WO2019054468A1

WO2019054468A1 - Method of freeing cells from cell-containing sample

Info

Publication number: WO2019054468A1
Application number: PCT/JP2018/034107
Authority: WO
Inventors: 林　真司
Original assignee: 株式会社カネカ
Priority date: 2017-09-15
Filing date: 2018-09-14
Publication date: 2019-03-21
Also published as: JPWO2019054468A1; JP7282679B2

Abstract

In order to free nucleated cells from a cell-containing sample such as fatty tissue with enzyme treatment, this method of freeing cells from a cell-containing sample by subjecting the cell-containing sample to enzyme treatment is characterized by involving a step for agitating a bag-shaped container 1 in which a gaseous phase 202 and a reaction mixture 201 containing the cell-containing sample and an enzyme are housed and sealed, wherein the ratio of the volume of the gaseous phase 202 is at least 7 vol% of the total volume of the gaseous phase 202 and the reaction mixture 201.

Description

Method of releasing cells from cell-containing sample

The present invention relates to a method in which a cell-containing sample liberates cells by enzymatically treating a cell-containing sample such as a living tissue.

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.

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.

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.

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.

International Publication No. 2008/018450 Japanese Patent Publication No. 2015-500031

In order to release cells from a cell-containing sample containing cells, such as a living tissue, a method of enzymatically treating the cell-containing sample has been conventionally performed.

However, the relationship between the target amount of recovered cells and the conditions such as the container for enzyme treatment for releasing cells from the cell-containing sample and the shaking method has not been sufficiently studied.

The present inventors have surprisingly found that a sufficient number of nucleated cells are obtained when enzyme treatment of a cell-containing sample is carried out while shaking in a conventional flexible tube, as in Patent Document 1. I found that I could not get it. The inventors also surprisingly found that the cell-containing sample and the enzyme solution were filled in a flexible bag-like container without using air in the usual manner, and the enzyme treatment was carried out while shaking. Also, we found that a sufficient number of nucleated cells could not be obtained. As a result of intensive studies, the present inventors have completed the following method.

(1) A method of enzymatically treating a cell-containing sample to release cells from the cell-containing sample,
A closed container comprising a reaction mixture containing a cell-containing sample and an enzyme and a gas phase is contained at a ratio of 7 vol% or more of the volume of the gas phase to the total volume of the reaction mixture and the gas phase. A method characterized in that it comprises the step of shaking.
(2) The method according to (1), wherein the cell-containing sample is a fat tissue (3) The width in the short direction of the container of the reaction mixture of the bag-like container and the gas phase The method according to (1) or (2), wherein A / B is 0.1 or more and 1 or less, where B is a longitudinal width.
The present specification includes the disclosure content of Japanese Patent Application No. 2017-178267 on which the priority of the present application is based.

According to the method of the present invention, the amount of cells released in the form of nucleated cells from a cell-containing sample such as adipose tissue can be increased.

An example of the bag for enzyme processing (bag-like container) for performing an enzyme processing of a cell containing sample is shown. It is a cross-sectional schematic diagram for demonstrating the state of the reaction liquid mixture containing a cell containing sample and an enzyme, and the gaseous phase in the bag for enzyme processing. The enzyme processing bag shown in FIG. 1 to which the reaction mixture containing the cell-containing sample and the enzyme and the gas phase (containing material) have been added is sealed with a clamp, and the container of the enzyme processing bag is contained. It is a schematic diagram for demonstrating that the volume of the part to accommodate was adjusted so that it might correspond with the volume of a container. The other example of the bag (bag-like container) for enzyme processing for performing an enzyme processing of a cell containing sample is shown. The other example of the bag (bag-like container) for enzyme processing for performing an enzyme processing of a cell containing sample is shown. The other example of the bag (bag-like container) for enzyme processing for performing an enzyme processing of a cell containing sample is shown.

Hereinafter, the present invention will be described in detail.
<Terms, Materials>
Examples of cell-containing samples include living tissues, and cell cultures prepared in vitro. 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.

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.
A nucleated cell is a cell having a nucleus in the cell.

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.

As an enzyme for releasing cells from a cell-containing sample, at least one degradation enzyme selected from collagenase, metalloprotease, dispase, trypsin, hyaluronidase, chymotrypsin, pepsin, aminopeptidase, lipase, amylase and their recombinants is used it can. When a living tissue, in particular an adipose tissue, is used as the cell-containing sample, at least one selected from collagenase, metalloprotease, dispase, trypsin and hyaluronidase from the viewpoint of degradation in a short time and minimally invasive manner as an enzyme Species degrading enzymes are preferred.

<Bag-like container>
The bag-like container used in the present invention is a bag-like container formed of a material having flexibility (also referred to as flexibility). It is preferable that the bag-like container is at least partially made of a transparent or translucent material, because the appearance of the cell suspension inside can be visually observed.

Specific examples of the flexible material forming the bag-like container include soft vinyl chloride, vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin such as polyethylene and polypropylene, styrene-butadiene-styrene copolymer Or a thermoplastic elastomer such as a hydrogenated substance thereof and a styrene-isoprene-styrene copolymer or a hydrogenated substance thereof, and a mixture of a thermoplastic elastomer and a softener such as a polyolefin and ethylene-ethyl acrylate, etc. The resin which it has is mentioned.

In the embodiment in which the bag-like container is formed of a resin sheet, the thickness of the resin sheet is not particularly limited, but can be, for example, 0.2 mm to 0.6 mm.

The bag-like container is preferably a container whose wall surface is formed of a flexible resin sheet, and particularly preferably, the flexible resin sheets are disposed in a double-layer facing manner and the peripheral portion is heat-sealed or It is a container joined by an adhesive and formed in a bag shape.

A specific embodiment of a bag-like container formed of two flexible resin sheets will be described with reference to FIG.

The container 1 of this embodiment includes a container body 10, a first inlet port 21, a second inlet port 22, a third inlet port 23, a first outlet port 31, and a second outlet port 32.

The first inlet port 21 includes a tube portion 211 forming a flow path of liquid, a female luer lock 212 which closes the inlet of the tube portion 211 so that the inlet can be communicated, and a removable lid 213 which protects the female luer lock 212. And the filter part 214 which filters the liquid which passes the pipe part 211. FIG.

The second inlet port 22 includes a pipe portion 221 that forms a flow path of liquid, a needleless port 222 that blocks the inlet of the pipe portion 221 so as to allow communication with it, and a removable lid 223 that protects the needleless port 222. Equipped with

The third inlet port 23 includes a tube portion 231 which forms a flow path of liquid, a needleless port 232 which closes the inlet of the tube portion 231 so as to allow communication with it, and a detachable lid 233 which protects the needleless port 232. Equipped with

The first outlet port 31 includes a pipe portion 311 forming a flow path of liquid, a needleless port 312 for sealing the outlet of the pipe portion 311 so as to allow communication with it, and a removable lid 313 for protecting the needleless port 312. Equipped with

The second outlet port 32 has a pipe portion 321 forming a flow path of liquid, a needleless port 322 which closes the outlet of the pipe portion 321 in a manner that allows communication with it, and a removable lid 323 which protects the needleless port 322. Equipped with

The container body 10 has a substantially rectangular first resin sheet 11 and a second resin sheet 12 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 11 and the second resin sheet 12 are joined by heat fusion is referred to as a fusion part 13. In the container body 10, a housing portion 14 is formed between the first resin sheet 11 and the second resin sheet 12. In the storage unit 14, an enzyme treatment described later is performed. The pipe portion 211 of the first inlet port 21, the pipe portion 221 of the second inlet port 22, and the pipe portion 231 of the third inlet port 23 are the sides on which the female luer lock 212 and the

needleless ports

222 and 232 are not disposed. The end portion of the first resin sheet 11 is disposed between the first resin sheet 11 and the second resin sheet 12 on the side of one end in the longitudinal direction of the container main body 10, and the first resin sheet 11 and the second resin sheet 12 around it. Are joined by heat fusion and integrated with the container body 10. Similarly, the tube portion 311 of the first outlet port 31 and the tube portion 321 of the second outlet port 32 have longitudinal ends on the side where the

needleless ports

312 and 322 are not disposed. The container body is disposed between the first resin sheet 11 and the second resin sheet 12 on the side of the other end, and the first resin sheet 11 and the second resin sheet 12 around it are joined by heat fusion to form the container body Integrated with 10 The pipe portion 211 of the first inlet port 21, the pipe portion 221 of the second inlet port 22, the pipe portion 231 of the third inlet port 23, the pipe portion 311 of the first outlet port 31, and the pipe portion 321 of the second outlet port 32 These are channels that respectively communicate the housing portion 14 of the container body 10 with the outside of the container body 10, and can communicate with other channels via the female luer lock 212 and the

needleless ports

222, 232, 312, 322. It is.

In the present embodiment, through

holes

15, 15 are formed in portions outside the fused portion 13 at both ends of the side where the first to

third inlet ports

21, 22, 23 of the container main body 10 are disposed. ing. The through hole 15 can be used to hook the container 1 on a hook or the like.

Another embodiment of a bag-like container formed of two flexible resin sheets is shown in FIG.
The container 1 of the present embodiment includes a container body 10, an inlet port 41 and an outlet port 51.

The inlet port 41 includes a pipe portion 411 forming a liquid flow path, a female luer lock 412 sealingly closing the inlet of the pipe portion 411 and a removable lid 413 protecting the female luer lock 412.

The outlet port 51 includes a tube portion 521 that forms a flow path of liquid, a needleless port 522 that closes the inlet of the tube portion 521 in a manner that allows communication with it, and a removable lid 523 that protects the needleless port 522. .

The container body 10 has a substantially square first resin sheet 11 and a second resin sheet 12 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 11 and the second resin sheet 12 are joined by heat fusion is referred to as a fusion part 13. The structures and functions of the container body 10, the inlet port 41 and the outlet port 51 in the embodiment shown in FIG. 4 are the same as those in the embodiment shown in FIG.

Another embodiment of a bag-like container formed of two flexible resin sheets is shown in FIG.
The container 1 of the present embodiment includes a container body 10 and an inlet port / outlet port 61.

The inlet port / outlet port 61 includes a pipe portion 611 which forms a flow path of liquid, a female luer lock 612 which closes the inlet of the tubular portion 611 so that the inlet can be communicated with it, and a removable lid 613 which protects the female luer lock 612. Equipped with

The container body 10 has a substantially rectangular first resin sheet 11 and a second resin sheet 12 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 11 and the second resin sheet 12 are joined by heat fusion is referred to as a fusion part 13. The structures and functions of the container body 10 and the inlet port / outlet port 61 in the embodiment shown in FIG. 5 are the same as those in the embodiment shown in FIG.

The container body 10 has a substantially rectangular first resin sheet 11 and a second resin sheet 12 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 11 and the second resin sheet 12 are joined by heat fusion is referred to as a fusion part 13. The structures and functions of the container body 10 and the inlet port / outlet port 61 in the embodiment shown in FIG. 5 are the same as those in the embodiment shown in FIG. The structure and function of the inlet port / outlet port 61 in the embodiment shown in FIG. 6 are the same as those of the embodiment shown in FIG.

Preferably, the bag-like container used in the present invention has A / B when the width in the width direction of the container containing the reaction mixture and the gas phase in plan view is A and the width in the longitudinal direction is B. Is 0.1 or more and 1 or less, more preferably 0.2 or more and 1 or less, more preferably 0.3 or more and 1 or less, and particularly preferably 0.5 or more and 1 or less. The use of a bag-like container with A / B in this range produces a surprising effect that the amount of cells released in the form of nucleated cells from the cell-containing sample is significantly increased. Although the cause is not necessarily clear, it is considered that the reaction mixture is easily stirred at the time of shaking.

Here, the plan view of the storage portion of the bag-like container is a plan view in a state where the empty bag-like container is spread on a plane so as to widen the area. It is preferable that A / B is the said range in the bag-like container which especially arrange | positions the sheet | seat of 2 sheets facing each other and it joins and forms like the bag-like container 1 shown in FIG. The plan view shape of the storage portion of the bag-like container is not limited to a substantially quadrangle as shown in FIG. 1, and may be any shape such as a circle, an ellipse, a triangle, or a pentagon or more polygon. 1 to 6 show the width A in the width direction and the width B in the longitudinal direction when the entire storage portion 14 is used as the storage portion in the bag-like container 1 whose plan view shape of the storage portion 14 is substantially square.

<Method of enzyme treatment>
The present invention
A method of enzymatically treating a cell-containing sample to release cells from the cell-containing sample,
A closed container comprising a reaction mixture containing a cell-containing sample and an enzyme and a gas phase is contained at a ratio of 7 vol% or more of the volume of the gas phase to the total volume of the reaction mixture and the gas phase. It is characterized by including a shaking step.

According to this method, the enzyme treatment of the cell-containing sample is carried out while shaking in a conventional flexible tube, or the cell-containing sample and the enzyme solution are contained in a flexible bag-like container by the usual method. This has the surprising effect that the amount of cells released in the form of nucleated cells from the cell-containing sample is significantly increased as compared to the case where the cells are packed without air and subjected to enzyme treatment while shaking. Although the cause is not necessarily clear, the wall surface of the bag-like container is flexible, so that even if free cells collide with the wall of the bag-like container during shaking, the impact is small and less likely to be damaged. It is presumed to be due to the fact that the reaction mixture is easily stirred by the gas phase during shaking because it contains a large amount of phases.

In the present method, the reaction mixture may be any one containing at least a cell-containing sample and an enzyme, but usually further contains a suitable aqueous medium. Examples of the aqueous medium include water, physiological saline, phosphate buffer, glucose solution, Ringer solution, Hanks solution, injection solution, culture medium, isotonic solution and the like.

In the reaction mixture containing an aqueous medium at the start of the enzyme treatment, the enzyme exists as a solution in which the enzyme is dissolved or suspended in the aqueous medium. This solution is called "enzyme solution". The enzyme solution is not limited to the one in which the whole amount of enzyme and aqueous medium are mixed and put into an enzyme solution and then put into the container, but the enzyme and the aqueous medium are separately put into the container and the enzyme solution is formed in the container Point to. The volume of the enzyme solution can be calculated by totaling the volumes of the enzyme, the enzyme solution and the aqueous medium which are introduced into the container in order to perform the enzyme treatment of the cell-containing sample. The ratio of the cell-containing sample to the enzyme solution in the reaction mixture contained in the bag-like container is preferably 50% of the volume of the enzyme solution with respect to the volume of the cell-containing sample such as a living tissue. It is 500%, more preferably 60% to 200%, more preferably 70% to 100%.

The gas phase may be a gas phase such as air, nitrogen, oxygen, inert gas and the like, but is preferably an air phase.

The ratio of the volume of the gas phase to the total volume of the reaction mixture and the gas phase is 7% by volume or more, preferably 9% by volume or more, more preferably 15% by volume or more, and more preferably 20% by volume or more. The upper limit is not particularly limited, but is preferably 80% by volume or less, more preferably 70% by volume or less, and more preferably 65% by volume or less.

2 stores the reaction mixture 201 and the gas phase 202 in the storage portion 14 of the bag-like container 1 shown in FIG. 1, and the side of the first inlet port 21, the second inlet port 22, and the third inlet port 23 is It shows a state where it is disposed vertically upward. Although not shown, when shaking the bag-like container 1 containing the reaction mixture 201 and the gas phase 202, the first inlet port 21, the second inlet port 22, the third inlet port 23, the first outlet port 31. The second outlet port 32 is sealed by the female luer lock 212, the needleless port 222, the needleless port 232, the needleless port 312, and the needleless port 322, respectively. The cell-containing sample, the enzyme, the aqueous medium, the air, and the like may be supplied to the storage unit 14 from any of the first inlet port 21, the second inlet port 22, and the third inlet port 23.

When the volume of the container consisting of the reaction mixture 201 and the gas phase 202 is much smaller than the internal volume of the container 14 of the bag-like container 1 (the maximum volume of the container which can be accommodated by the container 14) ( For example, in the case where the volume ratio is 50% or less), as shown in FIG. 3, the bag-like container 1 is sealed by the clamp 300, and the portion 14A of the storage portion 14 of the bag-like container 1 The internal volume of the container may be adjusted to be close to the volume of the contents. In addition, FIG. 3 is the figure which put the bag-like container 1 which accommodated the reaction liquid mixture 201 and the gaseous phase 202, sealed by the clamp 300 on the horizontal surface, and was planarly viewed. As shown in FIG. 3, when the reaction mixture and the gas phase are contained only in one portion without using the entire container of the bag-like container, the width A in the lateral direction and the width B in the longitudinal direction This refers to the width in the lateral direction and the width in the longitudinal direction in plan view of the portion containing the liquid and the gas phase. Specifically, as shown in FIG. 3, when the reaction liquid mixture and the gas phase are stored only in the portion 14A of the storage portion 14 formed by sealing the bag-like container 1 by the clamp 300, the storage portion 14 The lateral width of the portion 14A is A, and the longitudinal width is B.

As a method of shaking the bag-like container containing the reaction mixture and the gas phase at the above ratio and closed, the bag-like container is a straight line on a horizontal surface or a surface intersecting with the horizontal surface (particularly, a surface substantially perpendicular to the horizontal surface) Reciprocating motion, pivoting along a closed track, and / or a method of pivoting may be mentioned. These shaking methods allow cells to be released from cell-containing samples in the form of live cells. Here, a plane substantially perpendicular to the horizontal plane means a plane having an angle of, for example, 70 ° to 90 ° with the horizontal plane. In the case of shaking a bag-like container formed by arranging two sheets facing each other and bonding the periphery as in the bag-like container 1 shown in FIG. 1, a plane intersecting the horizontal plane or the horizontal plane, and two sheets It is preferable to arrange the bag-like container so that the direction in which the sheets face each other is perpendicular, and shake the bag-like container on the horizontal plane or a plane intersecting the horizontal plane.

The time and temperature of the step of shaking the bag-like container may be appropriately set according to the type and concentration of the cell-containing sample and the enzyme, and can be, for example, 25 to 40 ° C. for 10 to 40 minutes.

In the step of shaking the bag-like container containing the reaction mixture and a predetermined amount of gas phase, the cell-containing sample is enzymatically treated in the bag-like container and the cells are released in a living state. The released cells can be separated from the reaction mixture by a method such as filter filtration or centrifugation, and can be washed and recovered as necessary.

The adipose tissue from 5 persons (donor 1, donor 2, donor 3, donor 4, donor 5) was treated with collagenase and the steps of preparing the cell suspension under different conditions.

The following first enzyme treatment bag and second enzyme treatment bag are both specific examples of the bag-like container 1 shown in FIG.

As a first bag for enzyme treatment (bag-like container 1), two roughly rectangular

flexible sheets

11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm are disposed opposite to each other, and on one end side in the longitudinal direction A first inlet port 21, a second inlet port 22, and a third inlet port 23 are provided, and a first outlet port 31 and a second outlet port 32 are provided on the other end side, and the peripheral portion is fused by heat fusion. Forming a fusion-bonded portion 13 to form a bag, which is sandwiched between the two

sheets

11 and 12 and has a substantially rectangular housing portion 14 with an inner dimension of 114 mm wide and 196 mm long in an empty state Using. The internal volume (the maximum volume of the contents that can be accommodated by the accommodation portion 14) of the accommodation portion 14 of the first enzyme treatment bag is about 600 mL.

As a second bag for enzyme treatment (bag-like container 1), two roughly rectangular

flexible sheets

11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm are disposed opposite to each other, and one end side in the longitudinal direction A first inlet port 21, a second inlet port 22, and a third inlet port 23 are provided, and a first outlet port 31 and a second outlet port 32 are provided on the other end side, and the peripheral portion is fused by heat fusion. Forming a fusion-bonded portion 13 to form a bag, which is sandwiched between the two

sheets

11 and 12 and has a substantially rectangular storage portion 14 having an inner dimension of 67 mm in width and 116 mm in length in an empty state Using. The internal volume of the storage portion 14 of the second enzyme treatment bag is about 120 mL.

The third enzyme treatment bag (bag-like container 1) is shown in FIG. 4, the fourth enzyme treatment bag (bag-like container 1) in FIG. 5, and the fifth enzyme treatment bag (bag-like container 1) Each is shown in FIG.

In the third bag for enzyme treatment (bag-like container 1) shown in FIG. 4, two approximately square

flexible sheets

11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm are disposed opposite to each other, and on one side The inlet port 41 and the outlet port 51 are provided, and the peripheral portion is fusion-bonded by heat fusion to form the fusion-bonded portion 13 so that the two

sheets

11 and 12 are sandwiched between the two in an empty state. It is a bag in which a substantially square housing portion 14 having a size of 58 mm in width and 58 mm in length is formed. The internal volume of the container 14 for the third enzyme treatment bag is about 43 mL.

The fourth bag for enzyme treatment (bag-like container 1) shown in FIG. 5 has two substantially rectangular

flexible sheets

11 and 12 of soft polyvinyl chloride of 0.4 mm in thickness disposed opposite to each other, and one end in the longitudinal direction The inlet port / outlet port 61 is provided on the side of the sheet, and the peripheral part is fusion-bonded by heat fusion to form the fusion-bonded part 13 so that the two

sheets

11 and 12 are empty. In the bag, a substantially rectangular housing portion 14 having an inner dimension of 1323 mm and a length of 252 mm is formed. The internal volume of the container 14 for the fourth enzyme treatment bag is about 33 mL.

In the fifth bag for enzyme treatment (bag-like container 1) shown in FIG. 6, two approximately rectangular

flexible sheets

11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm are oppositely disposed, and one end in the longitudinal direction The inlet port / outlet port 61 is provided on the side of the sheet, and the peripheral part is fusion-bonded by heat fusion to form the fusion-bonded part 13 so that the two

sheets

11 and 12 are empty. The bag is a bag in which a substantially rectangular housing portion 14 having an inner dimension of 18 mm in width and 187.7 mm in length is formed. The internal volume of the container 14 for the fifth enzyme treatment bag is about 22 mL.

As a centrifuge tube, a 50 mL centrifuge tube made of polypropylene was used.
The first outlet port 31 and the second outlet port 32 of the first or second enzyme treatment bag 1 are closed with

lids

313 and 323, and 10 to 50 mL of fat tissue and 10 to 50 mL of fat tissue are contained in the storage unit 14. Collagenase solution (prepared by dissolving collagenase with physiological saline so that the concentration of collagenase is 0.1 w / v%) and the volume ratio of adipose tissue to collagenase solution are 1: 1 to 1: 3. The volume of the air phase 202 in the storage unit 14 is set as shown in Table 1 with respect to the volume of the storage unit 14 (that is, the total volume of the reaction mixture 201 and the air phase 202). Add air so as to become a percentage (%), the first inlet port 21, the second inlet port 22, and the third inlet port 24 of the first or second bag for enzyme treatment 1 with

lids

213, 223, 233. Sealed and sealed.

Similarly, the outlet port 51 of the third enzyme treatment bag 1 is closed with a lid 313, and 20 mL of adipose tissue and 20 mL of collagenase solution are accommodated in the accommodation portion 14 to form a reaction mixture solution 201, and is accommodated. 3 mL of air is added so that the volume of the air phase 202 in the section 14 becomes 7% of the volume of the accommodation section 14, and the inlet port 41 of the third enzyme treatment bag 1 is sealed with a lid 413 for sealing did.

Similarly, 15 mL of fat tissue and 15 mL of collagenase solution are stored in the storage portion 14 of the fourth enzyme processing bag 1 to form a reaction mixture 201, and the volume of the air phase 202 in the storage portion 14 is 2.2 mL of air was added so as to be 7% with respect to the volume of the housing portion 14, and the inlet port / outlet port 61 of the fourth enzyme treatment bag 1 was sealed with a lid 613 and sealed.

Similarly, 10 mL of fat tissue and 10 mL of collagenase solution are stored in the storage portion 14 of the fifth bag 1 for enzyme treatment to form a reaction liquid mixture 201, and the volume of the air phase 202 in the storage portion 14 is 1.5 mL of air was added so as to be 7% with respect to the volume of the housing portion 14, and the inlet port / outlet port 61 of the fifth enzyme treatment bag 1 was sealed with a lid 613 and sealed.

Here, in the test using adipose tissue from donor 1, donor 2 or donor 3, the first enzyme treatment bag was used as the enzyme treatment bag. In the test using fat tissue from the donor 4, a second enzyme treatment bag was used as the enzyme treatment bag. In the tests using adipose tissue from the donor 5, third to fifth enzyme treatment bags were used as enzyme treatment bags.

The internal volume of the first enzyme treatment bag is 600 mL, while the content of the reaction mixture containing fatty tissue and collagenase solution and the gas phase is at most 100 mL. The volume is small in comparison. For this reason, in the test using the first enzyme treatment bag, the first enzyme treatment bag containing the reaction mixture and the gas phase in the storage portion is partially sealed by the clamp 300 as shown in FIG. Then, in the accommodation portion 14 of the first enzyme treatment bag 1, the width direction width indicated by "A" in FIG. As shown in Table 1, it adjusted so that it might be set to 100 mm or 75 mm.

Such adjustment was not performed when using the second, third, fourth and fifth bags for enzyme treatment in which the internal volume of the container was about 120 mL, about 43 mL, about 33 mL and about 22 mL.

In the test using the centrifuge tube, the centrifuge tube is prepared by dissolving 10 to 23.3 mL of adipose tissue and 20 to 30 mL of collagenase solution (a concentration of collagenase is 0.1 w / v% with physiological saline). ) And the lid was closed and sealed so that the volume ratio of adipose tissue to collagenase solution was 1: 1 to 1: 3. At this time, the volume of the gas phase in the centrifuge tube was 20% or 7% with respect to the volume of the centrifuge tube (total volume of fat tissue, collagenase solution and gas phase).

The first to fifth enzyme treatment bags or centrifuge tubes containing fat tissue, collagenase solution and gas phase are subjected to conditions of 60 rpm (200 rpm when using donors 1 and 2 fat tissue samples) for 30 minutes at 37 ° C. The enzyme reaction was carried out by shaking with a shaker. When the first to fifth enzyme treatment bags are shaken, the bag is fixed so that one side of the two

sheets

11 and 12 constituting the bag is in contact with the installation surface of the shaker table, and the shaking is performed. The bag was shaken by shaking the platform in the direction along the mounting surface.

After the enzyme reaction for 30 minutes, the first to fifth enzyme treatment bags or centrifuge tubes are allowed to stand to separate the reaction mixture into an aqueous layer (lower layer) and an oil layer containing fat (upper layer), and the aqueous layer is separated. The number of nucleated cells and the number of adipose-derived stem cells in the aqueous layer were collected using a flow cytometer and BD TrucountTubes. The number of nucleated cells was calculated from the sum of the number of CD34 positive cells and the number of CD45 positive cells. Similarly, the number of adipose-derived stem cells was calculated from the number of CD34 positive / CD45 negative / CD31 negative cells.
The results are shown in Table 1.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A method of enzymatically treating a cell-containing sample to release cells from the cell-containing sample,
A closed container comprising a reaction mixture containing a cell-containing sample and an enzyme and a gas phase is contained at a ratio of 7 vol% or more of the volume of the gas phase to the total volume of the reaction mixture and the gas phase. A method characterized in that it comprises the step of shaking.
The method according to claim 1, wherein the cell-containing sample is adipose tissue.
A / B is 0.1 or more and 1 or less, where the width in a widthwise direction is A and the width in a longitudinal direction is B in plan view of the container containing the reaction mixture and the gas phase of the bag-like container The method according to claim 1 or 2.