WO2015163010A1

WO2015163010A1 - Morcellation device

Info

Publication number: WO2015163010A1
Application number: PCT/JP2015/056248
Authority: WO
Inventors: 武雄熊谷; 小林　明; 崇亘 ▲濱▼淵; 浩太郎吉村; 慎一郎久野
Original assignee: 株式会社カネカ; 国立大学法人東京大学
Priority date: 2014-04-23
Filing date: 2015-03-03
Publication date: 2015-10-29
Also published as: JP6592781B2; JPWO2015163010A1

Abstract

　The purpose of the present invention is to provide a morcellation device for efficiently preparing stem cells from a biological tissue. The morcellation device pertaining to the present invention has a cylindrical part (7), a rotary shaft (1) provided in the cylindrical part (7), a morcellation means (4) including a stirring member (3) and a rotary blade (2) provided to the rotary shaft (1), and a stabilizing member (6) provided to the rotary shaft (1) and having one or more through-paths (5).

Description

Shredding device

The present invention relates to an apparatus for efficiently preparing cells from a living tissue or the like.

The cells constituting the living body are broadly classified into differentiated cells, TA cells (Transient Amplifying Cell) and stem cells. Differentiated cells are also called terminally differentiated cells or terminally differentiated cells, and are not further differentiated into different types of cells, such as nerve cells and cells constituting organs, and hardly proliferate. TA cells are intermediate between differentiated cells and stem cells, and after differentiation, actively proliferate to become differentiated cells. Stem cells are defined as cells having both self-renewal ability and pluripotency, and can self-proliferate and differentiate into TA cells.

Stem cells are also pluripotent cells that can form individuals, such as fertilized eggs, pluripotent cells that can differentiate into any cell such as ES cells and iPS cells, and differentiable cell lines such as neural stem cells and hematopoietic stem cells However, it is classified as a pluripotent cell that can differentiate into various cell types. That is, since stem cells have the ability to differentiate into various cells, if stem cells are used, there is a possibility that they can be differentiated into target cells to regenerate damaged skin or organs and be useful for treatment.

However, ES cell production has an ethical problem in that it requires fertilized eggs and early embryos that can be individuals. In addition, proto-oncogenes are used for the production of iPS cells, and there is no guarantee that cells and tissues obtained by differentiating iPS cells will not become cancerous. On the other hand, mesenchymal stem cells that differentiate into mesenchymal cells such as ligament cells and cardiomyocytes are not isolated from the above problems, but have been isolated from the bone marrow and thus are highly invasive and can be obtained. The number of cells is limited. However, at present, it has been clarified that stem cells are present in large amounts in adipose tissue and umbilical cord matrix that can be collected with minimal invasiveness, and various techniques for isolating stem cells from these tissues have been studied.

For example, Patent Document 1 discloses an automated system and method for separating and concentrating regenerative cells from tissue, and collagenase or the like is used as an enzyme that degrades tissue. Patent Document 2 mentions collagenase as a condition for separating liposuction-derived material cells from adipose tissue. Furthermore, Patent Document 3 describes that an enzyme treatment is performed on a tissue piece in order to collect stem cells from the tissue piece. Thus, when stem cells are obtained from living tissue, most of the living tissue is mainly composed of extracellular matrix such as collagen. Therefore, in order to release the cells from the tissue, the extracellular matrix is separated by an enzyme such as collagenase. Decomposing is done.

As means for decomposing the main structure of the tissue other than the enzyme treatment,

Patent Documents

4 and 5 list scissors, scalpels, razor blades, and needles. Patent Document 6 exemplifies collagenase and hyperosmotic medium, as well as laser, lithotripsy, high flexion, phacoemulsification, sonication, high frequency wavelength, rotating blade, continuous filtration, forced screen filtration. Yes. Although there is no description of specific means in Patent Document 7, it is described that the adipose tissue is minced or sheared in addition to the treatment with an enzyme. Patent Document 8 describes an apparatus for pulverizing a fat sample charged in a syringe. Patent Document 9 discloses a method for isolating insulin-secreting cells from pancreatic strips, but this method is understood from the fact that trypsin inhibitor is added as necessary with the intention of avoiding excessive digestion. It uses enzymes secreted by the pancreas, and is limited to special organs such as the pancreas.

Special Table 2007-524396 Special table 2007-509601 gazette JP 2005-287479 A Special table 2007-508018 gazette JP 2012-80882 A Special table 2012-505658 gazette JP 2005-502712 A Korean Patent Application Publication No. 10-120963 Japanese Patent No. 3224164

Conventionally, collagen as a means of treating tissues to separate cells from biological tissues such as adipose tissue, umbilical cord matrix, cartilage, skin, muscle, heart muscle, tendon, liver, brain, blood vessels, etc. The method of disassembling was mainly adopted. For example, in

Patent Documents

4 and 5, collagenase treatment is performed after chopping adipose tissue with scissors.

In this way, the method using collagenase is the mainstream for obtaining stem cells from living tissues, etc., but generally sold collagenase is synthesized by microorganisms, and uses animal-derived components as raw materials, In addition to the risk of rejection due to contamination with substances derived from different organisms, it may cause infections such as prions. Therefore, especially for the purpose of regenerative medicine, it is desirable not to use enzymes such as collagenase if possible.

On the other hand, Patent Document 6 and Patent Document 8 disclose treatment means other than tissue degradation by an enzyme. However, Patent Document 6 aims to prepare adipose tissue containing a lot of blood vessel fractions, and Patent Document 8 aims to use adipose tissue obtained by grinding as a graft. Is not intended to use the cells themselves.

An object of the present invention is to provide a shredding device suitable for efficiently preparing cells from living tissue.

The present inventors used a rotary blade as a means for physically cutting biological tissue with the ultimate goal of separating cells by subdividing biological tissue without using enzymes. In order to efficiently separate cells from living tissue, we conducted extensive research on the configuration of the shredding device, such as the positional relationship with the rotary blade and other members. As a result, it has been found that cells can be separated from the living tissue by configuring the chopping means with a stirring member in addition to the rotary blade, and using this chopping means together with a stabilizing member having a through passage, and completed the present invention. .

That is, the shredding device of the present invention that has solved the above-described problem includes a cylindrical portion, a rotary shaft provided in the cylindrical portion, a rotary blade provided on the rotary shaft, and a stirring member. It has a shredding means and a stabilizing member provided on the rotating shaft and having one or more through passages. By using the rotary blade together with the stirring member, the biological tissue can be moved in the cylindrical portion with the rotation of the stirring member, and the biological tissue can be efficiently brought into contact with the rotary blade. In addition, although the detailed mechanism is not necessarily clear, when used together with a stabilizing member that abuts the inner wall of the cylindrical portion and has one or more through passages, the living tissue that has passed through the through passages can be captured well by the rotary blade. Conceivable.
Thereby, it is considered that the ratio of damaged cells can be reduced in total, and the cells can be efficiently separated from the living tissue without using an enzyme.

In the above-described shredding device, it is preferable that the rotating blade and the stirring member of the shredding means are arranged in the order of the rotating blade and the stirring member from the side closer to the stabilizing member.

In the above-described shredding device, the rotary blade has a base portion and an outer peripheral blade portion, and the thickness of the blade portion in the axial direction of the rotary shaft is the thickness of the stirring member in the axial direction of the rotary shaft. It is preferable that the thickness is smaller than the thickness.

In the shredding device described above, it is preferable that the blade portion is tilted in a direction opposite to the rotation direction of the shredding means.

In the above-described shredding device, it is also preferable that the blade portion is provided on the outer peripheral portion of the rotary blade.

In the above shredding device, the stirring member has one or more stirring blades, a vector direction from the rotating shaft to the tip of the stirring blade, and from the rotating shaft to the tip of the blade portion. It is preferable that an angle difference exists with respect to the vector direction.

In the above-described shredding device, it is preferable that the stirring blade is tilted in a direction opposite to the rotation direction of the shredding means.

In the above shredding device, the stirring member is preferably configured to be rotatable at the same angular velocity as the rotary blade.

In the above-described shredding device, it is preferable that the shredding means is configured to reciprocate in the axial direction of the rotating shaft.

In the above shredding device, it is preferable that the through passage is formed longer in the circumferential direction than in the radial direction of the stabilizing member.

It is preferable that the shredding device has two or more shredding means, and the stabilizing member is provided between at least two shredding means.

In the above-described shredding device, the stabilizing member can be implemented in a manner that does not follow the rotational movement of the rotary blade.

In the above shredding device, the cylindrical part has an inlet for introducing a biological tissue and a lead-out port for taking out a cell separated from the biological tissue, and the shredding means is the cylinder. It is preferable that it is provided in the shape part.

In the above-described shredding device, a filter is provided in the cylindrical portion so as to be separated into a first chamber and a second chamber, the introduction port communicates with the first chamber, and the outlet port communicates with the second chamber. It is preferable that the shredding means is provided in the first chamber.

In the above-described shredding device, it is also preferable that the stabilizing member is in contact with the inner wall of the cylindrical portion.

According to the present invention, cells such as stem cells can be safely and efficiently separated from a living tissue containing extracellular matrix and stem cells, specifically, adipose tissue, etc. without using an enzyme. Therefore, the present invention is very excellent in industry as a means for promoting the practical application of regenerative medicine.

FIG. 1 is an exploded view of a shredding device according to a first embodiment of the present invention. FIG. 2 is a plan view of shredding means in the shredding device according to the first embodiment of the present invention. FIG. 3 is a side view of the shredding means in the shredding device according to the first embodiment of the present invention. FIG. 4 is a plan view of the rotary blade in the shredding device according to the first embodiment of the present invention. FIG. 5 is a plan view of a stabilizing member in the shredding device according to the first embodiment of the present invention. FIG. 6 is a side view of the stabilizing member in the shredding device according to the first embodiment of the present invention. FIG. 7 is a plan view of another shredding means in the shredding device according to the first embodiment of the present invention. FIG. 8 is a side view of another shredding means in the shredding device according to the first embodiment of the present invention. FIG. 9 is a perspective view of the shredding device according to the first embodiment of the present invention. FIG. 10 is a cross-sectional view of the shredding device according to the second embodiment of the present invention. FIG. 11 is a perspective view of a filter in the shredding device according to the second embodiment of the present invention. FIG. 12 is a perspective view of a shredding device according to another embodiment of the present invention. FIG. 13 is a cross-sectional view of a shredding device according to yet another embodiment of the present invention. 14A is an exploded view of the shredding device in Comparative Example 1, FIG. 14B is an assembly view of FIG. 14A, and FIG. 14C is a view of the rotary blade after shredding in Comparative Example 1. It is a photograph showing the situation.

The shredding device according to the present invention includes a cylindrical part, a rotating shaft provided in the cylindrical part, a shredding means including a rotary blade and a stirring member provided on the rotating shaft, And a stabilizing member having one or more through passages. If the biological tissue is to be chopped only with the rotary blade, the biological tissue in the cylindrical portion is not sufficiently agitated and cannot be brought into contact with the rotary blade, leaving an unchopped tissue. Here, the rotary blade has one or a plurality of blade portions, and the blade portions are configured to be rotatable around a rotation axis. In the present invention, by using the rotary blade together with the stirring member, it is possible to move the non-chopped living tissue to contact the rotary blade. Further, although the detailed mechanism is not necessarily clear, it is considered that the living tissue flowing out from the through passage can be in good contact with the rotary blade by using it together with a stabilizing member having one or more through passages. That is, the living tissue is efficiently shredded by the cooperation of the through path, the stirring member, and the rotary blade, and the proportion of cells such as stem cells that receive great damage can be reduced in total. Therefore, according to the present invention, cells such as stem cells can be efficiently separated from living tissue without using an enzyme. The stabilizing member may be in contact with or separated from the inner wall of the cylindrical part. However, if the stabilizing member is in contact with the inner wall of the cylindrical part, the rotation of the rotary blade is further stabilized and the processing is performed. Since the internal biological tissue passes through the through passage without passing between the stabilizing member and the inner wall of the cylindrical portion, the biological tissue can be brought into contact with the rotary blade more reliably. On the other hand, if the stabilizing member and the inner wall of the cylindrical part are separated from each other, a biological tissue that is so large that it cannot pass through the through path can pass between the inner wall of the cylindrical part and the stabilizing member, thereby preventing the through path from being clogged. be able to. Further, since there is no contact resistance between the stabilizing member and the inner wall of the cylindrical portion, it is possible to suppress the torque applied to the rotating shaft. Hereinafter, more preferred embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1)
Hereinafter, a shredding device according to a first embodiment of the present invention will be described with reference to the drawings. FIG. 1 is an exploded view of a shredding device according to a first embodiment of the present invention. In FIG. 1, the rotary shaft 1 is provided with shredding means 4 and 14 including

rotary blades

2 and 12 and stirring

members

3 and 13, respectively. Hereinafter, in the explanation part where there is no difference between the rotary blade 2 and the rotary blade 12, there may be simply described as “rotary blade 2”. The same applies to the stirring member 3 and the shredding means 4 below. In FIG. 1, although the rotary blade 2 is comprised by the base 2a and the blade part 2b which are separate members, these may be formed integrally. In addition, it is preferable that the blade part 2b is provided in the outer peripheral part of the rotary blade 2 on the relationship of fine cutting efficiency. In addition, a stabilizing member 6 having a through passage 5 is provided between the chopping means 4 and 14.

As described above, the shredding means 4 and the stabilizing member 6 provided on the rotary shaft 1 are inserted into the cylindrical portion 7 to constitute a shredding device. Since the stabilizing member 6 is in contact with the inner wall of the cylindrical portion 7, it is prevented from swinging inside the cylindrical portion 7 even if the rotating shaft 1 rotates.

The through passage 5 is a passage provided through the stabilizing member 6 so that the living tissue inside the cylindrical portion 7 can move in the axial direction without being blocked by the stabilizing member 6. Therefore, the through-passage 5 may have any shape as long as it does not block the flow of the biological tissue, may be a hole passing through the stabilizing member 6, or may be formed on the side surface of the stabilizing member 6. It may be a groove formed.

2 and 3 are enlarged views of the shredding means 4, FIG. 2 is a plan view of the shredding means 4, and FIG. 3 is a side view of the shredding means 4 as viewed from the direction A in FIG. is there.
In FIG. 2, the shredding means 4 rotates in the direction of arrow B. As described above, the rotary blade 2 has the base portion 2a and the blade portion 2b, but these may be integrally formed. Further, the stirring member 3 includes stirring blades 3a at two locations. The number of stirring blades 3a may be one or plural. As an example, FIG. 2 shows an embodiment in which one agitating blade 3a is provided on a one-to-one basis for one blade portion 2b. Further, the shape of the stirring blade 3a is not particularly limited as long as it is a shape that can stir the biological tissue piece so as not to damage the cells. In FIG. An embodiment is shown. The biological tissue piece cut by the blade 2b is once separated from the blade 2b by the adjacent stirring blade 3a. As a result, the blade 2b can immediately chop the next biological tissue piece. That is, following the step of chopping the biological tissue piece with the blade portion 2b, the step of stirring the chopped biological tissue piece with the stirring blade 3a continuously occurs to effectively cut the biological tissue piece. And the efficiency of cell preparation can be improved. Furthermore, since the biological tissue piece is continuously agitated by the rotation of the stirring blade 3a, a state in which an unchopped biological tissue piece is biased in the cylindrical portion does not occur, and the biological tissue piece in the cylindrical portion is uniformly thinned. Can be cut.

In addition, the one where the blade angle (cutlery angle) of the blade part 2b is small is preferable. This is because if the blade angle is too large, the living tissue may be crushed, and the living tissue may be shredded, but the cells existing inside the living tissue may be damaged. . Therefore, the blade angle is preferably 45 degrees or less, more preferably 40 degrees or less, and even more preferably 35 degrees or less. On the other hand, the lower limit of the blade angle is not particularly limited. However, if the blade angle is too small, the cutting edge is quickly reduced. For example, it is 1 degree or more, more preferably 10 degrees or more, and further preferably 20 degrees or more. .

The vector direction from the center of the rotating shaft 1 to the cutting edge of the blade portion 2b and the vector direction from the center of the rotating shaft 1 to the tip of the stirring blade 3a make a certain angle α. That is, the rotary blade 2 and the stirring member 3 are in a fixed relationship, and when the rotary shaft 1 rotates, the stirring member 3 rotates at the same angular velocity as the rotary blade 2. The angle α is not particularly limited, and may be freely set according to the number of rotary blades or stirring blades. The step of cutting the biological tissue piece that has passed through the through-passage of the stable member by the blade portion 2b. Subsequently, it is preferable that a step of stirring the chopped biological tissue piece with the stirring blade 3a occurs, so that the position of the stirring blade 3b is delayed in phase with respect to the rotation direction with respect to one blade portion 2b, and It is desirable that the angle α is, for example, 1 ° or more, preferably 5 ° or more, more preferably 10 ° or more. The upper limit of the angle α is not particularly limited, but can be, for example, 60 ° or less, 50 ° or less, or 40 ° or less.

The rotation radius of the rotary blade 2, that is, the rotation radius of the blade portion 2b is not particularly limited. However, as the rotation radius is larger, more living tissues can be processed, and thus, for example, 4 mm or more, preferably 10 mm or more, more preferably 12 mm or more. More preferably, it is 15 mm or more. The smaller the rotation radius, the smaller the force required for rotation, and the more compact the device becomes, and the easier it is to handle the living tissue. Therefore, the rotation radius of the rotary blade 2 is, for example, 150 mm or less, preferably 100 mm. Hereinafter, it is more preferably 50 mm or less, and still more preferably 20 mm or less.

The rotation radius of the stirring blade 3a is preferably large enough to scrape away the living tissue that has escaped into the corner of the cylindrical portion and prevent the living tissue remaining in an unchopped state from remaining. , Preferably 10 mm or more, more preferably 12 mm or more, and even more preferably 15 mm or more. The smaller the radius of rotation, the smaller the force required for rotation, and the more compact the device becomes, and the easier it is to handle when processing biological tissue. Therefore, the radius of rotation of the stirring blade 3a is, for example, 150 mm or less, preferably 100 mm. Hereinafter, it is more preferably 50 mm or less, and still more preferably 20 mm or less.

The ratio of the rotation radius of the rotary blade 2 to the rotation radius of the stirring blade 3a is not particularly limited, but is, for example, 0.5 times or more, preferably 1 time or more, more preferably 1.2 times or more, and the upper limit is also set. Although there is no restriction | limiting in particular, For example, it is 2 times or less, Preferably it is 1.5 times or less, More preferably, it is 1.3 times or less.

As shown in FIG. 2, the stirring blade 3a is preferably tilted in the direction opposite to the direction of rotation of the shredding means 4 (arrow B). In order to reduce damage to cells by making the collision angle between the biological tissue piece and the stirring blade 3a shallow when the stirring member 3 is rotated and preventing the biological tissue piece from colliding perpendicularly with the stirring blade 3a. is there. Similarly, the blade portion 2b is preferably tilted in the direction opposite to the rotation direction (arrow B) of the shredding means 4. In the present invention, the state where the stirring blade or blade portion is “tilted in the direction opposite to the rotational direction” means that the straight line connecting the tip of the stirring blade or blade portion and the center of the rotating shaft is the stirring blade or A state having an angle larger than 0 ° in the direction opposite to the rotation direction (arrow B) than the straight line connecting the base of the front side and the center of the rotation axis with respect to the rotation direction (arrow B) of the blade portion. I mean.

3, the thickness of the blade portion 2b in the axial direction (arrow C) of the rotary shaft 1 is preferably thinner than the thickness of the stirring member 3 in the axial direction of the rotary shaft 1. This is from the viewpoint of improving the efficiency of shredding of living tissue, improving the stirring efficiency, and reducing damage to stem cells. There is no particular limitation on the thickness ratio of the stirring member 3 to the thickness of the blade portion 2b, but it is, for example, 2 times or more, preferably 4 times or more, more preferably 6 times or more, and the upper limit is not particularly limited. For example, it is 25 times or less, preferably 20 times or less, more preferably 15 times or less. The thickness of the blade is preferably thinner from the viewpoint of sharpness, for example, 1 mm or less, preferably 0.5 mm or less, more preferably 0.1 mm or less.

FIG. 4 is a plan view of the rotary blade 2 that can be observed when the stirring member 3 is removed from the shredding means 4 in FIG. As shown in FIG. 4, the blade portion 2b is fixed to the base portion 2a. The fixing method is not particularly limited, but may be screwed, or a shape that fits into the shape of the blade portion 2b may be provided in the base portion 2a, and both may be fitted. Thus, if the base part 2a and the blade part 2b are comprised by another member, even when the blade part 2b wears and the crushing performance of the rotary blade 2 falls, the blade part 2b can be replaced. Of course, the rotary blade 2 may be configured by integrally forming the blade portion 2b and the base portion 2a.

5 and 6 are enlarged views of the stabilizing member 6, FIG. 5 is a plan view of the stabilizing member 6, and FIG. 6 is a side view of the stabilizing member 6. FIG. In order to maintain airtightness between the stabilizing member 6 and the inner wall of the cylindrical portion 7, the O-ring member 6 a at a portion where the stabilizing member 6 abuts against the inner wall of the cylindrical portion 7 is maintained. When contacting the inner wall, it is preferable to use a silicone resin such as silicone rubber or a plastic. In the configuration as shown in FIG. 1, the stabilizing member 6 is fixed to the rotating shaft 1 and may be configured to rotate following the rotation of the rotating shaft 1. The structure which is fixed to and does not follow rotation of the rotating shaft 1 may be sufficient. By adopting a configuration in which the stable member 6 does not follow the rotation of the rotary shaft 1, a relative rotational relationship is generated between the stable member 6 and the rotary blade 2 that follows the rotation of the rotary shaft 1. The rotary blade 2 can effectively chop the living tissue coming out of 5. In addition, although there is no restriction | limiting in particular in the outer diameter (diameter) of the stable member 6, For example, it is 5 mm or more, Preferably it is 10 mm or more, More preferably, it is 15 mm or more, Although there is no restriction | limiting in particular about an upper limit, For example, 200 mm or less, Preferably it is 150 mm or less, More preferably, it is 100 mm or less. The inner diameter of the cylindrical portion 7 is set to be approximately the same as the outer diameter of the stabilizing member 6.

The shape and size of the through-passage 5 are not particularly limited, and may be appropriately set according to the type, size, property, and the like of the biological tissue to be processed. The circumferential direction of the stabilizing member 6 is formed so as to be longer than the radial direction of the stabilizing member 6. For example, the cross-sectional shape perpendicular to the passage direction of the through-passage 5 is not circular, but the radial direction of the stabilizing member is the same. By making the shape longer in the circumferential direction, the radius of the stabilizing member 6 can be reduced without changing the volume of the living tissue that can pass through the through-passage 5, and the entire shredding device can be made compact. Is possible.

7 and 8 show the shredding means 14 shown in FIG. 1, FIG. 7 is a plan view of the shredding means 14, and FIG. 8 is a side view. The shredding means 14 basically includes a rotary blade (12) having a base part (12a) and a blade part (12b), and a stirring blade (13a), similarly to the shredding means 4 shown in FIGS. And a stirring member (13). However, unlike the shredding means 4, the base 12 a of the rotary blade 12 is provided with a female screw portion 12 c for receiving the shaft tip of the rotary shaft 1. The screw formed on the inner wall of the female screw portion 12 c is cut in a direction in which the screwing is further reduced by the rotation of the rotary shaft 1.

As can be seen from FIG. 1, the rotary blade 2 and the stirring member 3 of the shredding means 4 are arranged in the order of the rotary blade 2 and the stirring member 3 with respect to the stabilizing member 6. That is, the rotary blade (2, 12) exists between the stirring member (3, 13) and the stabilizing member 6. By arranging in this way, it is possible to efficiently chop the living tissue immediately after flowing out of the through passage 5 with the blade portions (2b, 12b) of the rotary blade (2, 12). Moreover, the living tissue after being chopped by the rotary blade (2, 12) by arranging the rotary blade (2, 12) between the stirring member (3, 13) and the stabilizing member 6 The pieces can be efficiently stirred.

The distance between the entrance / exit of the through-passage 5 and the blade portion 12b is preferably 1 mm or less, more preferably 0.5 mm or less, and still more preferably 0.2 mm or less, from the viewpoint of efficient cutting of living tissue. . On the other hand, in order to ensure the clearance necessary for the rotation of the rotary blade 12, the distance between the entrance / exit of the through passage 5 and the blade portion 12b is preferably 0.01 mm or more, more preferably 0.03 mm or more, and still more preferably. Is 0.05 mm or more.

9 is a perspective view of the shredding device. After the shredding means 4 and the stabilizing member 6 provided on the rotary shaft 1 are inserted into the tubular part 7 as shown in FIG. The state sealed with the end cap 7a is shown. It is preferable to chop the living tissue in an aseptic state that is cut off from the outside. Therefore, it is preferable to provide the bellows sealing member 8 on the rotating shaft 1 in order to block the gap between the rotating shaft 1 and the opening of the end cap 7a from the outside. Thereby, the rotary shaft 1 can be reciprocated in the axial direction while keeping the inside of the cylindrical portion 7 in a sterile state.

As described above, according to the shredding device according to the first embodiment of the present invention, the living tissue flowing out from the through passage is efficiently shredded by the rotary blade, and the unsliced living tissue is agitated. It can be moved by a wing and brought into contact with the rotary blade.

(Embodiment 2)
FIG. 10 is a cross-sectional view of the shredding device according to the second embodiment of the present invention. Since the shredding device according to the second embodiment has basically the same configuration as the shredding device according to the first embodiment, the description of the overlapping parts is omitted. In FIG. 10, in addition to the configuration of the shredding device according to the first embodiment, a filter portion 9 that is sandwiched by an O-ring 15 from above and below may be provided inside the cylindrical portion 7. The filter unit 9 can increase cell separation efficiency by capturing and removing coarse residues remaining in the living tissue after chopping. The filter unit 9 divides the cylindrical part 7 into a first chamber 7b and a second chamber 7c. The cylindrical part 7 includes an introduction port 7d for introducing a living tissue into the first chamber 7b, and a filter. It has the outlet 7e for taking out the cell after passing the part 9 from the 2nd chamber 7c. The shredding means 4 is provided in the first chamber 7b. By configuring the shredding device as described above, it is possible to proceed in a sterile state that is blocked from the outside world, from shredding of living tissue, removal of residues by the filter unit 9, and removal of cells. As for the effect of creating an aseptic state blocked from the outside, the cylindrical portion 7 can be provided with the introduction port 7d and the outlet port 7e for taking out cells without the filter unit 9. It is enough.

Although not shown, a plurality of filter sections 9 can be obtained by arranging a plurality of filter sections 9. When a plurality of filter units 9 are arranged, the downstream filter has a smaller opening than the upstream filter. As a result, the cells can be separated more efficiently.

FIG. 11 is a perspective view of the filter unit 9 in the shredding device according to the second embodiment. As shown in FIG. 11, the filter unit 9 is configured in such a manner that the filter 9a is sandwiched between two upper and lower supports 9b. The reason why the support 9b is formed in a cross shape at the center is to suppress fluttering of the filter 9a.

As described above, the shredding device according to the second embodiment of the present invention, in addition to the effects of the shredding device according to the first embodiment, removes residues with the filter 9a, and a series of cell preparation steps. Can be performed under aseptic conditions.

(Other embodiments)
As mentioned above, although the shredding

apparatus concerning Embodiment

1, 2 of this invention was demonstrated separately, the shredding apparatus of this invention is provided in the cylindrical part, the rotating shaft, and this rotating shaft at least. The living tissue can be efficiently thinned if it has a chopping means including a rotating blade and a stirring member, and a stabilizing member provided on the rotating shaft and abutting against the inner wall of the cylindrical portion and having one or more through passages. Of course, it is possible to implement any combination of the members and methods further described in the first and second embodiments, and for example, the above-mentioned apparatus is used for chopping. The biological tissue may be further treated with an enzyme such as collagenase. In addition, the following various forms can be changed.

FIG. 12 is a perspective view of a shredding device according to another embodiment of the present invention. As shown in FIG. 12, instead of the cylindrical part 7 of the shredding device according to the first embodiment, a syringe 10 having a cylindrical part can be used in the same manner. The upper end portion of the syringe 10 is closed by a syringe cap 10a. When the syringe 10 is used, when the living tissue is shredded, that is, when the shredding means is rotated, the distal end portion of the syringe 10 is closed by, for example, a three-way valve 11, and the three-way valve 11 is opened after the shredding is completed. And the living tissue after chopping is taken out. The extracted biological tissue is subjected to, for example, a centrifuge to collect cells contained in the precipitate. In addition, even if the biological tissue after shredding is not taken out from the syringe 10, it can be applied to the centrifuge with the syringe 10 as it is. The three-way valve 11 has both functions of the inlet 7d and outlet 7e.

In the first and second embodiments, the example using two shredding means 4 has been described, but one shredding means 4 or three or more shredding means 4 may be used. Moreover, although the example using two blade parts 2b with respect to one rotary blade 2 was demonstrated, you may use one or three or more blade parts 2b. In the first and second embodiments, the cantilever having the rotating shaft 1 on only one side of the stabilizing member 6 has been described. However, in order to make the rotating operation more stable, as shown in FIG. It can be used even in the case of both ends having the rotary shaft 1 on both sides of the member 6. That is, the shredding device of FIG. 13 has a part in common with the shredding device shown in FIG. 10, but in the shredding device of FIG. 13, the rotating shaft 1 has a shredding means 14 (shown in a side view). And the filter portion 9 (shown in cross-sectional view) is rotatably fixed to the bottom portion of the tubular portion 7. Thus, by supporting the rotating shaft 1 not only on one side but on both sides, the operation of the rotating shaft 1 can be further stabilized. In addition, in order for the rotating shaft 1 to penetrate the filter part 9, it is necessary to provide the opening penetrated in the center part of the filter 9a and the support body 9b. Of course, the use of the filter unit 9 is not an essential requirement of the present invention.

In the first and second embodiments, the case where the side surface of the stirring member 3 is parallel to the rotating shaft 1 is shown. However, the side surface of the stirring member 3, particularly the side surface of the stirring blade 3 a, is It may be non-parallel, and the stirring blade 3a may act like a propeller so that the living tissue flows in the axial direction of the rotary shaft 1. In order to promote efficient shredding of living tissue and to satisfy the requirement of damaging stem cells as much as possible, the side surface of the stirring member 3 is rotated as in the first and second embodiments. It is desirable to be parallel to the axis 1. In the present invention, the biological tissue includes adipose tissue, umbilical cord matrix, cartilage, skin, muscle, myocardium, tendon, liver, and the like, excluding organ tissues that secrete special enzymes such as pancreas.

This application claims the benefit of priority based on Japanese Patent Application No. 2014-89583 filed on April 23, 2014. The entire contents of the specification of Japanese Patent Application No. 2014-89583 filed on April 23, 2014 are incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1
The dimensions, operations, and the like related to the rotary blade 2, the stirring member 3, the chopping means 4, the stabilizing member 6, and the cylindrical portion 7 used in this example are as follows. The rotation radius of the rotary blade 2 and the rotation radius of the rotary blade 12, the blade angle of the blade portion 2b and the blade angle of the blade portion 12b, the thickness of the blade portion 2b and the thickness of the blade portion 12b, the thickness of the stirring member 3 and the stirring The thickness of the member 13, the rotation radius of the stirring member 3, and the rotation radius of the stirring member 13 were designed to be the same value. 20 mL of adipose tissue is chopped using the above-mentioned chopping device to separate cells, and 3/20 of the number of cells obtained (number of cells obtained from 3 mL of adipose tissue) is converted into three φ90 petri dishes. Each was seeded and cultured for 12 days. Cell number respectively 1.82 × 10 ⁵ cells obtained after culturing, 2.09 × 10 ⁵ cells, was 1.92 × 10 ⁵ cells (mean 1.94 × 10 ⁵ cells).

Rotational radius of rotary blade 2: 12.9 mm
Blade angle of blade 2b: 22 °
Blade thickness 2b: 0.45 mm
Stirring member 3 thickness: 4 mm
Rotating radius of stirring member 3: 10.3 mm
Stability member 6 outer diameter: 27 mm
Inner diameter of cylindrical part 7: 27 mm

(Comparative Example 1)
Hereinafter, Comparative Example 1 of the present invention will be described. Although the shredding device in Comparative Example 1 has a part in common with the shredding device according to Example 1 (Embodiment 1), unlike the one in Example 1, it does not have the stirring member 3. . Fig.14 (a) is an exploded view of the shredding device in the comparative example 1, FIG.14 (b) is an assembly drawing of Fig.14 (a). As shown in FIGS. 14A and 14B, in the comparative example 1, as the rotary blade 2, a semi-cylindrical one was used. FIG. 14C is a photograph of the vicinity of the rotary blade 2 when a living tissue is shredded using the shredding device in Comparative Example 1. As can be seen from the photograph in FIG. 14 (c), the large biological tissue piece 16 remains on the rotary blade 2, and it has not been appropriately shredded, and the rotation of the blade was unstable. The operation to detach was stopped.

Rotating radius of rotary blade 2: 12.1 mm
Blade angle of blade part 2b: 45 °
Blade part 2b thickness: 3 mm
Stability member 6 outer diameter: 27 mm
Inner diameter of cylindrical part 7: 27 mm

(Comparative Example 2)
Hereinafter, Comparative Example 2 of the present invention will be described. The shredding device in Comparative Example 2 has parts in common with the shredding device according to Example 1 (Embodiment 1), but has

blade portions

2b and 12b unlike the example. Absent. Further, each of the stirring member 3 and the stirring member 13 has six stirring blades.

The dimensions and operations related to the stirring member 3, the stabilizing member 6, and the cylindrical portion 7 used in this comparative example are as follows.

Stirring member 3 thickness: 5 mm
Rotating radius of stirring member 3: 26.3 mm
Thickness of the stirring member 13: 2 mm
Rotating radius of stirring member 13: 26.3 mm
Stability member 6 outer diameter: 26.3 mm
Inner diameter of cylindrical part 7: 26.5 mm

20 mL of adipose tissue is minced using a shredding device to separate cells, and 3/20 of the number of cells obtained (number of cells obtained per 3 mL of adipose tissue) is divided into three φ90 dishes. Seed and cultured for 12 days. Cell number respectively 5.37 × 10 ³ cells obtained after culturing, 9.07 × 10 ³ cells, a 1.33 × 10 ⁴ cells (mean 9.25 × 10 ³ cells), 1 of Example 1 Only about / 20 was obtained.

DESCRIPTION OF SYMBOLS 1 Rotating shaft 2 Rotating blade 2a Base 2b Blade 3 Stirring member 3a Stirring blade 4 Shredding means 5 Through passage 6 Stabilizing member 6a O-ring member 7 Cylindrical portion

7a End cap

7b First chamber

7c Second chamber 7d Inlet 7e Outlet 8 Bellows sealing member 9 Filter 9a Filter 9b Support 10 Syringe 10a Syringe cap 11 Three-way valve 12 Rotary blade 12a Base 12b Blade 12c Female thread 13 Stirring member 13a Stirring blade 14 Shredder 15 O-ring 16 Living body Tissue fragment

Claims

A tubular portion;
A rotating shaft provided in the tubular portion;
A slitting means provided on the rotary shaft, including a rotary blade and a stirring member;
A stabilizing member provided on the rotating shaft and having one or more through passages;
A shredding device comprising:
The shredding device according to claim 1, wherein the rotating blade and the stirring member of the shredding means are arranged in the order of the rotating blade and the stirring member from the side closer to the stabilizing member.
The rotary blade has a base portion and a blade portion, and the thickness of the blade portion in the axial direction of the rotary shaft is smaller than the thickness of the stirring member in the axial direction of the rotary shaft. The shredding device according to claim 1 or 2.
4. The shredding device according to claim 3, wherein the blade portion is tilted in a direction opposite to a rotation direction of the shredding means.
The shredding device according to claim 3 or 4, wherein the blade portion is provided on an outer peripheral portion of the rotary blade.
The stirring member has one or more stirring blades, and is between a vector direction from the rotating shaft to the tip of the stirring blade and a vector direction from the rotating shaft to the tip of the blade. 6. The shredding device according to claim 3, wherein there is an angular difference.
The shredding device according to claim 6, wherein the stirring blade is tilted in a direction opposite to a rotation direction of the shredding means.
The shredding device according to any one of claims 1 to 7, wherein the stirring member is configured to be rotatable at the same angular velocity as the rotary blade.
The shredding device according to any one of claims 1 to 8, wherein the shredding means is configured to reciprocate in the axial direction of the rotating shaft.
10. The shredding device according to claim 1, wherein the through passage is formed longer in the circumferential direction than in the radial direction of the stabilizing member.
The shredding device according to any one of claims 1 to 10, wherein the shredding device has two or more shredding means, and the stabilizing member is provided between at least two shredding means.
12. The shredding device according to claim 1, wherein the stabilizing member does not follow the rotational movement of the rotary blade.
The cylindrical portion has an introduction port for introducing a biological tissue and a lead-out port for taking out a cell separated from the biological tissue, and the shredding means is provided in the cylindrical portion. The shredding device according to any one of claims 1 to 12.
A filter for separating the first chamber and the second chamber is provided in the cylindrical portion, the introduction port communicates with the first chamber, the outlet port communicates with the second chamber, and the shredding means 14. The shredding device according to claim 13, wherein is provided in the first chamber.
The shredding device according to any one of claims 1 to 14, wherein the stabilizing member is in contact with an inner wall of the cylindrical portion.