyme solution volume V 1. However, since it takes a long time to completely separate into two layers, there is a problem that cells are damaged during that time. Therefore, in step 2 of the present invention, without waiting until the volume of aqueous phase in the container is V 1, Once separated until such time as the volume of the aqueous layer does not exceed 0.93V 1, recovering the aqueous layer in the step 3 This shortens the standing time in step 2 and reduces the load on cells.
  • step 2 “when the volume of the aqueous layer does not exceed 0.93 V 1 ” is more preferably “when the volume of the aqueous layer does not exceed 0.92 V 1 ”, more preferably “the volume of the aqueous layer is a point "not exceeding 0.91 V 1, more preferably from” the time when the volume of the aqueous layer does not exceed 0.90 V 1 ", more preferably the volume of the" water layer does not exceed 0.89 V 1 time And more preferably "when the volume of the aqueous layer does not exceed 0.70 V 1 ".
  • the "volume of the aqueous layer 0.93V 1 (or, 0.92V 1, 0.91V 1, 0.90V 1, 0.89V 1, 0.70V 1) time not exceeding" the volume of the aqueous layer is preferably 0.20 V 1 or 0.93V 1 (or, 0.92V 1, 0.91V 1, 0.90V 1, 0.89V 1, 0.70V 1) or less, preferably 0.30 V 1 or 0 .93 (or, 0.92V 1, 0.91V 1, 0.90V 1, 0.89V 1, 0.70V 1) V 1 or less, 0.93V 1 (or more preferably 0.40 V 1 or more, 0 .92V 1, 0.91V 1, 0.90V 1 , 0.89V 1, 0.70V 1) or less, more preferably 0.50 V 1 or 0.93V 1 (or, 0.92 V 1, 0.91 V 1 , 0.90 V 1 , 0.89 V 1 , 0 .70V 1 ) or less.
  • step 2 if the volume V 1 of the enzyme solution by volume of the biological tissue in step 1 is not more than 200%, of the step 2 the volume of "water layer does not exceed 0.93V 1 "the volume of the" water layer is point "not exceeding 0.70 V 1, if the volume V 1 of the enzyme solution by volume of the biological tissue in step 1 is greater than 200%, step 2" of the aqueous layer “When the volume does not exceed 0.93 V 1 ” means “when the volume of the aqueous layer does not exceed 0.93 V 1 ”, “when the volume of the aqueous layer does not exceed 0.92 V 1 ”, “the volume of the aqueous layer Is not greater than 0.91 V 1 , “when the volume of the aqueous layer does not exceed 0.90 V 1 ”, or “when the volume of the aqueous layer does not exceed 0.89 V 1 ”.
  • the standing time in step 2 is preferably 3 minutes to 30 minutes, and more preferably 5 minutes to 15 minutes. By setting the standing time in step 2 to this range, it becomes easy to separate the water layer and the oil layer while suppressing the load on the cells.
  • Step 3 is a step of recovering the aqueous layer from the container and leaving the oil layer in the container when the volume of the aqueous layer is separated to a level not exceeding 0.93 V 1 in step 2.
  • FIG. 2B shows that the oil layer B and the aqueous layer C (volume is 0.93 V 1 or less, preferably 0.92 V 1 or less, preferably 0.91 V 1 or less, preferably 0.90 V 1 or less, in step 2) 0.89 V 1 or less, preferably an separated state to a 0.70 V 1 or less), FIG. 2 (C), in step 3, the container and the oil layer B with the process vessel 100 is recovered and the aqueous layer C The state of remaining in 100 is shown.
  • the container In the recovery of the water layer C, as shown in FIG. 2B, it is preferable to arrange the container so that the outlet of the container is positioned vertically below the water layer C, and recover the water layer through the outlet.
  • step 4 the washing solution is supplied into the container and mixed with the oil layer remaining in the container to form a mixed solution, and then the container is allowed to stand to separate the mixed solution into an oil layer and an aqueous layer.
  • This is a step of recovering the water layer and leaving the oil layer in the container.
  • the oil layer B remaining in the container is the target cell that should be originally contained in the aqueous layer C. Is likely to remain.
  • target cells remaining in the oil layer are recovered.
  • FIG. 2C shows a state in which the oil layer B remains in the processing container 100 after the step 3 is completed.
  • step 4 first, the cleaning liquid is supplied into the processing container 100 and mixed with the oil layer B to form a mixed liquid D as shown in FIG. 2 (D). Then, the mixed solution D is allowed to stand in the processing container 100 to be separated into the oil layer E and the water layer F as shown in FIG. 2 (E). Further, the aqueous layer F is recovered from the processing container 100, and the oil layer E is left in the processing container 100 as shown in FIG. 2 (F).
  • the aqueous layer recovered in step 4 contains the target cells, and can be combined with the aqueous layer recovered in step 3 to form a cell suspension.
  • the volume of the cleaning liquid supplied into the container in Step 4 is preferably 0.10 V 1 or more, more preferably 0.20 V 1 or more, more preferably 0.30 V 1 or more, more preferably 0.40 V 1 or more, more preferably Is 0.50 V 1 or more.
  • the volume of the cleaning liquid is preferably 3.00V 1 or less, and more preferably is 2.00V 1 below, and more preferably is 1.50 V 1 or less, a cell suspension consisting of collected aqueous layer, It is easy to carry out concentration washing treatment using a hollow fiber separation membrane.
  • the total volume of the volume V 1 of the enzyme solution used in step 1 and the volume of the washing solution used in step 4 is preferably By setting the concentration to 150% to 800%, and more preferably 200% to 500%, it is easy to prepare a relatively high density cell suspension under low load conditions for cells.
  • the time for which the container containing the mixed solution is allowed to stand is preferably 1/6 or more times as long as the standing time in the step 2, from the viewpoint of enhancing the collection efficiency of the cells to the aqueous layer.
  • the ratio is more preferably 3 times or more, more preferably 0.5 times or more, and particularly preferably 0.5 to 1.5 times.
  • the standing time in step 4 may also be a load on cells if it is too long, so it is preferably 3 minutes to 30 minutes, and more preferably 5 minutes to 15 minutes. By setting the standing time in step 2 to this range, it becomes easy to separate the water layer and the oil layer while suppressing the load on the cells.
  • Step 4 may be performed only once, or may be repeated twice, three times or more times.
  • the total of the standing time in step 2 and step 4 is preferably 60 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less.
  • ⁇ Filtration storage device> The aqueous layer separated by the method of the present invention is preferably recovered and stored in a filtration storage device 200 as shown in FIG.
  • the filtration storage device 200 of the embodiment shown in FIG. 3 includes a filter 210 and a storage container 220.
  • a pipe 231 and a pipe 232 are fluidly connected to the filter 210, and the liquid flowing from the pipe 231 passes through the mesh sheet 211 disposed in the filter 210 and is discharged to the pipe 232. Be done.
  • the opening of the mesh sheet 211 is set to a size that can allow target cells (for example, fat-derived stem cells) to pass through and unnecessary components (for example, fat cells) can not pass through.
  • a male connector 233 connectable to the needleless ports 133A and 133B of the processing container 100 is provided, and a lid 234 for protecting the male connector 233 is attached.
  • a roller clamp 235 for opening and closing the flow passage in the tube 231 is further provided.
  • the storage container 220 is a bag-like container, and one end of a pipe 232 is connected so as to be fluid-permeable.
  • the cell suspension that has passed through the filter 210 is stored in the storage container 220 through the pipe 232.
  • a pipe 236 is connected to the storage container 220 so as to be fluid-permeable.
  • a needleless port 237 is disposed, and a lid 238 for protecting the needleless port 237 is attached.
  • Adipose tissue from 3 human (donor 1, donor 2, donor 3) and 1 rabbit (donor 1) was treated with collagenase to prepare a cell suspension under the following conditions: .
  • a bag for enzyme treatment two approximately rectangular flexible sheets of soft polyvinyl chloride with a thickness of 0.4 mm are stacked, and three charging sections 120A, 120B, and 120C are provided on the side of one end in the longitudinal direction.
  • the outer edge is fusion-bonded by heat fusion except that two discharge parts 130A and 130B are provided on the edge side, the sheet is sandwiched between the two sheets, and the inner dimension is about 140 mm in width in an empty state,
  • a bag 100 (bag-like container) of the form shown in FIG. 1 was used, in which a generally rectangular receptacle having a length of about 196 mm was formed.
  • the internal volume of the receptacle of this bag is about 600 mL.
  • centrifuge tube As a centrifuge tube, a 50 mL centrifuge tube made of polypropylene was used.
  • the reaction mixture containing adipose tissue and collagenase solution is at most 210 mL, which is smaller than the internal volume.
  • the enzyme treatment bag containing the reaction mixture in the containing portion is partially sealed with a clamp, and the volume of the portion containing the reaction mixture in the containing portion of the enzyme treatment bag is the content Adjusted to approximate the volume of
  • the centrifuge tube contains 20 mL of adipose tissue and 20 mL of collagenase solution (dissolve with physiological saline so that the collagenase concentration is 0.1 w / v%, fat tissue and collagenase solution are 1: 1) and cover the lid. Closed and sealed.
  • the above enzyme treatment bag or centrifuge tube containing a mixture of adipose tissue and collagenase solution was placed on a shaker and shaken at 37 ° C. for 30 minutes under conditions of 60 rpm to carry out an enzyme reaction.
  • the cell suspension was recovered from the mixture in each container under the following conditions.
  • Condition 1 After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected.
  • 100% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed
  • the enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected.
  • 100% by volume of the initial amount of the enzyme solution (saline solution) is again supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution
  • the enzyme processing bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged to the discharge units 130A and 130B. Removed from one of the The aqueous layer recovered by the three recoverys was combined into one cell suspension.
  • 100% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed
  • the enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected. The aqueous layers recovered by the two recoverys were combined into one to form a cell suspension.
  • 50% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed
  • the enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected.
  • 50% by volume of the initial amount of the enzyme solution was again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction to form a mixed solution, and the mixed solution was stored.
  • 50% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed
  • the enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 5 minutes, and the aqueous layer (lower layer) after 5 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected.
  • 50% by volume of the initial amount of the enzyme solution was again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction to form a mixed solution, and the mixed solution was stored.
  • Condition 5 (comparative example): After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 30 minutes, and the aqueous layer (lower layer) after 30 minutes Were collected from one of the discharge units 130A and 130B and collected to obtain a cell suspension.
  • the volume of the recovered solution collected for the first time was measured, and the ratio (volume%) of the volume of the recovered solution to the volume of the initial amount of the enzyme solution was determined.
  • the number of cells in the cell suspension obtained under conditions 1 to 6 was measured using a flow cytometer and BD TrucountTubes.
  • the number of cells was calculated from the sum of the number of CD34 positive cells and the number of CD45 positive cells for human cells, and was measured using RetiticCount for rabbit cells.
  • the number of adipose-derived stem cells was calculated from the number of CD34 positive / CD45 negative / CD31 negative cells.
  • the number of cells was expressed as the number of cells per mL of the volume of adipose tissue used. The results are shown in Table 1.
  • Adipose tissue from human donor 4 was treated with collagenase to prepare a cell suspension under the following conditions.
  • the reaction mixture containing adipose tissue and collagenase solution is 40 mL, which is smaller than the internal volume.
  • the enzyme treatment bag containing the reaction mixture in the containing portion is partially sealed with a clamp, and the volume of the portion containing the reaction mixture in the containing portion of the enzyme treatment bag is the content Adjusted to approximate the volume of
  • the centrifuge tube contained 10 mL of adipose tissue and 30 mL of collagenase solution (see Experiment 1), and the lid was closed and sealed.
  • the above enzyme treatment bag or centrifuge tube containing a mixture of adipose tissue and collagenase solution was placed on a shaker and shaken at 37 ° C. for 30 minutes under conditions of 60 rpm to carry out an enzyme reaction.
  • the cell suspension was recovered from the mixture in each container under the following conditions.
  • Condition 11 After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected.
  • a washing solution (saline solution) equivalent to 20% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B, 120C and mixed with the remaining fraction and mixed
  • the enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B.
  • the aqueous layers recovered by the two recoverys were combined into one to form a cell suspension. The total volume of the aqueous layer recovered by the two rounds of recovery was taken as the total volume of recovered liquid.
  • Condition 12 After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected.
  • a washing solution (saline solution) equivalent to 20% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B, 120C and mixed with the remaining fraction and mixed
  • the enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B.
  • 6 mL of a washing solution (saline solution) equivalent to 20% by volume of the initial enzyme solution volume is again supplied into the storage unit via one of the input units 120A, 120B and 120C and mixed with the remaining fraction.
  • Condition 13 After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected.
  • a washing solution (saline solution) equivalent to 10% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction and mixed
  • the enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B.
  • the aqueous layers recovered by the two recoverys were combined into one to form a cell suspension. The total volume of the aqueous layer recovered by the two rounds of recovery was taken as the total volume of recovered liquid.
  • Condition 14 After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected.
  • 3 mL of a washing solution (saline solution) equivalent to 10% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction and mixed
  • the enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B.
  • 3 mL of a washing solution (saline solution) equivalent to 10% by volume of the initial enzyme solution volume is again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction.
  • Condition 15 (comparative example): After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 30 minutes, and the aqueous layer (lower layer) after 30 minutes Were collected from one of the discharge units 130A and 130B and collected to obtain a cell suspension. The amount of cell suspension collected was taken as the total collected liquid volume.
  • Condition 16 (comparative example): After 30 minutes of enzyme reaction, 12 mL of physiological saline is added to the enzyme treatment bag 100 containing the enzyme reaction solution and mixed, and the enzyme treatment bag 100 after mixing is such that the discharge parts 130A and 130B are vertically downward. The suspension was suspended for 30 minutes, and the aqueous layer (lower layer) after 30 minutes was taken out from one of the discharge parts 130A and 130B and collected to obtain a cell suspension. The amount of cell suspension collected was taken as the total collected liquid volume.
  • Condition 17 (comparative example): After the enzyme reaction for 30 minutes, the centrifuge tube containing the enzyme reaction solution was centrifuged under the conditions of 800 G for 10 minutes to recover the whole aqueous layer to obtain a cell suspension. The amount of cell suspension collected was taken as the total collected liquid volume.
  • the volume of the recovered solution collected for the first time was measured, and the ratio (volume%) of the volume of the recovered solution to the volume of the initial amount of the enzyme solution was determined.
  • the volume of the aqueous layer collected by centrifugation was measured, and the ratio (volume%) of the volume of the aqueous layer to the volume of the initial amount of enzyme liquid was determined.
  • the number of cells in the cell suspension obtained under conditions 1 to 17 was measured using a flow cytometer and BD TrucountTubes.
  • the number of cells was calculated from the sum of the number of CD34 positive cells and the number of CD45 positive cells for human cells, and was measured using RetiticCount for rabbit cells.
  • the number of adipose-derived stem cells was calculated from the number of CD34 positive / CD45 negative / CD31 negative cells.
  • the number of cells was expressed as the number of cells per mL of the volume of adipose tissue used. The results are shown in Table 2.

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Abstract

Provided is a method for enzymatically treating a biological tissue under low-load conditions for cells to separate and collect cells. The present invention relates to a method for separating and collecting cells from a biological tissue, the method including: step 1 of mixing the biological tissue with an enzyme solution in a volume of V1 in a container to perform an enzymatic treatment, thereby preparing an enzymatically treated solution containing free cells; step 2 of allowing the container to stand to cause the separation of the enzymatically treated solution into an oily layer and an aqueous layer until a point of time at which the volume of the aqueous layer does not exceed 0.93 V1; step 3 of collecting the aqueous layer from the container and causing the oily layer to remain in the container at the point of time mentioned in step 2; and step 4 of supplying a washing solution into the container, then mixing the resultant solution with the oily layer remaining in the container, then causing the resultant mixture to stand to cause the separation into an oily layer and an aqueous layer, and then collecting the aqueous layer from the container and causing the oily layer to remain in the container.

Description

生体組織からの細胞の分離回収方法Method of separating and collecting cells from living tissue
　本発明は、脂肪組織等の生体組織を酵素処理して細胞を分離させ回収する方法に関する。 The present invention relates to a method of enzymatically treating a living tissue such as adipose tissue to separate and recover cells.
　脂肪組織に含まれる有核細胞の少なくとも一部は生体組織幹細胞であり、それらは成熟脂肪細胞、骨細胞、軟骨細胞、筋芽細胞、血管内皮細胞等、様々な細胞へと分化可能であることが知られている。このような多分化能を有する脂肪由来生体組織幹細胞を効率良く分離・採取する方法は、再生医療発展の見地から極めて重要である。 At least a part of nucleated cells contained in adipose tissue are living tissue stem cells, which can be differentiated into various cells such as mature adipocytes, bone cells, chondrocytes, myoblasts, vascular endothelial cells, etc. It has been known. A method for efficiently separating and collecting such pluripotent adipose-derived living tissue stem cells is extremely important from the viewpoint of regenerative medicine development.
　脂肪組織等の、有用細胞を含む生体組織から、有用細胞を取得する方法として、消化酵素で生体組織を分解して有用細胞を遊離させ、その後に遠心分離、濾過等の分離工程を経て有用細胞を回収する方法が知られている。 As a method of obtaining useful cells from living tissue including useful cells such as adipose tissue, the living tissue is degraded with digestive enzymes to release useful cells, and then the cells are subjected to separation steps such as centrifugation, filtration and the like to obtain useful cells. Methods for recovering are known.
　例えば特許文献１の実施例１では、５０ｍＬ容の遠心チューブに、脂肪組織とコラゲナーゼ液とを収容し、３７℃、１２０回／ｍｉｎの条件で１時間振盪させて酵素反応を行い、その後にフィルターろ過及び遠心分離を行い、沈渣として沈降細胞集団（ＳＶＦ画分）を取得することが記載されている。 For example, in Example 1 of Patent Document 1, an adipose tissue and a collagenase solution are accommodated in a 50 mL centrifugal tube, shaken at 37 ° C., 120 times / min for 1 hour to perform an enzyme reaction, and then a filter It is described that filtration and centrifugation are performed to obtain a precipitated cell population (SVF fraction) as sediment.
　特許文献２には、脂肪組織試料から非脂肪細胞を分離するための装置として、第１シートの物質と、前記第１シートの物質に結合された第２シートの物質と、前記第１シートの物質と前記第２シートの物質との間で画成された複数のチャンバとを備える装置が開示されている。そして、複数のチャンバの１つである第１チャンバ内で、脂肪組織試料を、コラゲナーゼ等の酵素を含む解離溶液により処理して、脂肪組織試料を解離することが開示されている。 According to Patent Document 2, as a device for separating non-fat cells from a fat tissue sample, a substance of a first sheet, a substance of a second sheet bonded to the substance of the first sheet, and a substance of the first sheet An apparatus is disclosed comprising a plurality of chambers defined between a substance and the substance of the second sheet. Then, it is disclosed that the adipose tissue sample is treated with a dissociation solution containing an enzyme such as collagenase to dissociate the adipose tissue sample in a first chamber which is one of a plurality of chambers.
国際公開第２００８／０１８４５０号International Publication No. 2008/018450 特表２０１５－５０００３１号公報Japanese Patent Publication No. 2015-500031
　生体組織から、細胞を遊離させるために、生体組織を酵素処理する方法が従来から行われている。 In order to release cells from living tissue, methods for enzymatic treatment of living tissue have been conventionally performed.
　例えば、特許文献１に記載されているように、生体組織の酵素処後に遠心分離を行い、有用細胞を含む水層と、脂質等の油溶成分を含む油層に分離し、水層を回収する方法が公知である。しかしながら、遠心分離は細胞に対する負荷が大きいため、細胞が損傷し易く、有用細胞を回収する目的では好ましいものではない。 For example, as described in Patent Document 1, centrifugation is performed after enzymatic treatment of a living tissue to separate it into an aqueous layer containing useful cells and an oil layer containing oil-soluble components such as lipids, and the aqueous layer is recovered. Methods are known. However, centrifugation has a large load on cells, so the cells are easily damaged and is not preferable for the purpose of recovering useful cells.
　一方、脂肪組織等の生体組織を酵素処理して細胞を遊離させた酵素処理液が、細胞を含む水層と、脂質を含む油層とに完全に二層分離するには、長時間を要することが通常である。遠心分離を用いずに生体組織の酵素処理液から細胞を含む水層を回収しようとすると、長時間を要し、細胞に負荷がかかり易い。 On the other hand, it takes a long time to completely separate an aqueous solution containing cells and an oil layer containing lipids completely from an enzyme-treated solution prepared by enzymatic treatment of living tissues such as fat tissue to release cells. Is normal. If it is attempted to recover the aqueous layer containing cells from the enzyme-treated solution of a living tissue without using centrifugation, it takes a long time and the cells are likely to be loaded.
　本発明者らは、細胞に対し低負荷条件で、生体組織を酵素処理し細胞を分離し回収する方法の提供を目的として鋭意検討した結果、以下の発明を完成するに至った。
（１）生体組織から細胞を分離し回収する方法であって、
　容器内で、生体組織と体積Ｖ１の酵素液との混合物中で生体組織の酵素処理を行い、遊離細胞を含む酵素処理液を形成する工程１と、
　工程１後に酵素処理液を収容した容器を静置し、酵素処理液を油層と水層に、水層の体積が０．９３Ｖ１を超えない時点まで分離させる工程２と、
　前記時点で、容器から水層を回収するとともに油層を容器内に残留させる工程３と、
　洗浄液を容器内に供給し、容器内に残留した油層と混合して混合液を形成した後、容器を静置することで混合液を油層と水層とに分離させ、容器から水層を回収するとともに油層を容器内に残留させる工程４と
を含み、工程４を１回以上行うことを特徴とする方法。
（２）生体組織が脂肪組織である、（１）に記載の方法。
（３）工程４での洗浄液の体積が０．１０Ｖ１以上である、（１）又は（２）に記載の方法。
（４）工程４での静置時間が、工程２での静置時間の１／６倍以上の時間である、（１）～（３）のいずれかに記載の方法。
（５）工程２での静置時間が３分間以上３０分間以下である、（１）～（４）のいずれかに記載の方法。
（６）工程４での静置時間が３分間以上３０分間以下である、（１）～（５）のいずれかに記載の方法。
（７）容器が、液体の排出口が形成された容器であり、
　工程３及び４における水層の回収が、水層に対し鉛直下方に排出口が位置するように容器を配置し、排出口を通じて水層を回収することを含む、（１）～（６）のいずれかに記載の方法。
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０１７－１８０１７８号の開示内容を包含する。 The present inventors came to complete the following inventions as a result of earnestly examining for the purpose of providing the method of carrying out the enzyme processing of a biological tissue, and isolate | separating and collect | recovering cells with low load conditions with respect to cells.
(1) A method for separating and recovering cells from a living tissue, which comprises:
Carrying out the enzyme treatment of the living tissue in a mixture of the living tissue and the enzyme solution of volume V 1 in a container to form an enzyme-treated solution containing free cells;
And 2. leaving the container containing the enzyme-treated solution after step 1 and separating the enzyme-treated solution into an oil layer and an aqueous layer until the volume of the aqueous layer does not exceed 0.93 V 1 ;
Recovering the aqueous layer from the container and leaving the oil layer in the container at the time point;
The cleaning solution is supplied into the container and mixed with the oil layer remaining in the container to form a mixture, and then the mixture is allowed to stand to separate the mixture into an oil layer and an aqueous layer, and the water layer is recovered from the container And 4) leaving the oil layer in the container, and performing step 4 one or more times.
(2) The method according to (1), wherein the living tissue is fat tissue.
(3) The method according to (1) or (2), wherein the volume of the washing solution in step 4 is 0.10 V 1 or more.
(4) The method according to any one of (1) to (3), wherein the standing time in step 4 is 1/6 or more times the settling time in step 2.
(5) The method according to any one of (1) to (4), wherein the standing time in step 2 is 3 minutes or more and 30 minutes or less.
(6) The method according to any one of (1) to (5), wherein the standing time in step 4 is 3 minutes or more and 30 minutes or less.
(7) The container is a container in which a liquid outlet is formed,
The recovery of the aqueous layer in steps 3 and 4 includes disposing the container so that the outlet is positioned vertically below the aqueous layer, and recovering the aqueous layer through the outlet, (1) to (6) The method described in either.
The present specification includes the disclosure content of Japanese Patent Application No. 2017-180178 based on which the priority of the present application is based.
　本発明の方法によれば、細胞に対し低負荷条件で、生体組織を酵素処理し細胞を分離し回収することができる。 According to the method of the present invention, living tissue can be subjected to enzyme treatment to separate and recover cells under low load conditions for cells.
生体組織の酵素処理を行うための容器の一実施形態を示す。1 illustrates one embodiment of a container for enzymatic treatment of biological tissue. 生体組織の酵素処理液からの水層の回収の手順を説明するための模式図である。It is a schematic diagram for demonstrating the procedure of collection | recovery of the water layer from the enzyme processing liquid of a biological tissue. 回収された細胞含有水層を濾過し貯留するための濾過貯留デバイスの一実施形態を示す。FIG. 7 illustrates one embodiment of a filtration and storage device for filtering and storing the recovered cell-containing aqueous layer.
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
＜用語、材料＞
　生体組織は典型的には動物より採取した生体組織であり、例えば、脂肪、皮膚、血管、角膜、口腔、腎臓、肝臓、膵臓、心臓、神経、筋肉、前立腺、腸、羊膜、胎盤、臍帯などに由来する生体組織が挙げられる。本明細書で開示する方法は特に脂肪組織から間質血管画分（ＳＶＦ）細胞を取り出すのに有用である。 <Terms, Materials>
The biological tissue is typically a biological tissue collected from an animal, such as fat, skin, blood vessels, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine, amniotic membrane, placenta, umbilical cord, etc. And biological tissues derived from The methods disclosed herein are particularly useful for removing interstitial vascular fraction (SVF) cells from adipose tissue.
　脂肪組織とは典型的には哺乳動物の脂肪組織であり、例えば、ヒト由来の皮下脂肪、内臓脂肪、白色脂肪、褐色脂肪である。脂肪組織は、任意の形状であってよく、例えば、脂肪組織をハサミ等の鋭利な器具を用いて破砕したもの、濾し器等を用いてミンチ状にしたもの、脂肪吸引法を用いて分解したものであってよい。ここで脂肪吸引法とは、一般的な美容成形外科で行なわれている吸引法であれば特に限定されず、例えば、超音波脂肪吸引、カニューレ等を用いたパワードリポサクション、シリンジ吸引等による方法である。 The adipose tissue is typically mammalian adipose tissue, such as subcutaneous fat of human origin, visceral fat, white fat and brown fat. Adipose tissue may have any shape, for example, one obtained by crushing adipose tissue using a sharp instrument such as scissors, one obtained by mincing using a filter, etc., or one obtained by decomposition using liposuction. It may be Here, the liposuction method is not particularly limited as long as it is a suction method performed in general cosmetic surgery, and for example, a method by ultrasonic liposuction, powered liposome suction using a cannula or the like, syringe suction, etc. It is.
　本発明において遊離される細胞としては、生体組織幹細胞、白血球、単球、顆粒球、リンパ球、血管内皮細胞、血管内皮前駆細胞、周細胞等、細胞治療や実験等の目的で採取が必要とされる有核細胞が例示できる。生体組織幹細胞は、好ましくは、脂肪由来間葉系幹細胞、脂肪由来間質幹細胞であり、より好ましくは細胞表面のＣＤ３４、７３、９０、１０５、１０６、１３３、１６６から選ばれる少なくとも一つを発現している脂肪由来間葉系細胞、脂肪由来間質幹細胞である。 In the present invention, cells to be released in the present invention include stem cells, white blood cells, monocytes, granulocytes, lymphocytes, vascular endothelial cells, vascular endothelial precursor cells, pericytes, etc., which need to be collected for cell therapy and experiments. Nucleated cells can be exemplified. The living tissue stem cells are preferably fat-derived mesenchymal stem cells, fat-derived stromal stem cells, and more preferably express at least one selected from CD34, 73, 90, 105, 106, 133, 166 on the cell surface Fat-derived mesenchymal cells, fat-derived stromal stem cells.
　生体組織から細胞を遊離させるための酵素としては、コラゲナーゼ、メタロプロテアーゼ、ディスパーゼ、トリプシン、ヒアルロニダーゼ、キモトリプシン、ペプシン、アミノペプチダーゼ、リパーゼ、アミラーゼ及びそれらのリコンビナントから選ばれる少なくとも１種の分解酵素が使用できる。生体組織として脂肪組織を用いる場合には、酵素としては、短時間に、かつ低侵襲で分解するという観点から、コラゲナーゼ、メタロプロテアーゼ、ディスパーゼ、トリプシン及びヒアルロニダーゼから選択される少なくとも１種の分解酵素が好ましい。 As an enzyme for releasing cells from a living tissue, at least one degradation enzyme selected from collagenase, metalloprotease, dispase, trypsin, hyaluronidase, chymotrypsin, pepsin, aminopeptidase, lipase, amylase and their recombinants can be used. . When fat tissue is used as a living tissue, at least one degradable enzyme selected from collagenase, metalloprotease, dispase, trypsin and hyaluronidase from the viewpoint of degradation in a short time and with low invasiveness as an enzyme preferable.
　酵素を溶解または懸濁して酵素液を調製するための媒体、及び／又は、工程４で用いる洗浄液としては、水、生理食塩水、リン酸緩衝液、ブドウ糖液、リンゲル液、ハンクス液、注射溶液、培地、等張液等の水系媒体を用いることができる。 Water, saline, phosphate buffer, glucose solution, Ringer's solution, Hanks solution, injection solution, as a medium for dissolving or suspending the enzyme to prepare an enzyme solution and / or a washing solution used in step 4 An aqueous medium such as a culture medium or an isotonic solution can be used.
＜容器＞
　本発明の方法で酵素処理及び層分離に用いる容器（以下「処理容器」と称する場合がある）は、液体を収容することができる容器であれば特に限定されない。 <Container>
The container used for the enzyme treatment and the layer separation in the method of the present invention (hereinafter sometimes referred to as "processing container") is not particularly limited as long as it is a container capable of containing a liquid.
　本発明の方法は遠心分離を必要としないため、袋状容器のような、一般的には遠心分離処理が難しい形状の容器であっても使用することができる。 Since the method of the present invention does not require centrifugation, it is possible to use even containers that are generally difficult to centrifuge, such as bag-like containers.
　処理容器は、好ましくは、液体を排出するための排出口が形成された容器である。この場合、排出口を通じて水層を回収することが容易である。また、排出口を、回収しようとする水層の鉛直下方に位置させ、排出口を開放すると、重力により水層が排出されるため、ピペット等を用いて上方から水層を回収する方法と比べて油層と水層の界面を乱さず細胞に高負荷をかけずに水層を回収することが容易である。 The processing container is preferably a container in which a discharge port for discharging a liquid is formed. In this case, it is easy to recover the water layer through the outlet. In addition, when the discharge port is positioned vertically below the water layer to be collected and the discharge port is opened, the water layer is discharged by gravity, and this is compared to a method of collecting the water layer from the top using a pipette etc. Thus, it is easy to recover the aqueous layer without disturbing the interface between the oil layer and the aqueous layer and without applying a high load to the cells.
　処理容器は少なくとも一部が透明又は半透明であり、酵素処理液の層分離状態が確認できるものであることが好ましい。 It is preferable that at least a part of the processing container is transparent or translucent, and that the layer separation state of the enzyme processing solution can be confirmed.
　処理容器は、より好ましくは、柔軟性（可撓性）を有する樹脂のシートにより壁面が形成された袋状容器であり、特に好ましくは、柔軟性を有する樹脂シートを２枚重ねて対向配置し、周縁部を熱融着又は接着剤により接合して袋状に形成した容器である。 The processing container is more preferably a bag-like container having a wall surface formed of a flexible (flexible) resin sheet, and particularly preferably two opposing resin sheets having flexibility are disposed in an opposing manner. The container is formed in a bag-like shape by bonding the peripheral portion by heat fusion or an adhesive.
　袋状容器である処理容器の具体例である処理容器１００を、図１を参照して説明する。 A processing container 100 which is a specific example of a processing container which is a bag-like container will be described with reference to FIG.
　処理容器１００は、第１空間１１４を内包する容器本体１１０と、第１空間１１４への材料の投入口１２１Ａ、１２１Ｂ、１２１Ｃが形成された、投入部１２０Ａ、投入部１２０Ｂ、投入部１２０Ｃからなる投入部１２０と、第１空間１１４からの液体の排出口１３１Ａ、１３１Ｂが形成された、排出部１３０Ａ、排出部１３０Ｂからなる排出部１３０とを備える。 The processing container 100 includes a container main body 110 including the first space 114, and an input portion 120A, an input portion 120B, and an input portion 120C in which material inlets 121A, 121B, and 121C to the first space 114 are formed. The discharge unit 130 includes a discharge unit 130A and a discharge unit 130B. The discharge unit 130A includes a discharge unit 130 and discharge openings 131A and 131B of the liquid from the first space 114.
　投入部１２０Ａは、第１空間１１４への材料の投入口１２１Ａを形成する管部１２２Ａと、管部１２２Ａの入口を連通可能に封鎖するメスルアーロック１２３Ａと、メスルアーロック１２３Ａを保護する着脱自在の蓋部１２４Ａと、管部１２２Ａを通る液体を濾過するフィルター部１２５Ａとを備える。 The input portion 120A includes a pipe portion 122A that forms an input port 121A of the material into the first space 114, a female luer lock 123A that seals the inlet of the pipe portion 122A in a manner that allows communication with each other, and a removable type And a filter portion 125A for filtering the liquid passing through the pipe portion 122A.
　投入部１２０Ｂは、第１空間１１４への材料の投入口１２１Ｂを形成する管部１２２Ｂと、管部１２２Ｂの入口を連通可能に封鎖するニードルレスポート１２３Ｂと、ニードルレスポート１２３Ｂを保護する着脱自在の蓋部１２４Ｂとを備える。 The input portion 120B includes a pipe portion 122B that forms an input port 121B of the material into the first space 114, a needleless port 123B that blocks the inlet of the pipe portion 122B in communication, and a removable type that protects the needleless port 123B. And the lid portion 124B of the
　投入部１２０Ｃは、第１空間１１４への材料の投入口１２１Ｃを形成する管部１２２Ｃと、管部１２２Ｃの入口を連通可能に封鎖するニードルレスポート１２３Ｃと、ニードルレスポート１２３Ｃを保護する着脱自在の蓋部１２４Ｃとを備える。 The input portion 120C includes a pipe portion 122C that forms an input port 121C of the material into the first space 114, a needleless port 123C that blocks the inlet of the pipe portion 122C in communication with each other, and a removable port that protects the needleless port 123C. And a cover 124C of
　排出部１３０Ａは、第１空間１１４からの液体の排出口１３１Ａを形成する管部１３２Ａと、管部１３２Ａの出口を連通可能に封鎖するニードルレスポート１３３Ａと、ニードルレスポート１３３Ａを保護する着脱自在の蓋部１３４Ａとを備える。 The discharge part 130A includes a pipe part 132A which forms a discharge port 131A of the liquid from the first space 114, a needleless port 133A which closes the outlet of the pipe part 132A in a communicating manner, and a removable part which protects the needleless port 133A. And a lid 134A.
　排出部１３０Ｂは、第１空間１１４からの液体の排出口１３１Ｂを形成する管部１３２Ｂと、管部１３２Ｂの出口を連通可能に封鎖するニードルレスポート１３３Ｂと、ニードルレスポート１３３Ｂを保護する着脱自在の蓋部１３４Ｂとを備える。 The discharge unit 130B includes a pipe portion 132B that forms a discharge port 131B of the liquid from the first space 114, a needleless port 133B that seals the outlet of the pipe portion 132B in a communicating manner, and a removable port that protects the needleless port 133B. And a lid 134B.
　本実施形態では、処理容器１００は投入部１２０を３つ備えるが、１つのみ、２つのみまたは４つ以上の投入部を備えてもよい。投入部１２０は、処理容器１００の第１空間１１４での生体組織の酵素処理に用いる材料（例えば生体組織、酵素液等）、或いは、洗浄液を第１空間１１４に投入するために用いられる。また、投入部と排出部を別々に備えている必要はなく、投入部を備えず、後述する排出部が投入部として兼用できるように構成されていてもよい。 In the present embodiment, the processing container 100 is provided with three input units 120, but may be provided with only one, only two, or four or more input units. The input unit 120 is used to input, to the first space 114, a material (for example, a living tissue, an enzyme solution, etc.) used for enzyme treatment of a living tissue in the first space 114 of the processing container 100 or a cleaning solution. Further, the input unit and the discharge unit do not need to be separately provided. The input unit may not be provided, and a discharge unit described later may be used as the input unit.
　本実施形態では、処理容器１００は、排出部１３０を２つ備えるが、１つのみ又は３つ以上の排出部を備えてもよい。排出部１３０は、生体組織を酵素処理して形成される遊離細胞を含む酵素処理液の少なくとも一部を第１空間１１４から排出するために用いられる。 In the present embodiment, the processing container 100 includes two discharge units 130, but may include only one or three or more discharge units. The discharge unit 130 is used to discharge, from the first space 114, at least a part of an enzyme treatment solution containing free cells formed by enzyme treatment of a living tissue.
　容器本体１１０は、第１樹脂シート１１１と第２樹脂シート１１２とを対向配置し、周縁部を熱融着により接合して袋体としたものである。第１樹脂シート１１１と第２樹脂シート１１２が熱融着により接合した部分を融着部１１３とする。容器本体１１０には、第１樹脂シート１１１と第２樹脂シート１１２との間に第１空間１１４が形成されている。なお、図１は、第１空間１１４が空の状態の処理容器１００の平面模式図であり、図示しないが、第１空間１１４に生体組織及び酵素を含む反応混合液が収容されると、容器本体１１０は袋状に膨張する。第１空間１１４において生体組織の酵素処理が行われる。投入部１２０Ａ、１２０Ｂ、１２０Ｃの管部１２２Ａ、１２２Ｂ、１２２Ｃは、それらの一方の端部が、容器本体１１０の長手方向の一端の辺上の、第１樹脂シート１１１と第２樹脂シート１１２との間に配置され、その周りの第１樹脂シート１１１と第２樹脂シート１１２が熱融着により接合されて、容器本体１１０と一体化されている。同様に、排出部１３０Ａ、１３０Ｂの管部１３２Ａ、１３２Ｂは、それらの一方の端部が、容器本体１１０の長手方向の他端の辺上の、第１樹脂シート１１１と第２樹脂シート１１２との間に配置され、その周りの第１樹脂シート１１１と第２樹脂シート１１２が熱融着により接合されて、容器本体１１０と一体化されている。 The container body 110 has the first resin sheet 111 and the second resin sheet 112 disposed opposite to each other, and peripheral portions thereof are joined by heat fusion to form a bag. A portion where the first resin sheet 111 and the second resin sheet 112 are joined by heat fusion is referred to as a fusion part 113. In the container body 110, a first space 114 is formed between the first resin sheet 111 and the second resin sheet 112. FIG. 1 is a schematic plan view of the processing container 100 in which the first space 114 is empty, and although not shown, when the reaction mixture containing biological tissue and enzymes is accommodated in the first space 114, the container is not shown. The main body 110 expands in a bag shape. In the first space 114, enzyme treatment of a living tissue is performed. The first resin sheet 111 and the second resin sheet 112 of the pipe sections 122A, 122B, 122C of the input sections 120A, 120B, 120C have one end thereof on the side of one end of the container body 110 in the longitudinal direction. The first resin sheet 111 and the second resin sheet 112 are joined by heat fusion to be integrated with the container body 110. Similarly, in the pipe sections 132A and 132B of the discharge sections 130A and 130B, the first resin sheet 111 and the second resin sheet 112 are disposed at one end of the pipe sections 132A and 132B on the other end of the container body 110 in the longitudinal direction. The first resin sheet 111 and the second resin sheet 112 are joined by heat fusion to be integrated with the container body 110.
　本実施形態では容器本体１１０の投入部１２０が配置される側の辺の両端の、融着部１１３よりも外側の部分に、貫通孔１１５、１１５が形成されている。貫通孔１１５はフックなどに掛けるために用いることができる。 In the present embodiment, through holes 115, 115 are formed in portions outside the fusion bonded portion 113 on both sides of the side of the container main body 110 where the input portion 120 is disposed. The through hole 115 can be used to hook on a hook or the like.
　第１樹脂シート１１１及び第２樹脂シート１１２並びに各管部１２２Ａ、１２２Ｂ、１２２Ｃ、１３２Ａ、１３２Ｂは、柔軟性を有する材料により形成されることが好ましい。柔軟性を有する材料の具体例としては、軟質塩化ビニル、塩化ビニル、ポリウレタン、エチレン－酢酸ビニル共重合体、ポリエチレンやポリプロピレンのようなポリオレフィン、スチレン－ブタジエン－スチレン共重合体又はその水添物及びスチレン－イソプレン－スチレン共重合体またはその水添物等の熱可塑性エラストマー、ならびに、熱可塑性エラストマーとポリオレフィン及びエチレン－エチルアクリレート等の軟化剤との混合物等の、柔軟性を有する樹脂が挙げられる。第１樹脂シート１１１及び第２樹脂シート１１２は、少なくとも一部を透明または半透明の材料からなるものとすることが好ましい。第１樹脂シート１１１及び第２樹脂シート１１２の内側面は、ナシジ加工されていると、水層の排出時に残液を少なくすることができるため好ましい。 It is preferable that the first resin sheet 111 and the second resin sheet 112 and the respective tube portions 122A, 122B, 122C, 132A, and 132B be formed of a material having flexibility. Specific examples of the material having flexibility include soft vinyl chloride, vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin such as polyethylene and polypropylene, styrene-butadiene-styrene copolymer or hydrogenated product thereof Examples thereof include flexible resins such as thermoplastic elastomers such as styrene-isoprene-styrene copolymer or a hydrogenated product thereof and mixtures of thermoplastic elastomers and softeners such as polyolefin and ethylene-ethyl acrylate. It is preferable that at least a part of the first resin sheet 111 and the second resin sheet 112 be made of a transparent or translucent material. It is preferable that the inner side surfaces of the first resin sheet 111 and the second resin sheet 112 be subjected to a sashi process because residual liquid can be reduced when the water layer is discharged.
＜生体組織から細胞を分離し回収する方法＞
　本発明の方法の工程１～４の具体的な一実施形態を、図２を参照して説明する。図２は、処理容器として図１に示す処理容器１００を例示するが、他の容器も使用できることは上述の通りである。 <Method for separating and recovering cells from living tissue>
One specific embodiment of steps 1 to 4 of the method of the present invention will be described with reference to FIG. Although FIG. 2 exemplifies the processing container 100 shown in FIG. 1 as a processing container, as described above, other containers can also be used.
　工程１は、容器内で、生体組織と体積Ｖ１の酵素液との混合物中で生体組織の酵素処理を行い、遊離細胞を含む酵素処理液を形成する工程である。 Step 1, in a vessel, subjected to the enzymatic treatment of the living tissue in a mixture of an enzyme solution of the biological tissue and the volume V 1, a step of forming an enzyme treatment solution containing free cells.
　生体組織及び酵素の具体例は既述の通りである。 Specific examples of living tissues and enzymes are as described above.
　「酵素液」は、酵素が水系媒体中に溶解又は懸濁した液を指す。酵素液は、予め酵素と水系媒体とを全量混合して酵素液としてから容器に投入する必要はなく、酵素、水系媒体を別々に容器に投入し、容器内で酵素液を形成してもよい。このため酵素液の体積Ｖ１は、酵素処理を行うために容器内に投入された酵素、酵素液、水系媒体の体積を合計して算出することができる。 An "enzyme solution" refers to a solution in which an enzyme is dissolved or suspended in an aqueous medium. The enzyme solution does not have to be mixed in advance with the whole amount of the enzyme and the aqueous medium to form an enzyme solution, and then the enzyme solution and the aqueous medium may be separately charged into the container to form the enzyme solution in the container. . Therefore the volume V 1 of the enzyme solution can be calculated by summing enzyme loaded into the container in order to carry out the enzyme treatment, the enzyme solution, the volume of the aqueous medium.
　生体組織と酵素液との体積比は、特に限定されないが、工程２、工程４での層分離のためには、生体組織の体積に対して、酵素液の体積Ｖ１が、好ましくは３０％～５００％、より好ましくは３０％～３００％、より好ましくは５０％～２００％、より好ましくは７０％～１３０％である。 The volume ratio of the living tissue to the enzyme solution is not particularly limited, but for layer separation in Step 2 and Step 4, the volume V 1 of the enzyme solution is preferably 30% with respect to the volume of the living tissue. It is about -500%, more preferably 30% to 300%, more preferably 50% to 200%, more preferably 70% to 130%.
　工程１では、生体組織と酵素液を含む混合物を収容した容器を振盪させるか容器内で混合物を撹拌することにより、生体組織の酵素処理を促進することができる。酵素処理の温度、時間は適宜調節することができる。 In the step 1, the enzyme treatment of the living tissue can be promoted by shaking the container containing the mixture containing the living tissue and the enzyme solution or stirring the mixture in the container. The temperature and time of the enzyme treatment can be adjusted appropriately.
　工程１により、遊離細胞を含む酵素処理液Ａが容器内に形成される（図２（Ａ）参照）。 According to step 1, an enzyme treatment solution A containing free cells is formed in the container (see FIG. 2 (A)).
　工程２は、工程１後に酵素処理液を収容した容器を静置し、酵素処理液を油層と水層に、水層の体積が０．９３Ｖ１を超えない時点まで分離させる工程である。図２（Ｂ）に示すように、工程１で形成された生体組織の酵素処理液は、脂質等の成分を含む油層Ｂと、水系媒体中に遊離細胞を含む水層Ｃとに、時間経過とともに層分離する。生体組織が脂肪組織である場合、脂肪細胞は油層Ｂに含まれ、脂肪由来幹細胞は水層Ｃに含まれる。工程１終了後は酵素処理液Ａとして全体が濁った状態であったものが、時間経過とともに油層Ｂと水層Ｃがそれぞれ明確になり、最終的には境界が明確な二層に分離する。完全に二層分離したときの水層の体積は、酵素液量Ｖ１に近い体積となる。しかし二層に完全に分離するには長時間を要するため、その間に細胞がダメージを受けるという問題がある。そこで本発明の工程２では、容器中の水層の体積がＶ１となるまで待たず、水層の体積が０．９３Ｖ１を超えない時点まで分離させたら、工程３において水層を回収することで、工程２での静置時間を短縮し、細胞への負荷を軽減する。工程２において「水層の体積が０．９３Ｖ１を超えない時点」は、より好ましくは「水層の体積が０．９２Ｖ１を超えない時点」であり、より好ましくは「水層の体積が０．９１Ｖ１を超えない時点」であり、より好ましくは「水層の体積が０．９０Ｖ１を超えない時点」であり、より好ましくは「水層の体積が０．８９Ｖ１を超えない時点」であり、より好ましくは「水層の体積が０．７０Ｖ１を超えない時点」である。「水層の体積が０．９３Ｖ１（或いは、０．９２Ｖ１、０．９１Ｖ１、０．９０Ｖ１、０．８９Ｖ１、０．７０Ｖ１）を超えない時点」とは、水層の体積が好ましくは０．２０Ｖ１以上０．９３Ｖ１（或いは、０．９２Ｖ１、０．９１Ｖ１、０．９０Ｖ１、０．８９Ｖ１、０．７０Ｖ１）以下、好ましくは０．３０Ｖ１以上０．９３（或いは、０．９２Ｖ１、０．９１Ｖ１、０．９０Ｖ１、０．８９Ｖ１、０．７０Ｖ１）Ｖ１以下、より好ましくは０．４０Ｖ１以上０．９３Ｖ１（或いは、０．９２Ｖ１、０．９１Ｖ１、０．９０Ｖ１、０．８９Ｖ１、０．７０Ｖ１）以下、より好ましくは０．５０Ｖ１以上０．９３Ｖ１（或いは、０．９２Ｖ１、０．９１Ｖ１、０．９０Ｖ１、０．８９Ｖ１、０．７０Ｖ１）以下となる時点である。 Step 2 is to stand a container containing the enzyme treatment solution after the step 1, the enzyme treatment solution into an oil layer and an aqueous layer, the volume of the aqueous layer is a step of separating to the point not exceeding 0.93V 1. As shown in FIG. 2 (B), the enzyme-treated solution of the living tissue formed in step 1 is time-lapsed to an oil layer B containing components such as lipids and an aqueous layer C containing free cells in an aqueous medium. Separate with the When the living tissue is fat tissue, fat cells are contained in oil layer B, and fat-derived stem cells are contained in water layer C. After the completion of step 1, although the whole of the enzyme treatment liquid A is in a turbid state, the oil layer B and the water layer C become clear as time passes, and finally they are separated into two layers with clear boundaries. Volume of water layer when fully separated two layers, the volume close to the enzyme solution volume V 1. However, since it takes a long time to completely separate into two layers, there is a problem that cells are damaged during that time. Therefore, in step 2 of the present invention, without waiting until the volume of aqueous phase in the container is V 1, Once separated until such time as the volume of the aqueous layer does not exceed 0.93V 1, recovering the aqueous layer in the step 3 This shortens the standing time in step 2 and reduces the load on cells. In step 2, “when the volume of the aqueous layer does not exceed 0.93 V 1 ” is more preferably “when the volume of the aqueous layer does not exceed 0.92 V 1 ”, more preferably “the volume of the aqueous layer is a point "not exceeding 0.91 V 1, more preferably from" the time when the volume of the aqueous layer does not exceed 0.90 V 1 ", more preferably the volume of the" water layer does not exceed 0.89 V 1 time And more preferably "when the volume of the aqueous layer does not exceed 0.70 V 1 ". The "volume of the aqueous layer 0.93V 1 (or, 0.92V 1, 0.91V 1, 0.90V 1, 0.89V 1, 0.70V 1) time not exceeding" the volume of the aqueous layer is preferably 0.20 V 1 or 0.93V 1 (or, 0.92V 1, 0.91V 1, 0.90V 1, 0.89V 1, 0.70V 1) or less, preferably 0.30 V 1 or 0 .93 (or, 0.92V 1, 0.91V 1, 0.90V 1, 0.89V 1, 0.70V 1) V 1 or less, 0.93V 1 (or more preferably 0.40 V 1 or more, 0 .92V 1, 0.91V 1, 0.90V 1 , 0.89V 1, 0.70V 1) or less, more preferably 0.50 V 1 or 0.93V 1 (or, 0.92 V 1, 0.91 V 1 , 0.90 V 1 , 0.89 V 1 , 0 .70V 1 ) or less.
　工程２のより好ましい実施形態では、工程１において生体組織の体積に対して酵素液の体積Ｖ１が２００％以下である場合、工程２の「水層の体積が０．９３Ｖ１を超えない時点」は「水層の体積が０．７０Ｖ１を超えない時点」であり、工程１において生体組織の体積に対して酵素液の体積Ｖ１が２００％を超える場合、工程２の「水層の体積が０．９３Ｖ１を超えない時点」は、「水層の体積が０．９３Ｖ１を超えない時点」、「水層の体積が０．９２Ｖ１を超えない時点」、「水層の体積が０．９１Ｖ１を超えない時点」、「水層の体積が０．９０Ｖ１を超えない時点」、又は「水層の体積が０．８９Ｖ１を超えない時点」である。 Time In a more preferred embodiment of step 2, if the volume V 1 of the enzyme solution by volume of the biological tissue in step 1 is not more than 200%, of the step 2 the volume of "water layer does not exceed 0.93V 1 "the volume of the" water layer is point "not exceeding 0.70 V 1, if the volume V 1 of the enzyme solution by volume of the biological tissue in step 1 is greater than 200%, step 2" of the aqueous layer “When the volume does not exceed 0.93 V 1 ” means “when the volume of the aqueous layer does not exceed 0.93 V 1 ”, “when the volume of the aqueous layer does not exceed 0.92 V 1 ”, “the volume of the aqueous layer Is not greater than 0.91 V 1 , “when the volume of the aqueous layer does not exceed 0.90 V 1 ”, or “when the volume of the aqueous layer does not exceed 0.89 V 1 ”.
　工程２での静置時間は３分間以上３０分間以下であることが好ましく、５分間以上１５分間以下であることが特に好ましい。工程２での静置時間をこの範囲とすることで、細胞への負荷を抑制しつつ水層と油層を分離することが容易となる。 The standing time in step 2 is preferably 3 minutes to 30 minutes, and more preferably 5 minutes to 15 minutes. By setting the standing time in step 2 to this range, it becomes easy to separate the water layer and the oil layer while suppressing the load on the cells.
　工程３は、工程２において水層の体積が０．９３Ｖ１を超えない程度にまで分離した時点で、容器から水層を回収するとともに油層を容器内に残留させる工程である。図２（Ｂ）は、工程２において油層Ｂと水層Ｃ（体積は０．９３Ｖ１以下、好ましくは０．９２Ｖ１以下、好ましくは０．９１Ｖ１以下、好ましくは０．９０Ｖ１以下、好ましくは０．８９Ｖ１以下、好ましくは０．７０Ｖ１以下とする）に分離した状態を示し、図２（Ｃ）は、工程３において、処理容器１００から水層Ｃを回収するとともに油層Ｂを容器１００内に残留させた状態を示す。 Step 3 is a step of recovering the aqueous layer from the container and leaving the oil layer in the container when the volume of the aqueous layer is separated to a level not exceeding 0.93 V 1 in step 2. FIG. 2B shows that the oil layer B and the aqueous layer C (volume is 0.93 V 1 or less, preferably 0.92 V 1 or less, preferably 0.91 V 1 or less, preferably 0.90 V 1 or less, in step 2) 0.89 V 1 or less, preferably an separated state to a 0.70 V 1 or less), FIG. 2 (C), in step 3, the container and the oil layer B with the process vessel 100 is recovered and the aqueous layer C The state of remaining in 100 is shown.
　水層Ｃの回収は、図２（Ｂ）に示すように、水層Ｃの鉛直下方に容器の排出口が位置するように容器を配置し、排出口を通じて水層を回収することが好ましい。 In the recovery of the water layer C, as shown in FIG. 2B, it is preferable to arrange the container so that the outlet of the container is positioned vertically below the water layer C, and recover the water layer through the outlet.
　工程４は、洗浄液を容器内に供給し、容器内に残留した油層と混合して混合液を形成した後、容器を静置することで混合液を油層と水層とに分離させ、容器から水層を回収するとともに油層を容器内に残留させる工程である。上記の通り、工程１～３では水層Ｃの体積が０．９３Ｖ１を上回る前に水層Ｃを回収するため、容器に残留する油層Ｂには、本来水層Ｃに含まれるべき目的細胞が残留している可能性が高い。そこで工程４により、油層中に残留している目的細胞を回収する。 In step 4, the washing solution is supplied into the container and mixed with the oil layer remaining in the container to form a mixed solution, and then the container is allowed to stand to separate the mixed solution into an oil layer and an aqueous layer. This is a step of recovering the water layer and leaving the oil layer in the container. As described above, in steps 1 to 3, since the aqueous layer C is recovered before the volume of the aqueous layer C exceeds 0.93 V 1 , the oil layer B remaining in the container is the target cell that should be originally contained in the aqueous layer C. Is likely to remain. Thus, in step 4, target cells remaining in the oil layer are recovered.
　図２（Ｃ）には、工程３終了後に、処理容器１００内に油層Ｂが残留している状態を示す。工程４では、まず、処理容器１００内に洗浄液を供給し油層Ｂと混合して、図２（Ｄ）に示すように、混合液Ｄを形成する。そして、処理容器１００内で、混合液Ｄを静置して、図２（Ｅ）に示すように、油層Ｅと水層Ｆとに分離させる。更に、処理容器１００から水層Ｆを回収し、図２（Ｆ）に示すように、油層Ｅを処理容器１００内に残留させる。工程４により回収された水層は、目的とする細胞を含んでおり、工程３で回収した水層と併せて細胞懸濁液とすることができる。 FIG. 2C shows a state in which the oil layer B remains in the processing container 100 after the step 3 is completed. In step 4, first, the cleaning liquid is supplied into the processing container 100 and mixed with the oil layer B to form a mixed liquid D as shown in FIG. 2 (D). Then, the mixed solution D is allowed to stand in the processing container 100 to be separated into the oil layer E and the water layer F as shown in FIG. 2 (E). Further, the aqueous layer F is recovered from the processing container 100, and the oil layer E is left in the processing container 100 as shown in FIG. 2 (F). The aqueous layer recovered in step 4 contains the target cells, and can be combined with the aqueous layer recovered in step 3 to form a cell suspension.
　工程４において容器内に供給する洗浄液の体積は、好ましくは０．１０Ｖ１以上、より好ましくは０．２０Ｖ１以上、より好ましくは０．３０Ｖ１以上、より好ましくは０．４０Ｖ１以上、より好ましくは０．５０Ｖ１以上である。洗浄液の体積は大きいほど油層からの細胞の回収には好適である。また、洗浄液の体積が好ましくは３．００Ｖ１以下、より好ましくは２．００Ｖ１以下であると、より好ましくは１．５０Ｖ１以下であると、回収した水層からなる細胞懸濁液を、中空糸分離膜を用いて濃縮洗浄処理することが容易である。
　生体組織の体積に対して、工程１で用いる酵素液の体積Ｖ１と工程４で用いる洗浄液の体積（工程４を複数回行う場合は使用した洗浄液の合計体積）との合計体積を、好ましくは１５０％以上８００％以下であり、より好ましくは２００％以上５００％以下とすることにより、比較的高密度の細胞懸濁液を、細胞に対し低負荷条件で調製することが容易である。 The volume of the cleaning liquid supplied into the container in Step 4 is preferably 0.10 V 1 or more, more preferably 0.20 V 1 or more, more preferably 0.30 V 1 or more, more preferably 0.40 V 1 or more, more preferably Is 0.50 V 1 or more. The larger the volume of the washing solution, the better the recovery of cells from the oil layer. The volume of the cleaning liquid is preferably 3.00V 1 or less, and more preferably is 2.00V 1 below, and more preferably is 1.50 V 1 or less, a cell suspension consisting of collected aqueous layer, It is easy to carry out concentration washing treatment using a hollow fiber separation membrane.
Preferably, the total volume of the volume V 1 of the enzyme solution used in step 1 and the volume of the washing solution used in step 4 (the total volume of the washing solution used multiple times for step 4) is preferably By setting the concentration to 150% to 800%, and more preferably 200% to 500%, it is easy to prepare a relatively high density cell suspension under low load conditions for cells.
　工程４において、混合液を収容した容器を静置する時間は、細胞の水層への回収効率を高める観点から、工程２での静置時間の１／６倍以上であることが好ましく、０．３倍以上であることがより好ましく、０．５倍以上であることがより好ましく、０．５～１．５倍であることが特に好ましい。工程４での静置時間もまた、長すぎると細胞に負荷となる可能性があるから、３分間以上３０分間以下であることが好ましく、５分間以上１５分間以下であることが特に好ましい。工程２での静置時間をこの範囲とすることで、細胞への負荷を抑制しつつ水層と油層を分離することが容易となる。 In the step 4, the time for which the container containing the mixed solution is allowed to stand is preferably 1/6 or more times as long as the standing time in the step 2, from the viewpoint of enhancing the collection efficiency of the cells to the aqueous layer. The ratio is more preferably 3 times or more, more preferably 0.5 times or more, and particularly preferably 0.5 to 1.5 times. The standing time in step 4 may also be a load on cells if it is too long, so it is preferably 3 minutes to 30 minutes, and more preferably 5 minutes to 15 minutes. By setting the standing time in step 2 to this range, it becomes easy to separate the water layer and the oil layer while suppressing the load on the cells.
　工程４は１回のみ行ってもよいし、２回、３回又はそれ以上の回数繰り返し行ってもよい。 Step 4 may be performed only once, or may be repeated twice, three times or more times.
　工程２及び工程４での静置時間の累計は、細胞に対する負荷を小さくする観点から、好ましくは６０分間以下、より好ましくは４０分間以下、より好ましくは３５分間以下である。 From the viewpoint of reducing the load on cells, the total of the standing time in step 2 and step 4 is preferably 60 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less.
＜濾過貯留デバイス＞
　本発明の方法で分離された水層は、図３に示すような濾過貯留デバイス２００に回収し貯留することが好ましい。 <Filtration storage device>
The aqueous layer separated by the method of the present invention is preferably recovered and stored in a filtration storage device 200 as shown in FIG.
　図３に示す実施形態の濾過貯留デバイス２００は、濾過器２１０と、貯留容器２２０とを備える。濾過器２１０には、管２３１と、管２３２とが通液可能に接続されており、管２３１から流入した液体は、濾過器２１０内に配置されたメッシュシート２１１を通過して管２３２に排出される。メッシュシート２１１の目開は、目的とする細胞（例えば脂肪由来幹細胞）が通過することができ、不要な成分（例えば脂肪細胞）が通過することができない寸法に設定されている。 The filtration storage device 200 of the embodiment shown in FIG. 3 includes a filter 210 and a storage container 220. A pipe 231 and a pipe 232 are fluidly connected to the filter 210, and the liquid flowing from the pipe 231 passes through the mesh sheet 211 disposed in the filter 210 and is discharged to the pipe 232. Be done. The opening of the mesh sheet 211 is set to a size that can allow target cells (for example, fat-derived stem cells) to pass through and unnecessary components (for example, fat cells) can not pass through.
　管２３１の上流側には、処理容器１００が備えるニードルレスポート１３３Ａ、１３３Ｂと接続可能なオスコネクター２３３が設けられ、オスコネクター２３３を保護する蓋部２３４が取り付けられている。管２３１の外側には更に、管２３１内の流路を開閉するためのローラークランプ２３５が設置されている。 On the upstream side of the pipe 231, a male connector 233 connectable to the needleless ports 133A and 133B of the processing container 100 is provided, and a lid 234 for protecting the male connector 233 is attached. At the outside of the tube 231, a roller clamp 235 for opening and closing the flow passage in the tube 231 is further provided.
　貯留容器２２０は、袋状容器であって、管２３２の一端が通液可能に接続されている。濾過器２１０を通過した細胞懸濁液は管２３２を通って貯留容器２２０に貯留される。 The storage container 220 is a bag-like container, and one end of a pipe 232 is connected so as to be fluid-permeable. The cell suspension that has passed through the filter 210 is stored in the storage container 220 through the pipe 232.
　貯留容器２２０には更に、管２３６の一端が通液可能に接続されている。管２３６の他端にはニードルレスポート２３７が配置され、更にニードルレスポート２３７を保護する蓋部２３８が取り付けられている。 Further, one end of a pipe 236 is connected to the storage container 220 so as to be fluid-permeable. At the other end of the tube 236, a needleless port 237 is disposed, and a lid 238 for protecting the needleless port 237 is attached.
　処理容器１００のニードルレスポート１３３Ａ、１３３Ｂと、濾過貯留デバイス２００のオスコネクター２３３とを接続し、処理容器１００、濾過器２１０、貯留容器２２０がこの順で鉛直方向の上から下になるように配置する。そして、本発明の方法の工程３及び工程４において水層の回収を行うときに、ローラークランプ２３５を操作して管２３１内の流路を開放し、水層を、濾過器２１０を通過させて貯留容器２２０に貯留する。 Connect the needleless ports 133A and 133B of the processing container 100 and the male connector 233 of the filtration storage device 200 so that the processing container 100, the filter 210 and the storage container 220 are in this order from the top to the bottom in the vertical direction. Deploy. Then, when the aqueous layer is recovered in step 3 and step 4 of the method of the present invention, the roller clamp 235 is operated to open the flow path in the pipe 231, and the aqueous layer is allowed to pass through the filter 210. It stores in the storage container 220.
＜実験１＞ <Experiment 1>
　３名のヒト（ドナー１、ドナー２、ドナー３）及び１匹のウサギ（ドナー１）からの脂肪組織を、コラゲナーゼにより処理し、細胞懸濁液を調製する工程を以下の各条件で行った。 Adipose tissue from 3 human (donor 1, donor 2, donor 3) and 1 rabbit (donor 1) was treated with collagenase to prepare a cell suspension under the following conditions: .
　酵素処理用バッグとして、厚さ０．４ｍｍの軟質ポリ塩化ビニルの概ね長方形の柔軟なシートを２枚重ね合わせ、長手方向の一端の辺上に３つの投入部１２０Ａ、１２０Ｂ、１２０Ｃを設け、他端の辺上に２つの排出部１３０Ａ，１３０Ｂを設ける以外は外縁部を熱融着により融着することで、前記２枚のシートに挟まれた、空の状態で内寸が幅約１４０ｍｍ、長さ約１９６ｍｍの概ね長方形の収容部が形成された図１に示す形態のバッグ１００（袋状容器）を用いた。このバッグの前記収容部の内容積は約６００ｍＬである。 As a bag for enzyme treatment, two approximately rectangular flexible sheets of soft polyvinyl chloride with a thickness of 0.4 mm are stacked, and three charging sections 120A, 120B, and 120C are provided on the side of one end in the longitudinal direction. The outer edge is fusion-bonded by heat fusion except that two discharge parts 130A and 130B are provided on the edge side, the sheet is sandwiched between the two sheets, and the inner dimension is about 140 mm in width in an empty state, A bag 100 (bag-like container) of the form shown in FIG. 1 was used, in which a generally rectangular receptacle having a length of about 196 mm was formed. The internal volume of the receptacle of this bag is about 600 mL.
　遠沈管として、ポリプロピレン製の内容量が５０ｍＬの遠沈管を用いた。 As a centrifuge tube, a 50 mL centrifuge tube made of polypropylene was used.
　前記酵素処理用バッグの収容部内に、５５～１０５ｍＬの脂肪組織と、５５～１０５ｍＬのコラゲナーゼ液（コラゲナーゼ濃度０．１ｗ／ｖ％となるように生理食塩液で溶解して調製した）とを、脂肪組織とコラゲナーゼ液の体積比が１：１となるように収容した。このときのコラゲナーゼ液の体積を「初期酵素液量」とする。 55 to 105 mL of adipose tissue and 55 to 105 mL of collagenase solution (prepared by dissolving in physiological saline so as to have a collagenase concentration of 0.1 w / v%) in the container of the enzyme treatment bag, It was stored such that the volume ratio of adipose tissue to collagenase solution was 1: 1. The volume of the collagenase solution at this time is referred to as "initial enzyme solution volume".
　酵素処理用バッグの収容部の内容積は６００ｍＬであるのに対して、脂肪組織とコラゲナーゼ液とを含む反応混合液は最大でも２１０ｍＬであり内容積と比較して少ない。このため、収容部に反応混合液を収容した酵素処理用バッグをクランプにより一部を封止して、酵素処理用バッグの収容部のうち、反応混合液を収容する部分の容積を、収容物の体積に近づけるように調節した。 While the internal volume of the container for enzyme treatment bag is 600 mL, the reaction mixture containing adipose tissue and collagenase solution is at most 210 mL, which is smaller than the internal volume. For this reason, the enzyme treatment bag containing the reaction mixture in the containing portion is partially sealed with a clamp, and the volume of the portion containing the reaction mixture in the containing portion of the enzyme treatment bag is the content Adjusted to approximate the volume of
　前記遠沈管に、２０ｍＬの脂肪組織と、２０ｍＬのコラゲナーゼ液（コラゲナーゼ濃度０．１ｗ／ｖ％となるように生理食塩液で溶解、脂肪組織とコラゲナーゼ液は１：１）とを収容し蓋を閉じて密閉した。 The centrifuge tube contains 20 mL of adipose tissue and 20 mL of collagenase solution (dissolve with physiological saline so that the collagenase concentration is 0.1 w / v%, fat tissue and collagenase solution are 1: 1) and cover the lid. Closed and sealed.
　脂肪組織及びコラゲナーゼ液からなる混合物を収容した前記の酵素処理用バッグ又は遠沈管を、シェーカーに設置し、３７℃、３０分間、６０ｒｐｍの条件で振盪させ酵素反応を行った。 The above enzyme treatment bag or centrifuge tube containing a mixture of adipose tissue and collagenase solution was placed on a shaker and shaken at 37 ° C. for 30 minutes under conditions of 60 rpm to carry out an enzyme reaction.
　３０分の酵素反応後に以下の条件で各容器内の混合液から細胞懸濁液を回収した。 After the enzyme reaction for 30 minutes, the cell suspension was recovered from the mixture in each container under the following conditions.
条件１（実施例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の１００体積％の洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の１００体積％の洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に再び供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。３回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。 Condition 1 (Example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 100% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed The enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected. Next, 100% by volume of the initial amount of the enzyme solution (saline solution) is again supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, The enzyme processing bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged to the discharge units 130A and 130B. Removed from one of the The aqueous layer recovered by the three recoverys was combined into one cell suspension.
条件２（実施例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の１００体積％の洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。２回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。 Condition 2 (Example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 100% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed The enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected. The aqueous layers recovered by the two recoverys were combined into one to form a cell suspension.
条件３（実施例）
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の５０体積％の洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の５０体積％の洗浄液を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に再び供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。３回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。 Condition 3 (Example)
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 50% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed The enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 10 minutes, and the aqueous layer (lower layer) after 10 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected. Next, 50% by volume of the initial amount of the enzyme solution was again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction to form a mixed solution, and the mixed solution was stored. Suspend the enzyme processing bag 100 so that the discharge units 130A and 130B are vertically downward and let it stand again for 10 minutes, and take out the aqueous layer (lower layer) after 10 minutes from one of the discharge units 130A and 130B. It was collected. The aqueous layer recovered by the three recoverys was combined into one cell suspension.
条件４（実施例）
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして５分間静置し、５分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の５０体積％の洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び５分間静置し、５分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の５０体積％の洗浄液を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に再び供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び５分間静置し、５分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の５０体積％の洗浄液を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に再び供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び５分間静置し、５分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。４回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。 Condition 4 (Example)
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and left for 5 minutes, and the aqueous layer (lower layer) after 5 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 50% by volume of the initial amount of enzyme solution (saline solution) is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction to form a mixed solution, and mixed The enzyme treatment bag 100 containing the solution is suspended so that the discharge units 130A and 130B are vertically downward, and left still for 5 minutes, and the aqueous layer (lower layer) after 5 minutes is discharged from the discharge units 130A and 130B. It was taken out from one and collected. Next, 50% by volume of the initial amount of the enzyme solution was again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction to form a mixed solution, and the mixed solution was stored. Suspend the enzyme processing bag 100 so that the discharge units 130A and 130B are vertically downward and let it stand for 5 minutes again, and take out the aqueous layer (lower layer) after 5 minutes from one of the discharge units 130A and 130B. It was collected. Next, 50% by volume of the initial amount of the enzyme solution was again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction to form a mixed solution, and the mixed solution was stored. Suspend the enzyme processing bag 100 so that the discharge units 130A and 130B are vertically downward and let it stand for 5 minutes again, and take out the aqueous layer (lower layer) after 5 minutes from one of the discharge units 130A and 130B. It was collected. The aqueous layer recovered by the four recoverys was combined into one cell suspension.
条件５（比較例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして３０分間静置し、３０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収し、細胞懸濁液とした。 Condition 5 (comparative example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 30 minutes, and the aqueous layer (lower layer) after 30 minutes Were collected from one of the discharge units 130A and 130B and collected to obtain a cell suspension.
条件６（比較例）：
　３０分の酵素反応後に酵素反応液を収容した遠沈管を８００Ｇ、１０分の条件で遠心分離し水層を全量回収し、細胞懸濁液とした。 Condition 6 (comparative example):
After the enzyme reaction for 30 minutes, the centrifuge tube containing the enzyme reaction solution was centrifuged under the conditions of 800 G for 10 minutes to recover the whole aqueous layer to obtain a cell suspension.
　条件１～５で初回に回収された回収液の体積を測定し、回収液の体積の初期酵素液量の体積に対する割合（体積％）を求めた。 Under the conditions 1 to 5, the volume of the recovered solution collected for the first time was measured, and the ratio (volume%) of the volume of the recovered solution to the volume of the initial amount of the enzyme solution was determined.
　条件６では、遠心分離で回収された水層の体積を測定し、水層の体積の初期酵素液量の体積に対する割合（体積％）を求めた。 Under condition 6, the volume of the aqueous layer collected by centrifugation was measured, and the ratio (volume%) of the volume of the aqueous layer to the volume of the initial amount of enzyme liquid was determined.
　また、条件１～６で得られた細胞懸濁液中の細胞数をフローサイトメーターおよびＢＤ　ＴｒｕｃｏｕｎｔＴｕｂｅｓを用いて測定した。細胞数は、ヒト細胞についてはＣＤ３４陽性細胞数とＣＤ４５陽性細胞数の和から算出し、ウサギ細胞についてはＲｅｔｉｔｉｃＣｏｕｎｔを用いて測定した。脂肪由来幹細胞数は、ＣＤ３４陽性／ＣＤ４５陰性／ＣＤ３１陰性の細胞数から算出した。細胞数は、使用した脂肪組織の体積１ｍＬ当たりの細胞数で表した。
　結果を表１に示す。 In addition, the number of cells in the cell suspension obtained under conditions 1 to 6 was measured using a flow cytometer and BD TrucountTubes. The number of cells was calculated from the sum of the number of CD34 positive cells and the number of CD45 positive cells for human cells, and was measured using RetiticCount for rabbit cells. The number of adipose-derived stem cells was calculated from the number of CD34 positive / CD45 negative / CD31 negative cells. The number of cells was expressed as the number of cells per mL of the volume of adipose tissue used.
The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜実験２＞
　ヒトドナー４からの脂肪組織を、コラゲナーゼにより処理し、細胞懸濁液を調製する工程を以下の各条件で行った。 <Experiment 2>
Adipose tissue from human donor 4 was treated with collagenase to prepare a cell suspension under the following conditions.
　酵素処理用バッグ及び遠沈管は、実験１で使用したものと同じものを用いた。 As the bag for enzyme treatment and the centrifuge tube, the same one as used in Experiment 1 was used.
　前記酵素処理用バッグの収容部内に、１０ｍＬの脂肪組織と、３０ｍＬのコラゲナーゼ液（実験１参照）とを収容した。下記の条件１１～１５、１７では、このときのコラゲナーゼ液の体積を「初期酵素液量」とした。下記の条件１６では、コラゲナーゼ液３０ｍＬに生理食塩水１２ｍＬを加えた体積４２ｍＬを「初期酵素液量」とした。 10 mL of adipose tissue and 30 mL of collagenase solution (see Experiment 1) were accommodated in the container for the enzyme treatment bag. Under the following conditions 11 to 15 and 17, the volume of the collagenase solution at this time was defined as the "initial enzyme solution volume". Under the following condition 16, a volume of 42 mL obtained by adding 12 mL of physiological saline to 30 mL of collagenase solution was defined as "initial enzyme solution volume".
　酵素処理用バッグの収容部の内容積は６００ｍＬであるのに対して、脂肪組織とコラゲナーゼ液とを含む反応混合液は４０ｍＬであり内容積と比較して少ない。このため、収容部に反応混合液を収容した酵素処理用バッグをクランプにより一部を封止して、酵素処理用バッグの収容部のうち、反応混合液を収容する部分の容積を、収容物の体積に近づけるように調節した。 While the internal volume of the container for enzyme treatment bag is 600 mL, the reaction mixture containing adipose tissue and collagenase solution is 40 mL, which is smaller than the internal volume. For this reason, the enzyme treatment bag containing the reaction mixture in the containing portion is partially sealed with a clamp, and the volume of the portion containing the reaction mixture in the containing portion of the enzyme treatment bag is the content Adjusted to approximate the volume of
　前記遠沈管に、１０ｍＬの脂肪組織と、３０ｍＬのコラゲナーゼ液（実験１参照）とを収容し蓋を閉じて密閉した。 The centrifuge tube contained 10 mL of adipose tissue and 30 mL of collagenase solution (see Experiment 1), and the lid was closed and sealed.
　脂肪組織及びコラゲナーゼ液からなる混合物を収容した前記の酵素処理用バッグ又は遠沈管を、シェーカーに設置し、３７℃、３０分間、６０ｒｐｍの条件で振盪させ酵素反応を行った。 The above enzyme treatment bag or centrifuge tube containing a mixture of adipose tissue and collagenase solution was placed on a shaker and shaken at 37 ° C. for 30 minutes under conditions of 60 rpm to carry out an enzyme reaction.
　３０分の酵素反応後に以下の条件で各容器内の混合液から細胞懸濁液を回収した。 After the enzyme reaction for 30 minutes, the cell suspension was recovered from the mixture in each container under the following conditions.
条件１１（実施例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の２０体積％に相当する６ｍＬの洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。２回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。２回の回収により回収された水層の合計量を総回収液量とした。 Condition 11 (Example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 6 mL of a washing solution (saline solution) equivalent to 20% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B, 120C and mixed with the remaining fraction and mixed The enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B. The aqueous layers recovered by the two recoverys were combined into one to form a cell suspension. The total volume of the aqueous layer recovered by the two rounds of recovery was taken as the total volume of recovered liquid.
条件１２（実施例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の２０体積％に相当する６ｍＬの洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の２０体積％に相当する６ｍＬの洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に再び供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。３回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。３回の回収により回収された水層の合計量を総回収液量とした。 Condition 12 (Example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 6 mL of a washing solution (saline solution) equivalent to 20% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B, 120C and mixed with the remaining fraction and mixed The enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B. Next, 6 mL of a washing solution (saline solution) equivalent to 20% by volume of the initial enzyme solution volume is again supplied into the storage unit via one of the input units 120A, 120B and 120C and mixed with the remaining fraction. Suspend the enzyme treatment bag 100 containing the mixed solution so that the mixed solution is contained, with the discharge units 130A and 130B vertically downward, and let stand for 10 minutes again, and discharge the aqueous layer (lower layer) after 10 minutes It took out and collect | recovered from one of the part 130A, 130B. The aqueous layer recovered by the three recoverys was combined into one cell suspension. The total volume of the aqueous layer recovered by the three rounds of recovery was taken as the total volume of recovered fluid.
条件１３（実施例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の１０体積％に相当する３ｍＬの洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。２回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。２回の回収により回収された水層の合計量を総回収液量とした。 Condition 13 (Example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 3 mL of a washing solution (saline solution) equivalent to 10% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction and mixed The enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B. The aqueous layers recovered by the two recoverys were combined into one to form a cell suspension. The total volume of the aqueous layer recovered by the two rounds of recovery was taken as the total volume of recovered liquid.
条件１４（実施例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の１０体積％に相当する３ｍＬの洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。次に、前記初期酵素液量の１０体積％に相当する３ｍＬの洗浄液（生理食塩液）を投入部１２０Ａ、１２０Ｂ、１２０Ｃの１つを介して収容部内に再び供給し残存画分と混合して混合液とし、混合液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして再び１０分間静置し、１０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収した。３回の回収により回収された水層は１つにまとめて、細胞懸濁液とした。３回の回収により回収された水層の合計量を総回収液量とした。 Condition 14 (Example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 10 minutes, and the aqueous layer (lower layer) after 10 minutes Were taken out from one of the discharge parts 130A, 130B and collected. Next, 3 mL of a washing solution (saline solution) equivalent to 10% by volume of the initial enzyme solution volume is supplied into the storage unit through one of the input units 120A, 120B and 120C and mixed with the remaining fraction and mixed The enzyme treatment bag 100 containing the mixed solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand again for 10 minutes, and the water layer (lower layer) after 10 minutes is discharged It was taken out and collected from one of 130A and 130B. Next, 3 mL of a washing solution (saline solution) equivalent to 10% by volume of the initial enzyme solution volume is again supplied into the storage unit through one of the input units 120A, 120B, and 120C and mixed with the remaining fraction. Suspend the enzyme treatment bag 100 containing the mixed solution so that the mixed solution is contained, with the discharge units 130A and 130B vertically downward, and let stand for 10 minutes again, and discharge the aqueous layer (lower layer) after 10 minutes It took out and collect | recovered from one of the part 130A, 130B. The aqueous layer recovered by the three recoverys was combined into one cell suspension. The total volume of the aqueous layer recovered by the three rounds of recovery was taken as the total volume of recovered fluid.
条件１５（比較例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして３０分間静置し、３０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収し、細胞懸濁液とした。回収された細胞懸濁液の量を総回収液量とした。 Condition 15 (comparative example):
After the enzyme reaction for 30 minutes, the enzyme treatment bag 100 containing the enzyme reaction solution is suspended so that the discharge units 130A and 130B are vertically downward, and allowed to stand for 30 minutes, and the aqueous layer (lower layer) after 30 minutes Were collected from one of the discharge units 130A and 130B and collected to obtain a cell suspension. The amount of cell suspension collected was taken as the total collected liquid volume.
条件１６（比較例）：
　３０分の酵素反応後に酵素反応液を収容した酵素処理用バッグ１００に、１２ｍＬの生理食塩水を加えて混合し、混合後の酵素処理用バッグ１００を、排出部１３０Ａ，１３０Ｂが鉛直下方になるように吊るして３０分間静置し、３０分間経過時点での水層（下層）を排出部１３０Ａ，１３０Ｂの１つから取り出し回収し、細胞懸濁液とした。回収された細胞懸濁液の量を総回収液量とした。 Condition 16 (comparative example):
After 30 minutes of enzyme reaction, 12 mL of physiological saline is added to the enzyme treatment bag 100 containing the enzyme reaction solution and mixed, and the enzyme treatment bag 100 after mixing is such that the discharge parts 130A and 130B are vertically downward. The suspension was suspended for 30 minutes, and the aqueous layer (lower layer) after 30 minutes was taken out from one of the discharge parts 130A and 130B and collected to obtain a cell suspension. The amount of cell suspension collected was taken as the total collected liquid volume.
条件１７（比較例）：
　３０分の酵素反応後に酵素反応液を収容した遠沈管を８００Ｇ、１０分の条件で遠心分離し水層を全量回収し、細胞懸濁液とした。回収された細胞懸濁液の量を総回収液量とした。 Condition 17 (comparative example):
After the enzyme reaction for 30 minutes, the centrifuge tube containing the enzyme reaction solution was centrifuged under the conditions of 800 G for 10 minutes to recover the whole aqueous layer to obtain a cell suspension. The amount of cell suspension collected was taken as the total collected liquid volume.
　条件１１～１６で初回に回収された回収液の体積を測定し、回収液の体積の初期酵素液量の体積に対する割合（体積％）を求めた。 Under the conditions 11 to 16, the volume of the recovered solution collected for the first time was measured, and the ratio (volume%) of the volume of the recovered solution to the volume of the initial amount of the enzyme solution was determined.
　条件１７では、遠心分離で回収された水層の体積を測定し、水層の体積の初期酵素液量の体積に対する割合（体積％）を求めた。 Under the condition 17, the volume of the aqueous layer collected by centrifugation was measured, and the ratio (volume%) of the volume of the aqueous layer to the volume of the initial amount of enzyme liquid was determined.
　また、条件１～１７で得られた細胞懸濁液中の細胞数をフローサイトメーターおよびＢＤ　ＴｒｕｃｏｕｎｔＴｕｂｅｓを用いて測定した。細胞数は、ヒト細胞についてはＣＤ３４陽性細胞数とＣＤ４５陽性細胞数の和から算出し、ウサギ細胞についてはＲｅｔｉｔｉｃＣｏｕｎｔを用いて測定した。脂肪由来幹細胞数は、ＣＤ３４陽性／ＣＤ４５陰性／ＣＤ３１陰性の細胞数から算出した。細胞数は、使用した脂肪組織の体積１ｍＬ当たりの細胞数で表した。
　結果を表２に示す。 In addition, the number of cells in the cell suspension obtained under conditions 1 to 17 was measured using a flow cytometer and BD TrucountTubes. The number of cells was calculated from the sum of the number of CD34 positive cells and the number of CD45 positive cells for human cells, and was measured using RetiticCount for rabbit cells. The number of adipose-derived stem cells was calculated from the number of CD34 positive / CD45 negative / CD31 negative cells. The number of cells was expressed as the number of cells per mL of the volume of adipose tissue used.
The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (7)

  1. 　生体組織から細胞を分離し回収する方法であって、
    　容器内で、生体組織と体積Ｖ１の酵素液との混合物中で生体組織の酵素処理を行い、遊離細胞を含む酵素処理液を形成する工程１と、
    　工程１後に酵素処理液を収容した容器を静置し、酵素処理液を油層と水層に、水層の体積が０．９３Ｖ１を超えない時点まで分離させる工程２と、
    　前記時点で、容器から水層を回収するとともに油層を容器内に残留させる工程３と、
    　洗浄液を容器内に供給し、容器内に残留した油層と混合して混合液を形成した後、容器を静置することで混合液を油層と水層とに分離させ、容器から水層を回収するとともに油層を容器内に残留させる工程４と
    を含み、工程４を１回以上行うことを特徴とする方法。     A method of separating and recovering cells from a living tissue, comprising
    Carrying out the enzyme treatment of the living tissue in a mixture of the living tissue and the enzyme solution of volume V 1 in a container to form an enzyme-treated solution containing free cells;
    And 2. leaving the container containing the enzyme-treated solution after step 1 and separating the enzyme-treated solution into an oil layer and an aqueous layer until the volume of the aqueous layer does not exceed 0.93 V 1 ;
    Recovering the aqueous layer from the container and leaving the oil layer in the container at the time point;
    The cleaning solution is supplied into the container and mixed with the oil layer remaining in the container to form a mixture, and then the mixture is allowed to stand to separate the mixture into an oil layer and an aqueous layer, and the water layer is recovered from the container And 4) leaving the oil layer in the container, and performing step 4 one or more times.
  2. 　生体組織が脂肪組織である、請求項１に記載の方法。 The method according to claim 1, wherein the living tissue is a fat tissue.
  3. 　工程４での洗浄液の体積が０．１０Ｖ１以上である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the volume of the washing solution in step 4 is 0.10 V 1 or more.
  4. 　工程４での静置時間が、工程２での静置時間の１／６倍以上の時間である、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the settling time in step 4 is at least 1/6 times the settling time in step 2.
  5. 　工程２での静置時間が３分間以上３０分間以下である、請求項１～４のいずれか１項に記載の方法。 The method according to any one of claims 1 to 4, wherein the standing time in step 2 is 3 minutes or more and 30 minutes or less.
  6. 　工程４での静置時間が３分間以上３０分間以下である、請求項１～５のいずれか１項に記載の方法。 The method according to any one of claims 1 to 5, wherein the standing time in step 4 is 3 minutes or more and 30 minutes or less.
  7. 　容器が、液体の排出口が形成された容器であり、
    　工程３及び４における水層の回収が、水層に対し鉛直下方に排出口が位置するように容器を配置し、排出口を通じて水層を回収することを含む、請求項１～６のいずれか１項に記載の方法。     The container is a container in which a liquid outlet is formed,
    7. Recovery of the aqueous layer in steps 3 and 4 comprises arranging the container so that the outlet is located vertically below the aqueous layer, and recovering the aqueous layer through the outlet. Method according to paragraph 1.
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