JP7282679B2 - Method for releasing cells from cell-containing sample - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method of liberating cells from a cell-containing sample such as biological tissue by enzymatically treating the cell-containing sample.

At least part of the nucleated cells contained in adipose tissue are biological tissue stem cells, and they are capable of differentiating into various cells such as mature adipocytes, osteocytes, chondrocytes, myoblasts, and vascular endothelial cells. It has been known. A method for efficiently isolating and collecting such pluripotent adipose-derived biological tissue stem cells is extremely important from the standpoint of the development of regenerative medicine.

As a method for obtaining useful cells from a biological tissue containing useful cells, such as adipose tissue, the biological tissue is degraded with a digestive enzyme to release the useful cells, and then the useful cells are subjected to a separation process such as centrifugation and filtration. is known.

For example, in Example 1 of Patent Document 1, adipose tissue and a collagenase solution are placed in a 50 mL centrifuge tube, shaken at 37° C. and 120 times/min for 1 hour to perform an enzymatic reaction, and then filtered. Filtration and centrifugation are performed to obtain a sedimented cell population (SVF fraction) as a sediment.

Patent Document 2 discloses a device for separating non-adipocytes from an adipose tissue sample, which comprises a first sheet of material, a second sheet of material bonded to the first sheet of material, and the first sheet of material. A device is disclosed comprising a substance and a plurality of chambers defined between said second sheet of substance. Then, in one of the plurality of chambers, a first chamber, the adipose tissue sample is treated with a dissociation solution containing an enzyme such as collagenase to dissociate the adipose tissue sample.

WO2008/018450 Japanese translation of PCT publication No. 2015-500031

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

However, it cannot be said that the relationship between the amount of the desired cells to be collected and the conditions such as the vessel, shaking method, etc. during the enzymatic treatment for releasing the cells from the cell-containing sample has been sufficiently investigated so far.

The present inventors surprisingly found that when the enzymatic treatment of a cell-containing sample is performed while shaking in a normal inflexible centrifuge tube as in Patent Document 1, a sufficient number of nucleated cells are generated. I found that I can't get it. Surprisingly, the present inventors also filled a flexible bag-shaped container with a cell-containing sample and an enzyme solution in a conventional manner so as not to contain air, and performed enzyme treatment while shaking. Also in this case, we found that a sufficient number of nucleated cells could not be obtained. As a result of intensive studies, the inventors of the present invention completed the following method.

(1) A method of enzymatically treating a cell-containing sample to release cells from the cell-containing sample,
A closed bag-like container containing a reaction mixture containing a cell-containing sample and an enzyme and a gas phase in such a ratio that the volume of the gas phase is 7% by volume or more of the total volume of the reaction mixture and the gas phase. A method comprising the step of shaking.
(2) The method according to (1), wherein the cell-containing sample is adipose tissue; The method according to (1) or (2), wherein A/B is 0.1 or more and 1 or less, where B is the width in the longitudinal direction.
This specification includes the disclosure content of Japanese Patent Application No. 2017-178267, which is the basis of priority of this application.

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

An example of an enzyme treatment bag (bag-like container) for enzymatically treating a cell-containing sample is shown. FIG. 3 is a schematic cross-sectional view for explaining the state of a reaction mixture containing a cell-containing sample and an enzyme and a gas phase in an enzyme treatment bag. The enzyme treatment bag shown in FIG. 1 to which the reaction mixture containing the cell-containing sample and the enzyme and the gas phase (contents) are added is sealed with a clamp, and the contents are removed from the storage part of the enzyme treatment bag. FIG. 10 is a schematic diagram for explaining that the volume of the portion to be accommodated is adjusted to match the volume of the object to be accommodated; Another example of an enzyme treatment bag (bag-like container) for enzymatically treating a cell-containing sample is shown. Another example of an enzyme treatment bag (bag-like container) for enzymatically treating a cell-containing sample is shown. Another example of an enzyme treatment bag (bag-like container) for enzymatically treating a cell-containing sample is shown.

The present invention will be described in detail below.
<Terms, materials>
Examples of cell-containing samples include biological tissues, cell cultures prepared in vitro, and the like. A biological tissue is typically a biological tissue collected from an animal, for example, fat, skin, blood vessel, 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 stromal vascular fraction (SVF) cells from adipose tissue.

Adipose tissue is typically mammalian adipose tissue, for example, human-derived subcutaneous fat, visceral fat, white fat, and brown fat. The adipose tissue may be of any shape. For example, adipose tissue crushed using a sharp instrument such as scissors, minced using a strainer or the like, or decomposed using liposuction. can be anything. Here, the liposuction method is not particularly limited as long as it is a suction method that is commonly performed in cosmetic plastic surgery. is.
A nucleated cell is a cell having a nucleus within the cell.

Cells released in the present invention include biological tissue stem cells, leukocytes, monocytes, granulocytes, lymphocytes, vascular endothelial cells, vascular endothelial progenitor cells, pericytes, etc., which need to be collected for purposes such as cell therapy and experiments. nucleated cells can be exemplified. Biological tissue stem cells are preferably adipose-derived mesenchymal stem cells and adipose-derived stromal stem cells, and more preferably express at least one selected from cell surface CD34, 73, 90, 105, 106, 133, and 166. adipose-derived mesenchymal cells and adipose-derived stromal stem cells.

At least one degrading enzyme selected from collagenase, metalloprotease, dispase, trypsin, hyaluronidase, chymotrypsin, pepsin, aminopeptidase, lipase, amylase, and recombinants thereof is used as the enzyme for releasing cells from the cell-containing sample. can. When a biological tissue, particularly adipose tissue, is used as a cell-containing sample, the enzyme is at least one selected from collagenase, metalloprotease, dispase, trypsin, and hyaluronidase, from the viewpoint of short-time and minimally invasive decomposition. Seed degrading enzymes are preferred.

<Bag-like container>
The bag-like container used in the present invention is a bag-like container made of a material having softness (also referred to as flexibility). The bag-like container is preferably made of a transparent or semi-transparent material at least in part because the state of the cell suspension inside can be visually observed.

Specific examples of materials having flexibility for forming the bag-like container include soft vinyl chloride, vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefins such as polyethylene and polypropylene, and styrene-butadiene-styrene copolymer. or thermoplastic elastomers such as hydrogenated products thereof and styrene-isoprene-styrene copolymers or hydrogenated products thereof, and mixtures of thermoplastic elastomers with polyolefins and softeners such as ethylene-ethyl acrylate. resins having

In an 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 having a wall surface formed of a flexible resin sheet, and particularly preferably, two flexible resin sheets are arranged facing each other, and the peripheral edges are heat-sealed or bonded. It is a container formed into a bag shape by bonding with an adhesive.

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 that forms a liquid flow path, a female luer lock 212 that blocks the inlet of the tube portion 211 so as to allow communication, and a detachable lid portion 213 that protects the female luer lock 212. , and a filter portion 214 for filtering the liquid passing through the tube portion 211 .

The second inlet port 22 includes a tube portion 221 forming a liquid flow path, a needleless port 222 communicably blocking the inlet of the tube portion 221, and a detachable lid portion 223 protecting the needleless port 222. Prepare.

The third inlet port 23 includes a tube portion 231 forming a liquid flow path, a needleless port 232 communicably blocking the inlet of the tube portion 231, and a detachable lid portion 233 protecting the needleless port 232. Prepare.

The first outlet port 31 includes a tube portion 311 that forms a liquid flow path, a needleless port 312 that communicably blocks the outlet of the tube portion 311, and a detachable lid portion 313 that protects the needleless port 312. Prepare.

The second outlet port 32 includes a tube portion 321 that forms a liquid flow path, a needleless port 322 that communicably blocks the outlet of the tube portion 321, and a detachable lid portion 323 that protects the needleless port 322. Prepare.

The container body 10 is formed by arranging a substantially rectangular first resin sheet 11 and a second resin sheet 12 so as to face each other, and joining the peripheral edges thereof by thermal fusion to form a bag. A portion where the first resin sheet 11 and the second resin sheet 12 are joined by heat-sealing is referred to as a fused portion 13 . A housing portion 14 is formed between the first resin sheet 11 and the second resin sheet 12 in the container body 10 . An enzymatic treatment, which will be described later, is performed in the container 14 . The tube portion 211 of the first inlet port 21, the tube portion 221 of the second inlet port 22, and the tube portion 231 of the third inlet port 23 are arranged on the side where the female luer lock 212 and the needleless ports 222, 232 are not arranged. is arranged between the first resin sheet 11 and the second resin sheet 12 on one end side in the longitudinal direction of the container body 10, and the first resin sheet 11 and the second resin sheet 12 around it. are joined by heat sealing to be integrated with the container body 10 . Similarly, the pipe portion 311 of the first outlet port 31 and the pipe portion 321 of the second outlet port 32 have their ends on the side where the needleless ports 312 and 322 are not arranged, extending in the longitudinal direction of the container body 10 . It is arranged between the first resin sheet 11 and the second resin sheet 12 on the other end side, and the first resin sheet 11 and the second resin sheet 12 around it are joined by heat sealing to form a container body. 10 are integrated. 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 are , are flow paths that communicate between the housing portion 14 of the container body 10 and the outside of the container body 10, and can communicate with other flow paths via the female luer lock 212 and the needleless ports 222, 232, 312, 322. is.

In this embodiment, through holes 15, 15 are formed in portions outside the fused portion 13 at both ends of the sides of the container body 10 on which the first to third inlet ports 21, 22, 23 are arranged. ing. Through hole 15 can be used to hang container 1 on a hook or the like.

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

The inlet port 41 includes a tube portion 411 that forms a liquid flow path, a female luer lock 412 that communicably blocks the inlet of the tube portion 411 , and a detachable lid portion 413 that protects the female luer lock 412 .

The outlet port 51 includes a tube portion 521 that forms a liquid flow path, a needleless port 522 that communicably blocks the inlet of the tube portion 521, and a detachable lid portion 523 that protects the needleless port 522. .

The container body 10 is formed by arranging a substantially square first resin sheet 11 and a second resin sheet 12 so as to face each other, and joining the peripheral edges thereof by thermal fusion to form a bag. A portion where the first resin sheet 11 and the second resin sheet 12 are joined by heat-sealing is referred to as a fused portion 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 this embodiment comprises a container body 10 and an inlet/outlet port 61 .

The inlet/outlet port 61 includes a tube portion 611 that forms a liquid flow path, a female luer lock 612 that blocks the inlet of the tube portion 611 so as to be able to communicate with it, and a detachable lid portion 613 that protects the female luer lock 612. Prepare.

The container body 10 is formed by arranging a substantially rectangular first resin sheet 11 and a second resin sheet 12 so as to face each other, and joining the peripheral edges thereof by thermal fusion to form a bag. A portion where the first resin sheet 11 and the second resin sheet 12 are joined by heat-sealing is referred to as a fused portion 13 . The structure and function of the container body 10 and the inlet/outlet port 61 in the embodiment shown in FIG. 5 are the same as those in the embodiment shown in FIG.

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

The container body 10 is formed by arranging a substantially rectangular first resin sheet 11 and a second resin sheet 12 so as to face each other, and joining the peripheral edges thereof by thermal fusion to form a bag. A portion where the first resin sheet 11 and the second resin sheet 12 are joined by heat-sealing is referred to as a fused portion 13 . The structure and function of the container body 10 and the inlet/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/outlet port 61 in the embodiment shown in FIG. 6 are the same as those in the embodiment shown in FIG. 5, and the description thereof is omitted.

The bag-like container used in the present invention preferably has a ratio of A/B, where A is the width in the lateral direction and B is the width in the longitudinal direction of the storage portion that stores the reaction mixture and the gas phase in plan view. 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. By using a bag-like container with A/B in this range, there is a surprising effect that the amount of cells released in the state of nucleated cells from a cell-containing sample is remarkably increased. The reason for this is not entirely clear, but it is believed that the reaction mixture is easily agitated during shaking.

Here, the planar view of the accommodating portion of the bag-like container is a planar view of the empty bag-like container spread out on a plane so as to have the largest area. In particular, like the bag-like container 1 shown in FIG. 1, it is preferable that A/B is within the above range in a bag-like container formed by arranging two sheets facing each other and joining the periphery thereof. The plan view shape of the storage portion of the bag-like container is not limited to the substantially quadrangular shape shown in FIG. 1, and may be any desired shape such as substantially circular, elliptical, triangular, or polygonal with pentagons or more. 1 to 6 show width A in the lateral direction and 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 approximately square.

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

According to this method, a cell-containing sample is subjected to enzymatic treatment while being shaken in an ordinary rigid centrifuge tube, or a cell-containing sample and an enzyme solution are placed in a flexible bag-like container by a conventional method. As compared with the case where the enzyme treatment is performed while the container is filled without containing air and shaken, the amount of cells released in the state of nucleated cells from the cell-containing sample is remarkably increased, which is a surprising effect. The reason for this is not entirely clear, but the walls of the bag-like container are flexible, so even if the free cells collide with the walls of the bag-like container during shaking, the impact is small and they are less likely to be damaged. It is presumed that this is due to the fact that the reaction mixture is likely to be stirred by the gas phase during shaking due to the presence of many phases.

In this method, the reaction mixture may contain at least the cell-containing sample and the enzyme, but usually further contains an appropriate aqueous medium. Examples of aqueous media include water, physiological saline, phosphate buffer, glucose solution, Ringer's solution, Hank's solution, injection solutions, media, isotonic solutions, and the like.

In the reaction mixture containing the aqueous medium at the start of enzyme treatment, the enzyme exists as a solution in which the enzyme is dissolved or suspended in the aqueous medium. This liquid is called "enzyme liquid". The enzyme solution is not limited to the one in which the entire amount of the enzyme and the aqueous medium are mixed in advance to prepare the enzyme solution and then put into the container, but the enzyme solution and the aqueous medium are separately put into the container and the enzyme solution is formed in the container. Point. The volume of the enzyme solution can be calculated by totaling the volumes of the enzyme, the enzyme solution, and the aqueous medium put into the container for enzymatic treatment of the cell-containing sample. The ratio of the cell-containing sample and the enzyme solution in the reaction mixture contained in the bag-like container is preferably from 50% to 50% of the volume of the cell-containing sample such as biological tissue. 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, etc., 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, more preferably 20% by volume or more, Although the upper limit is not particularly limited, it is preferably 80% by volume or less, more preferably 70% by volume or less, and more preferably 65% by volume or less.

In FIG. 2, the reaction mixture 201 and the gas phase 202 are accommodated in the accommodating portion 14 of the bag-like container 1 shown in FIG. It shows a state in which it is arranged 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 are blocked by a female luer lock 212, a needleless port 222, a needleless port 232, a needleless port 312, and a needleless port 322, respectively. A cell-containing sample, an enzyme, an aqueous medium, air, or the like may be supplied to the container 14 from any one of the first inlet port 21, the second inlet port 22, and the third inlet port 23.

When the volume of the content consisting of the reaction mixture 201 and the gas phase 202 is significantly smaller than the internal volume of the storage portion 14 of the bag-like container 1 (maximum volume of the content that the storage portion 14 can accommodate) ( For example, when the volume ratio is 50% or less), as shown in FIG. The internal volume of the container may be adjusted to be close to the volume of the contents. FIG. 3 is a plan view of the bag-like container 1 containing the reaction mixture 201 and the gas phase 202 and sealed with a clamp 300 placed on a horizontal plane. As shown in FIG. 3, when the reaction mixed liquid and the gas phase are contained in only a part of the containing portion of the bag-shaped container without using the entire containing portion, the width A in the lateral direction and the width B in the longitudinal direction are It refers to the width in the lateral direction and the width in the longitudinal direction of the portion that accommodates the liquid and gas phase when viewed from above. Specifically, as shown in FIG. 3, when the reaction mixture and the gas phase are contained only in a portion 14A of the container 14 formed by sealing the bag-like container 1 with a clamp 300, the container 14 Let A be the width in the lateral direction of the portion 14A, and B be the width in the longitudinal direction.

As a method for shaking a sealed bag-like container containing the reaction mixture and the gas phase at the above ratio, the bag-like container is placed on a horizontal plane or a plane intersecting the horizontal plane (especially a plane substantially perpendicular to the horizontal plane), and shaken in a straight line. Methods include reciprocating motion, pivoting motion along a closed track, and/or pivoting. These shaking methods make it possible to release the cells in the viable state from the cell-containing sample. Here, the plane substantially perpendicular to the horizontal plane refers to a plane forming 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 joining their periphery like the bag-like container 1 shown in FIG. It is preferable to arrange the bag-like container so that the direction in which the sheets face each other is perpendicular, and to 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 enzyme, and examples include 10 to 40 minutes and 25 to 40°C.

By shaking the bag-shaped container containing the reaction mixture and a predetermined amount of the gas phase, the cell-containing sample is treated with enzymes in the bag-shaped container, and the cells are released in a viable state. The liberated cells can be separated from the reaction mixture by a method such as filter filtration or centrifugation, and washed and collected as necessary.

Adipose tissue from 5 individuals (Donor 1, Donor 2, Donor 3, Donor 4, Donor 5) was treated with collagenase and the process of preparing cell suspensions was performed 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 the first enzymatic treatment bag (bag-shaped container 1), two roughly rectangular flexible sheets 11 and 12 made of soft polyvinyl chloride with a thickness of 0.4 mm are placed opposite each other, and placed on one end in the longitudinal direction. A first inlet port 21, a second inlet port 22 and a third inlet port 23 are provided, a first outlet port 31 and a second outlet port 32 are provided on the other end side, and the peripheral edges are fused by heat fusion. By forming the fusion-bonded portion 13, a bag having a substantially rectangular storage portion 14 with an inner dimension of 114 mm in width and 196 mm in length in an empty state sandwiched between the two sheets 11 and 12 is formed. Using. The internal volume of the storage portion 14 of the first enzyme treatment bag (the maximum volume of the contents that the storage portion 14 can store) is about 600 mL.

As a second enzymatic treatment bag (bag-like container 1), two roughly rectangular flexible sheets 11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm are arranged opposite each other, and are placed on one end in the longitudinal direction. A first inlet port 21, a second inlet port 22 and a third inlet port 23 are provided, a first outlet port 31 and a second outlet port 32 are provided on the other end side, and the peripheral edges are fused by heat fusion. By forming the fusion-bonded portion 13, a bag in which a substantially rectangular storage portion 14 having an inner dimension of 67 mm in width and 116 mm in length in an empty state sandwiched between the two sheets 11 and 12 is formed. Using. The content 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) is shown in FIG. 5, and the fifth enzyme treatment bag (bag-like container 1) is shown in FIG. FIG. 6 shows each.

The third enzymatic treatment bag (bag-shaped container 1) shown in FIG. An inlet port 41 and an outlet port 51 are provided in the inner portion, and the peripheral portion is fused by heat-sealing to form a fused portion 13, so that the inner portion is sandwiched between the two sheets 11 and 12 in an empty state. It is a bag in which a substantially square containing portion 14 having dimensions of 58 mm in width and 58 mm in length is formed. The content of the storage portion 14 of the third enzyme treatment bag is about 43 mL.

The fourth enzymatic treatment bag (bag-like container 1) shown in FIG. 5 comprises two generally rectangular flexible sheets 11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm and arranged facing each other. An inlet port/outlet port 61 is provided on the side of , and the peripheral edge is fused by heat-sealing to form a fused portion 13, thereby sandwiching the two sheets 11 and 12 in an empty state. This is a bag in which a substantially rectangular containing portion 14 having internal dimensions of 1323 mm in width and 252 mm in length is formed. The content of the storage portion 14 of the fourth enzyme treatment bag is about 33 mL.

The fifth enzymatic treatment bag (bag-like container 1) shown in FIG. 6 comprises two generally rectangular flexible sheets 11 and 12 of soft polyvinyl chloride having a thickness of 0.4 mm and arranged facing each other. An inlet port/outlet port 61 is provided on the side of , and the peripheral edge is fused by heat-sealing to form a fused portion 13, thereby sandwiching the two sheets 11 and 12 in an empty state. The bag is formed with a substantially rectangular containing portion 14 having internal dimensions of 18 mm in width and 187.7 mm in length. The content of the storage portion 14 of the fifth enzyme treatment bag is about 22 mL.

As the centrifuge tube, a centrifuge tube made of polypropylene and having an internal capacity of 50 mL 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 adipose tissue and 10 to 50 mL of Collagenase solution (prepared by dissolving collagenase in physiological saline so that the collagenase concentration is 0.1 w/v%) so that the volume ratio of adipose tissue and collagenase solution is 1: 1 to 1: 3 The reaction mixture 201 is accommodated, and the volume of the air phase 202 in the accommodation portion 14 is the predetermined value shown in Table 1 with respect to the volume of the accommodation portion 14 (that is, the total volume of the reaction mixture 201 and the air phase 202). Air is added to the ratio (%), and the first inlet port 21, the second inlet port 22, and the third inlet port 24 of the first or second enzyme treatment bag 1 are closed with lids 213, 223, and 233. Closed 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 section 14 to form a reaction mixture 201. 3 mL of air is added so that the volume of the air phase 202 in the part 14 becomes 7% of the volume of the storage part 14, and the inlet port 41 of the third enzyme treatment bag 1 is sealed with a lid 413. bottom.

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

Similarly, 10 mL of adipose tissue and 10 mL of collagenase solution are stored in the storage section 14 of the fifth enzyme treatment bag 1 to form a reaction mixture 201, and the volume of the air phase 202 in the storage section 14 is 1.5 mL of air was added so as to be 7% of the volume of the container 14, and the inlet/outlet port 61 of the fifth enzyme treatment bag 1 was closed with a lid 613 to seal it.

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

While the internal volume of the storage portion of the first enzyme treatment bag is 600 mL, the maximum volume of the storage containing the reaction mixture containing the adipose tissue and the collagenase solution and the gas phase is 100 mL. relatively small in volume. 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 container was partially sealed with a clamp 300 as shown in FIG. 3, the width of the portion 14A for containing the contents (reaction mixture and gas phase) in the containing portion 14 of the first enzyme treatment bag 1 is As shown in Table 1, it was adjusted to be 100 mm or 75 mm.

No such adjustments were made when using the second, third, fourth and fifth enzymatic treatment bags with container volumes of about 120 mL, about 43 mL, about 33 mL and about 22 mL.

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

The 1st to 5th enzyme treatment bags or centrifuge tubes containing adipose tissue, collagenase solution and gas phase were heated at 37° C. for 30 minutes at 60 rpm (200 rpm when using adipose tissue samples from donors 1 and 2). Enzyme reaction was carried out by shaking with a shaker. When shaking the first to fifth bags for enzymatic treatment, 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 shaking table of the shaker, and shaken. The bag was shaken by shaking the table in a direction along its mounting surface.

After the enzymatic reaction for 30 minutes, the first to fifth enzyme treatment bags or centrifuge tubes are left to stand to separate the reaction mixture into a water layer (lower layer) and an oil layer containing fat (upper layer), and the water layer is separated. After harvesting, the number of nucleated cells and the number of adipose-derived stem cells in the aqueous layer were measured using a flow cytometer and BD Trucount Tubes. 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.
Table 1 shows the results.

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

Claims (4)

A method of enzymatically treating a cell-containing sample to release cells from the cell-containing sample, comprising:
A bag-like container in which a reaction mixture containing a cell-containing sample and an enzyme and a gas phase are contained and closed at a ratio such that the volume of the gas phase is 20 % by volume or more with respect to the total volume of the reaction mixture and the gas phase. including the step of shaking,
A method characterized in that the bag-like container has flexibility. The method according to claim 1, wherein the bag-like container is made of a resin sheet with a thickness of 0.2 mm to 0.6 mm. 3. The method of claim 1 or 2, wherein the cell-containing sample is adipose tissue. A/B is not less than 0.1 and not more than 1, where A is the width in the lateral direction and B is the width in the longitudinal direction of the storage portion for storing the reaction mixture and the gas phase in the bag-like container in plan view. , the method according to any one of claims 1 to 3.

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