JP2009038998A - Cell-separating device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compact cell-separating device capable of cleaning, decomposing and concentrating a biomedical tissue, having improved operability and providing an aseptically concentrated tissue-originated cell without transfer between devices. <P>SOLUTION: The cell-separating device 1 is equipped with: a storing part 2 for storing a mixed liquid W containing an adipose tissue A; a container 3 installed in the storing part 2 and having two opening parts 3a and 3b; a first filter 4 arranged at the position for closing one opening part 3a of the container 3 and prohibiting the permeation of the adipose tissue A; a second filter 5 arranged at the position for closing the other opening part 3b of the container 3, prohibiting the permeation of the adipose tissue A and allowing the permeation of a cell originated from fat; a collecting tube 6 one terminal of which is connected to the opening part 3b at the second filter 5 side; a third filter 7 arranged at the position for closing the other terminal of the collecting tube 6, prohibiting the permeation of the cell originated from the fat, and allowing the permeation of erythrocyte; and a flow direction-switching mechanism for switching the flow direction of the mixed liquid W in the container 3. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell separation device.

  Conventionally, in order to decompose a biological tissue and separate tissue-derived stem cells, the biological tissue is washed and decomposed by stirring in a digestive enzyme solution, and the obtained cell suspension is concentrated by a centrifuge. The method is known (for example, refer nonpatent literature 1).

K. Yoshimura et al., "Characterization of Freshly Isolated and Cultured Cells Derived Fromthe Fatty and Fluid Portions of Liposuction Aspirates", J. Cellular Physiology 208, pp64-76, 2006

However, in the method of Patent Document 1, it is necessary to perform the steps of cleaning, decomposing, and concentrating living tissue in separate devices, and the entire device is enlarged, and the living tissue and cell suspension are transferred between the steps. There is an inconvenience of having to change.
The present invention has been made in view of the above-described circumstances, and can clean, disassemble and concentrate biological tissue with a compact device, improve operability, and eliminate transfer between devices. It aims at providing the cell separation apparatus which can obtain the tissue origin cell concentrated aseptically.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a container including a container that contains a mixed solution containing adipose tissue, two openings provided in the container, and a position of the fat tissue that is disposed at a position that closes one opening of the container. A first filter that prohibits passage, a second filter that is disposed at a position that closes the other opening of the container, prohibits passage of adipose tissue, and allows passage of fat-derived cells; A collection tube having one end connected to the opening on the filter side, a third filter disposed at a position to close the other end of the collection tube, prohibiting passage of fat-derived cells and allowing passage of red blood cells; A cell separation device comprising a flow direction switching mechanism for switching the flow direction of the mixed solution in the container is provided.

  According to the present invention, the liquid mixture containing adipose tissue is accommodated in the container accommodating portion, and the liquid mixture is set by setting the flow direction of the liquid mixture toward the first filter by the operation of the flow direction switching mechanism. Inner adipose tissue can be captured by the first filter and smaller material can be passed through the first filter for removal. For example, by washing the adipose tissue with a washing liquid and filtering it with a first filter, dust such as tissue residue and blood components adhering to the adipose tissue can be removed. Thereafter, by mixing the digestive enzyme solution and the adipose tissue, the adipose tissue is decomposed in the accommodating portion, and a cell suspension containing adipose-derived cells is generated.

  Then, by operating the flow direction switching mechanism and switching the flow direction of the cell suspension to the direction toward the second filter, the decomposed fat-derived cells and smaller red blood cells pass through the second filter. And a larger adipose tissue is captured by the second filter and maintained in the container. The adipose-derived cells that have passed through the second filter are collected in a collection tube connected to the opening on the second filter side, and captured by a third filter arranged at a position that closes the other end of the collection tube. Is done. On the other hand, red blood cells or the like having a particle size smaller than that of fat-derived cells pass through the third filter and are separated from the fat-derived cells.

  That is, according to the present invention, adipose tissue can be easily washed, digested and concentrated without transferring the adipose tissue or cell suspension to a separate container. Therefore, the operability can be improved, the contamination of bacteria and the like accompanying the transfer can be prevented, and fat-derived cells can be aseptically separated and recovered. In addition, fat-derived cells can be concentrated and separated more easily with a compact configuration without using a centrifuge for separation.

In the above invention, the cleaning liquid supply unit that supplies the cleaning liquid into the storage unit and the waste liquid discharge unit that discharges the waste liquid after cleaning may be connected to the opening provided with the first filter. Good.
Thus, when the cleaning liquid is supplied from the cleaning liquid supply unit, the cleaning liquid is supplied into the storage unit through the opening provided with the first filter, and the adipose tissue stored in the storage unit is cleaned. Can do. And the waste liquid after washing | cleaning is discharged | emitted from a waste-liquid discharge part, A washing | cleaning fat tissue is made to capture | acquire by a 1st filter, and dust, such as a tissue residue and blood component adhering to a fat tissue, are 1st. It can be removed by passing through a filter.

Moreover, in the said invention, the said cleaning liquid supply part is good also as supplying gas with a cleaning liquid.
By doing so, the adipose tissue and the cleaning liquid in the housing part can be well agitated by the action of gas, and dust and the like adhering to the adipose tissue can be efficiently removed.

Moreover, in the said invention, the enzyme supply port which supplies a digestive enzyme liquid to the accommodating part of the said container is good also as being provided.
By doing in this way, by supplying the digestive enzyme solution into the accommodating part from the enzyme supply port provided in the container accommodating part, the digestive enzyme solution is allowed to act on the fatty tissue in the accommodating part to decompose the fatty tissue, Adipose-derived cells can be isolated.

  Moreover, in the said invention, the said flow direction switching mechanism is arrange | positioned with the opening provided with the 1st filter facing down at the time of washing | cleaning and digestion of an adipose tissue, and 2nd after completion | finish of digestion The container may be rotated so that the opening provided with the filter is disposed downward.

  In this way, by operating the flow direction switching mechanism, the container is arranged with the first filter facing downward, and cleaning is performed, whereby the adipose tissue is captured by the first filter, and the cleaning liquid and dust are removed. The first filter can be discharged downward and discharged. Also, by disassembling the container with the first filter facing downward, the adipose tissue is efficiently decomposed in the digestive enzyme solution stored in the container, and the cell suspension in which the fat-derived cells float is suspended. A suspension can be obtained.

  Then, by the operation of the flow direction switching mechanism, the cell suspension is caused to flow through the second filter by rotating the container and directing the opening provided with the second filter to the lower side. The adipose tissue can be captured by this filter, and fat-derived cells having a sufficiently small particle size can be passed therethrough. Thereby, fat-derived cells and a substance having a smaller particle diameter can be guided into the collection tube provided with the third filter and separated from the adipose tissue. Then, fat-derived cells can be captured by the third filter, and erythrocytes or the like having a smaller particle diameter can pass through the third filter and be discharged to the outside.

In the above invention, a valve for opening and closing the recovery tube is provided in the vicinity of the opening on the second filter side, and the cleaning medium is supplied from the opposite side of the recovery tube with the third filter interposed therebetween. A cleaning medium supply unit may be provided.
By doing in this way, after a fat-derived cell is collect | recovered in a collection | recovery tube, a collection | recovery tube can be cut | disconnected from an accommodating part by closing a valve | bulb. In this state, by supplying the cleaning medium from the third filter side by the operation of the cleaning medium supply unit, the fat-derived cells captured by the third filter are peeled off from the third filter and put into the collection tube. It can be recovered.
In this case, the cleaning medium may be a cleaning liquid and / or a gas.

In the above invention, one end of another collection tube is connected to the opening on the first filter side, and passage of fat-derived cells is prohibited at a position where the other end of the other collection tube is closed. A fourth filter that allows passage of red blood cells may be provided.
In this way, for example, it is possible to prevent the fat-derived cells from being discharged through the first filter during cleaning using the cleaning liquid.

  According to the present invention, it is possible to clean, decompose and concentrate biological tissue with a compact device, improve operability, and eliminate tissue-transferred cells that are aseptically concentrated without transfer between devices. There is an effect that it can be obtained.

Hereinafter, a cell separation device 1 according to an embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, the cell separation device 1 according to the present embodiment includes a substantially cylindrical storage portion 2 that stores adipose tissue, and a container 3 that includes openings 3 a and 3 b that are open at both ends. A first filter 4 disposed at a position closing the first opening 3 a at one end of the container 3 and a second filter disposed at a position closing the second opening 3 b at the other end of the container 3. A filter 5, a collection tube 6 having one end connected to the second opening 3 b, a third filter 7 disposed at a position closing the other end of the collection tube 6, and a second of the collection tube 6 And a pinch valve 8 for opening and closing the opening on the filter 5 side.

The container 2 is provided with an inlet 9 for opening / closing the living tissue or injecting a digestive enzyme solution described later.
The first opening 3a of the container 3 is provided with a cleaning liquid supply unit 10 for supplying a cleaning liquid and nitrogen gas, and a waste liquid discharging unit 11 for discharging the waste liquid after cleaning.
In addition, a gas supply port 12 for supplying nitrogen gas and a waste liquid discharge unit 13 for discharging waste liquid are provided outside the third filter 7 of the recovery tube 6.

  The collection tube 6 is made of a material that can be easily cut by heat, such as polyethylene. Thereby, the aseptic cutting process which obstruct | occludes a cross section, cut | disconnecting the collection | recovery tube 6 with a heat | fever can be performed by a well-known method.

The container 3 is provided to be rotatable around a horizontal axis C extending in the radial direction.
The first filter 4 has a through hole of 5 to 25 μm, and preferably has a through hole of 10 to 15 μm.
The second filter 5 has a through hole of 30 to 150 μm, and preferably has a through hole of 40 to 70 μm.
The third filter 7 has 3 to 10 μm through holes, and preferably 5 to 8 μm through holes.

The operation of the cell separation device 1 according to the present embodiment configured as described above will be described below.
In order to separate and collect the adipose-derived cells B from the adipose tissue A using the cell separation device 1 according to the present embodiment, first, as shown in FIG. 2, the first filter 4 is arranged on the lower side. The container 3 is disposed in the container 3, the adipose tissue A is stored in the container 2 of the container 3, and the cleaning liquid W and nitrogen gas are supplied from the cleaning liquid supply unit 10. The cleaning liquid W is stored in the container 3, and nitrogen gas is supplied in a state where the fat tissue A is immersed in the cleaning liquid W, whereby the fat tissue A is stirred in the cleaning liquid W and efficiently cleaned. It will be.

  Then, the supply of the cleaning liquid W and the nitrogen gas is stopped, and as shown in FIG. Dust such as tissue residues and blood components smaller than that passes through the first filter 4 and is discharged from the waste liquid discharger 11 to the outside. By repeating this step a plurality of times, the adipose tissue A can be more reliably washed. In addition, by supplying the cleaning liquid W and nitrogen gas into the housing portion 2 from below the first filter 4, dust such as tissue residues and blood components captured by the first filter 4 is backwashed. It is possible to prevent the filter 4 from being clogged.

  Then, after the waste liquid of the cleaning liquid W is discharged from the waste liquid discharge section 11, the waste liquid discharge section 11 is closed by a valve (not shown) or the like, and the inlet 9 provided in the container 3 as shown in FIG. The digestive enzyme solution D is supplied into the storage unit 2. As a result, the digestive enzyme solution D is stored in the storage part 2, and the adipose tissue A is immersed in the digestive enzyme solution D. Isolated. At this time, the adipose tissue A and the digestive enzyme solution D can be sufficiently mixed by supplying nitrogen gas from the cleaning liquid supply unit 10. When a predetermined time elapses, a cell suspension is formed in which a large number of isolated fat-derived cells B float in the digestive enzyme solution D.

  In this state, as shown in FIG. 5, the container 3 is rotated around the horizontal axis C so that the first filter 4 is disposed on the upper side and the second filter 5 is disposed on the lower side. Place the container 3. As a result, the cell suspension flows downward through the second filter 5. Then, the adipose tissue A contained in the cell suspension is captured by the second filter 5 and stays in the storage unit 2, and fat-derived cells B and erythrocytes smaller than that pass through the second filter 5. Flow into the collection tube 6.

  Since the third filter 7 is provided at the other end of the collection tube 6, a relatively large fat-derived cell B out of the cell suspension that has flowed into the collection tube 6 as shown in FIG. Is captured by the third filter 7, while relatively small red blood cells pass through the third filter 7 and are discharged from the waste liquid discharger 13 to the outside. Thereby, since the desired fat-derived cells B remain on the third filter 7 in a concentrated state, the pinch valve 8 provided on the collection tube 6 is closed, and the container as shown in FIG. 3 is rotated again around the horizontal axis C to be reversed so that the third filter 7 is disposed above.

  In this state, the Ringer solution is supplied from the gas supply port 12 to separate the fat-derived cells B captured by the third filter 7, and the third filter 7 is backwashed by supplying nitrogen gas. It collects in the collection tube 6 closed by the pinch valve 8. And the process (aseptic cutting process) which obstruct | occludes the edge part in the 3rd filter 7 side of the collection | recovery tube 6 and the edge part (dashed line part E in the figure) between the pinch valve 8 and the container 3 while cutting | disconnecting with heat. ) Can be sealed in the collection tube 6.

  Thus, according to the cell separation device 1 according to the present embodiment, while using the three filters 4, 5, and 7 having different diameters of the through holes, the flow direction of the liquid is switched only by reversing the container 3, Washing and degradation of adipose tissue A and concentration of adipose-derived cells B can be performed easily. That is, the cleaning process, the decomposition process, and the concentration process that have been conventionally performed while being accommodated in separate containers can be easily performed by a single device. Accordingly, the operability can be improved by omitting the replacement work.

  In addition, since the replacement is not accompanied, the substance in the storage unit 2 does not come into contact with the outside air until the fat-derived cells B are concentrated after the fat tissue A is stored in the storage unit 2. There is an advantage that contamination can be prevented more reliably and aseptically processed.

  In the present embodiment, the waste liquid generated during the cleaning of the adipose tissue A is discharged from the waste liquid discharge unit 11 via the first filter 4, but as shown in FIG. One end of another collection tube 6 ′ may be connected to the opening 3 a and the other end opening may be closed by a fourth filter 14 having a through hole equivalent to the third filter 7. In this way, when the waste liquid that has passed through the first filter 4 is allowed to pass through the fourth filter 14, the fat-derived cells B that are present in the waste liquid are captured by the fourth filter 14. The Thereby, it is possible to prevent the fat-derived cells B and blood-derived cells isolated from the adipose tissue during washing from being discharged together with the waste liquid, and to increase the recovery efficiency of the fat-derived cells B and blood-derived cells. .

In this embodiment, nitrogen gas is used for backwashing the filters 4 and 7, but the present invention is not limited to this, and any other inert gas or any gas harmless to the fat-derived cells B is used. May be used.
Moreover, although the opening parts 3a and 3b were provided in the both ends of the cylindrical container 3, you may decide to provide an opening part in the arbitrary positions around the horizontal axis line C instead.

It is a whole lineblock diagram showing the cell separation device concerning one embodiment of the present invention. It is a figure explaining the washing | cleaning process by the cell separation apparatus of FIG. It is a figure explaining the state after completion | finish of the washing | cleaning process by the cell separation apparatus of FIG. It is a figure explaining the digestion process by the cell separation apparatus of FIG. It is a figure explaining the concentration process after completion | finish of the digestion process of FIG. In the concentration process of FIG. 4, it is a figure which shows the state by which the fat origin cell was concentrated by the 3rd filter. It is a figure explaining the collection | recovery method of the fat origin cell concentrated on the 3rd filter in the concentration process of FIG. It is a whole block diagram which shows the modification of the cell separation apparatus of FIG.

Explanation of symbols

A Adipose tissue B Adipose-derived cells C Axis 1 Cell separation device 2 Container 3 Container 3a First opening 3b Second opening 4 First filter 5 Second filter 6 Collection tube 6 ′ Other collection tube 7 Third filter 8 Pinch valve (valve)
9 Enzyme Supply Port 10 Cleaning Liquid Supply Unit 11 Waste Liquid Discharge Unit 12 Cleaning Medium Supply Unit 14 Fourth Filter

Claims (8)

A container comprising a container for containing a mixed liquid containing adipose tissue, and two openings provided in the container;
A first filter disposed at a position to close one opening of the container and prohibiting passage of adipose tissue;
A second filter disposed at a position to close the other opening of the container and prohibiting passage of adipose tissue and allowing passage of fat-derived cells;
A collection tube having one end connected to the opening on the second filter side;
A third filter disposed at a position to close the other end of the collection tube, prohibiting passage of fat-derived cells and allowing passage of red blood cells;
A cell separation apparatus comprising: a flow direction switching mechanism that switches a flow direction of the mixed liquid in the container.   The cell separation according to claim 1, wherein a cleaning liquid supply unit that supplies a cleaning liquid into the housing unit and a waste liquid discharge unit that discharges the waste liquid after cleaning are connected to the opening provided with the first filter. apparatus.   The cell separation device according to claim 2, wherein the cleaning liquid supply unit supplies gas together with the cleaning liquid.   The cell separation device according to claim 1, wherein an enzyme supply port for supplying a digestive enzyme solution is provided in the container housing part.   In the flow direction switching mechanism, the container is arranged with the opening provided with the first filter facing downward when the adipose tissue is washed and digested, and the opening provided with the second filter after digestion is completed. The cell separation device according to any one of claims 1 to 4, wherein the container is rotated such that the container is disposed facing downward. A valve for opening and closing the recovery tube in the vicinity of the opening on the second filter side;
The cell separation device according to claim 1, further comprising a cleaning medium supply unit that supplies a cleaning medium from a side opposite to the collection tube with the third filter interposed therebetween.   The cell separation device according to claim 6, wherein the cleaning medium is a cleaning liquid and / or a gas. One end of another recovery tube is connected to the opening on the first filter side,
The fourth filter for prohibiting passage of fat-derived cells and allowing passage of red blood cells is provided at a position where the other end of the other collection tube is blocked. Cell separation device.

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