WO2019059278A1 - Procédé pour la séparation et le recueil de cellules à partir de tissu biologique - Google Patents

Procédé pour la séparation et le recueil de cellules à partir de tissu biologique 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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container
solution
volume
enzyme
aqueous layer
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PCT/JP2018/034815
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English (en)
Japanese (ja)
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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

L'invention concerne un procédé pour le traitement enzymatique d'un tissu biologique dans des conditions de faible charge pour des cellules pour séparer et recueillir des cellules. Le procédé pour la séparation et le recueil de cellules à partir d'un tissu biologique selon la présente invention comprend : l'étape 1 consistant à mélanger le tissu biologique avec une solution d'enzyme en un volume V1 dans un récipient pour effectuer un traitement enzymatique, ce qui permet de préparer une solution traitée par voie enzymatique contenant des cellules libres ; l'étape 2 consistant à laisser le récipient reposer pour provoquer la séparation de la solution traitée par voie enzymatique en une phase huileuse et une phase aqueuse jusqu'à un moment auquel le volume de la phase aqueuse ne dépasse pas 0,93V1 ; l'étape 3 consistant à recueillir la phase aqueuse à partir du récipient et amener la phase huileuse à rester dans le récipient au moment mentionné dans l'étape 2 ; et l'étape 4 consistant à introduire une solution de lavage dans le récipient, puis mélanger la solution ainsi obtenue avec la phase huileuse restant dans le récipient, puis amener le mélange ainsi obtenu à reposer pour provoquer la séparation en une phase huileuse et une phase aqueuse, puis recueillir la phase aqueuse à partir du récipient et amener la phase huileuse à rester dans le récipient.
PCT/JP2018/034815 2017-09-20 2018-09-20 Procédé pour la séparation et le recueil de cellules à partir de tissu biologique WO2019059278A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007185157A (ja) * 2006-01-16 2007-07-26 Kaneka Corp 脂肪組織の酵素処理液から有核細胞を採取する方法
JP2007289076A (ja) * 2006-04-25 2007-11-08 Kaneka Corp 脂肪組織から幹細胞を採取するのに適した細胞分離装置、およびその方法
JP2011010583A (ja) * 2009-06-30 2011-01-20 Kaneka Corp 液性油成分除去方法、細胞分離方法及び細胞分離キット
WO2011111386A1 (fr) * 2010-03-11 2011-09-15 株式会社カネカ Procédé permettant de concentrer/récupérer des cellules et liquide d'élution cellulaire
JP2015519067A (ja) * 2012-12-14 2015-07-09 鄭▲ちゅう▼ＺＨＥＮＧ Ｃｈｏｎｇ 遠心動的濾過装置及びそれを用いた細胞分離システム

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007185157A (ja) * 2006-01-16 2007-07-26 Kaneka Corp 脂肪組織の酵素処理液から有核細胞を採取する方法
JP2007289076A (ja) * 2006-04-25 2007-11-08 Kaneka Corp 脂肪組織から幹細胞を採取するのに適した細胞分離装置、およびその方法
JP2011010583A (ja) * 2009-06-30 2011-01-20 Kaneka Corp 液性油成分除去方法、細胞分離方法及び細胞分離キット
WO2011111386A1 (fr) * 2010-03-11 2011-09-15 株式会社カネカ Procédé permettant de concentrer/récupérer des cellules et liquide d'élution cellulaire
JP2015519067A (ja) * 2012-12-14 2015-07-09 鄭▲ちゅう▼ＺＨＥＮＧ Ｃｈｏｎｇ 遠心動的濾過装置及びそれを用いた細胞分離システム

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NAKAYAMA, TAKAYUKI ET AL.: "CELL THERAPY USING ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS : CURRENT STATUS AND PERSPECTIVES", JAPANESE JOURNAL OF TRANSFUSION AND CELL THERAPY, vol. 59, no. 3, 2013, pages 450 - 456, XP055586411 *

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