JP2010088376A - Cell recovering device and liquid containing adipose-derived cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To recover cells in high yield while avoiding trouble of inclusion of fat in the recovered cells as an impurity. <P>SOLUTION: The cell recovering device 1 is provided with a cell capturing filter 2 to capture adipose-derived cells: a cell source container 3 holding a cell suspension A containing a fat tissue and a digestive enzyme liquid and a washing liquid container 4 holding a washing liquid B and connected through an alternatively switchable first changeover valve 9 to pipes 8, 10 connected to one side of the filter: a fat capturing filter 6 placed in the pipe 8 and capturing a fat component included in the cell suspension A supplied from the cell source container 3: a recovering vessel 7 connected through an alternatively switchable second changeover valve 11 to the pipe 10 between the fat capturing filter 6 and the cell capturing filter 2: and a waste liquid container 12 to hold waste liquid and an eluate supplying means 13 to supply an eluate C and connected to the other side of the cell capturing filter 2 through an alternatively switchable third changeover valve 14. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell recovery apparatus and a fat-derived cell-containing liquid.

  Conventionally, a filter that separates mononuclear cells such as leukocytes from bone marrow, peripheral blood, umbilical cord blood or the like is known (see Patent Document 1). Moreover, the method of collect | recovering fat origin cells by a high yield is disclosed by using such a filter with respect to the adipose tissue digestive liquid containing the enzyme which digests adipose tissue (for example, refer patent document 2). ).

Japanese Patent No. 39389973 JP 2007-289076 A

  However, when the adipose tissue digestion fluid is separated by a filter, the fat content released from the fat cells contained in the adipose tissue digestion fluid is captured by the filter that captures the fat-derived cells, and the final recovery is performed. There is an inconvenience of being collected together with the desired fat-derived cells.

  This invention is made | formed in view of the situation mentioned above, Comprising: The fat origin cell can be collect | recovered in high yield avoiding the problem that fat is contained as an impurity in the fat origin cell collect | recovered. The object is to provide a cell recovery device.

In order to achieve the above object, the present invention provides the following means.
That is, the present invention relates to a cell capture filter that captures fat-derived cells and a pipe connected to one side of the cell capture filter via a first switching valve that can be alternatively switched. A cell source container for containing a cell suspension containing a tissue and a digestive enzyme solution, a washing liquid container for containing a washing solution, and a cell suspension provided in the pipe and supplied from the cell source vessel A fat-capturing filter that traps the fat contained in the water, and the pipe between the fat-capturing filter and the cell-capturing filter via a second switching valve that is alternatively switchable In order to accommodate the waste liquid that passes through the cell capture filter connected to the cell capture filter via a third switching valve that can be selectively switched, on the other side of the collection container and the cell capture filter Waste container and cell recovery Providing a cell harvester and a elution solution supply means for supplying an eluate.

  In the cell collection device of the present invention, a cell source container is disposed above the cell trapping filter, and the first source valve is operated to connect the cell source container to a pipe continuing to the inlet side (one side) of the cell trapping filter. And the second switching valve is switched to the oil trapping filter side, while the third switching valve is operated to connect the waste liquid container to the outlet side (other side) of the cell trapping filter. When the cell suspension is passed through the cell trapping filter by gravity or the like, the cell suspension first passes through the fat trapping filter, and the fat and some fat-derived cells are trapped. The cell suspension containing most of the fat-derived cells passes through the fat trapping filter and flows into the cell trapping filter.

The fat-derived cells contained in the cell suspension are captured by the cell trapping filter, and the other liquid passes through the cell trapping filter and flows into the waste liquid container via the third switching valve. Some digestive enzyme solutions adhere to the cell capture filter.
In this state, the first switching valve is switched to the cleaning liquid container side to allow the cleaning liquid to flow into the fat trap filter. The fat that has been trapped in the fat trapping filter is maintained in the trapped state, and some of the fat-derived cells that have also been trapped are washed away by the washing solution and flowed into the downstream cell trapping filter, Captured by cell capture filter.

  Thereafter, the second switching valve is switched to the collection container side, the third switching valve is switched to the eluate supply means side, and the eluate supply means is operated, so that the eluate is supplied to the cell capture filter and the outlet side thereof. Supply from. As a result, fat-derived cells are peeled off from the cell trapping filter and flow into the collection container and collected. In this manner, fat-derived cells from which the fat and digestive enzyme solution have been sufficiently removed can be collected in the collection container.

In the said invention, the said fat content capture filter is a nonwoven fabric, Comprising: It is good also as an average fiber diameter being 20 micrometers or more and 100 micrometers or less.
Moreover, it is preferable that an average fiber diameter is 30 micrometers or more and 50 micrometers or less.
By doing in this way, the fat is more efficiently captured by the nonwoven fabric made of fibers having a relatively large average fiber diameter, and the fat and digestive enzyme solution are sufficiently reduced from the fat-derived cells that are finally recovered. can do. That is, by setting the average fiber diameter to preferably 20 μm or more, more preferably 30 μm or more, the fat content can easily enter the fat capture filter more efficiently, while the fat-derived cells can capture the fat content. It becomes easy to pass through the filter. In addition, when the average fiber diameter is preferably 100 μm or less, more preferably 50 μm or less, the fat captured by the fat trapping filter is more easily retained in the filter.

In the present invention, the fat trapping filter may be a porous material having a large number of through pores, and the average pore diameter may be 20 μm or more and 100 μm or less.
Moreover, it is preferable that an average pore diameter is 25 micrometers or more and 40 micrometers or less.
By doing in this way, fat content can be efficiently captured, and the fat content and digestive enzyme solution can be sufficiently reduced from the finally recovered fat-derived cells. That is, by setting the average pore diameter to preferably 20 μm or more, more preferably 25 μm or more, the fat content can easily enter the fat capture filter more efficiently, while the fat-derived cells can capture the fat content. It becomes easy to pass through the filter. In addition, when the average pore diameter is preferably 100 μm or less, more preferably 40 μm or less, the fat captured by the fat trapping filter is more easily retained in the filter.

According to the cell collection device of the present invention, the fat-derived cells to be collected and the fat-containing cell-containing liquid containing the same can avoid the inconvenience that fats are contained as impurities, and the cells can be collected in high yield. There is an effect that can be.
In addition, in the fat-derived cell-containing liquid recovered by the cell recovery device of the present invention, as described above, the amount of fat and digestive enzyme solution is sufficiently eliminated, for example, when transplanted into a living body, etc. The adverse effect of the enzyme on the living body is sufficiently reduced.

A cell recovery apparatus 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the cell collection device 1 according to this embodiment includes a cell capture filter 2 that captures fat-derived cells, a cell source container 3 that is disposed on the inlet side of the cell capture filter 2, and a washing liquid container. 4, a first switching valve that switches the mesh chamber 5, the fat-capturing filter 6 and the recovery container 7, and the cell source container 3 and the cleaning liquid container 4 to selectively connect to the flow path 8 to the mesh chamber 5. 9, a second switching valve 11 for selectively connecting the flow path 10 from the fat trapping filter 6 and the collection container 7 to the inlet 2 a side of the cell trapping filter 2, and the outlet 2 b side of the cell trapping filter 2 And a third switching valve 14 for selectively connecting the waste liquid container 12 and the eluate supply means 13 to the outlet 2b side of the cell trapping filter 2. Have .

As the cell trapping filter 2, a laminate of non-woven fabric filters having an average fiber diameter of about 0.01 to 10 μm containing polyester or polypropylene is employed.
The fat trapping filter 6 is also formed by laminating a nonwoven fabric filter having an average fiber diameter of about 20 μm to 100 μm, preferably 30 μm to 50 μm, including polyester or polypropylene.

The mesh chamber 5 captures a relatively large piece of adipose tissue and prevents inflow into the cell capture filter 2. The mesh chamber 5 and the fat trapping filter 6 are arranged in series in the flow path 10 between the first switching valve 9 and the cell trapping filter 2.
Further, the cell source container 3, the washing liquid container 4, the collection container 7 and the waste liquid container 12 may all be containers made of a material such as flexible polyester such as a blood transfusion bag. Connected.

The cell source container 3 contains a cell suspension A obtained by mixing a finely cut adipose tissue and a digestive enzyme solution for isolating the adipose tissue to isolate adipose-derived cells.
The cleaning liquid container 4 contains, for example, a cleaning liquid B such as physiological saline or lactated Ringer's solution.
Further, nothing is accommodated in the recovery container 7 and the waste liquid container 12 in the initial state.

The first to third switching valves 9, 11, and 14 are constituted by, for example, three-way valves.
The eluate supply means 13 is an eluate C that has little degeneration of fat-derived cells, for example, a physiological saline, a buffer solution such as Dulbecco's phosphate buffer (D-PBS), Hanks's solution, a medium such as RPMI 1640, etc. Is a syringe accommodated.

The operation of the cell recovery apparatus 1 according to this embodiment configured as described above will be described below.
To separate and recover adipose-derived cells from a cell suspension A containing adipose tissue and digestive enzyme solution using the cell recovery apparatus 1 according to the present embodiment, first, as shown in FIG. The first switching valve 9 is placed on the cell source container 3 side, the second switching valve 11 is placed on the flow channel 10 side from the fat trapping filter, the second 3 is switched to the waste liquid container 12 side.

  In this way, after the cell suspension A in the cell source container 3 is passed through the mesh chamber 5 and the oil trap 6 through the first switching valve 9 by gravity, The liquid is passed through the cell trapping filter 2 through the two switching valves 11. By being passed through the mesh chamber 5, relatively large adipose tissue or the like in the cell suspension A is captured in the mesh chamber 5, and fat-derived cells, fat and digestive enzyme liquid isolated from the fat tissue are contained in the fat. The liquid is passed through the capture filter 6.

  The fat trapping filter 6 mainly traps fat. Most of the liquid components such as fat-derived cells and digestive enzyme solution contained in the cell suspension A pass through, but some of them adhere and remain. The fat-derived cells and the like that have passed through the fat-capturing filter 6 flow into the cell-capturing filter 2 that is arranged immediately thereafter.

  In the cell trapping filter 2, fat-derived cells are mainly trapped. Most of the liquid components such as the digestive enzyme solution pass through, but some of them also adhere and remain. The liquid that has passed through the cell trapping filter 2 is collected in the waste liquid container 12 as the waste liquid D through the third switching valve 14.

When all the cell suspensions A in the cell source container 3 are discharged, the first switching valve 9 is switched to the cleaning liquid container 4 side as shown in FIG. As a result, the cleaning liquid B in the cleaning liquid container 4 flows into the mesh chamber 5, the fat capturing filter 6 and the cell capturing filter 2 by gravity and is allowed to pass through.
By flowing the cleaning liquid B, the fat-derived cells and the digestive enzyme liquid adhering to the mesh chamber 5 are pushed away toward the downstream fat-capturing filter 6. Further, the fat-derived cells and the digestive enzyme solution adhering to the fat trapping filter 6 are pushed toward the cell trapping filter 2. Furthermore, the digestive enzyme solution adhering to the cell trapping filter 2 is also washed away and flows into the waste liquid container 12.

That is, only the fat-derived cells are mainly captured by the cell trapping filter 2 in a state where unnecessary fat and digestive enzyme solution are removed by the operation of circulating the cleaning liquid B.
When almost all of the cleaning liquid B is discharged from the cleaning liquid container 4, as shown in FIG. 4, the second switching valve 11 is switched to the collection container 7 side, and the third switching valve 14 is switched to the eluate supply means 13. By switching to the side and operating the eluate supply means 13, the eluate C is supplied to the cell capture filter 2 from the outlet 2b side. Thereby, the fat-derived cells captured on the inlet 2a side of the cell trapping filter 2 are peeled off from the cell trapping filter 2.
The fat-derived cells peeled in this way flow into the collection container 7 through the second switching valve 11 together with the eluate C and are collected.

  As described above, according to the cell collection device 1 according to the present embodiment, the cell suspension A containing the adipose tissue and the digestive enzyme solution is not directly flowed into the cell trapping filter 2 but is captured before the inflow. Since the filter 6 is passed, it is possible to prevent the occurrence of inconvenience that unnecessary fat is trapped by the cell trapping filter 2. Moreover, since the fat-derived cells captured by the cell trapping filter 2 are not collected as they are, but washed once with the washing liquid B, the amount of digestive enzyme solution mixed in the collected fat-derived cells can be reduced. As a result, fat-derived cells with little damage can be collected and maintained in a healthy state.

In the present embodiment, the first to third switching valves 9, 11, and 14 have been exemplified and described as three-way valves that are selectively switched to a route at any position, but the present invention is not limited thereto. Instead of this, the path may alternatively be selected by a clamp such as a pinch cock.
In this embodiment, the cell suspension A containing fat-derived cells is passed through the cell trapping filter due to the influence of gravity. However, the method of passing the fat-derived cells through the filter is limited to this. For example, the liquid may be passed using a peristaltic pump, a pressurized device, or the like.
Further, as the fat trapping filter 6, a layer in which a sponge-like filter made of polyester or polypropylene and having an average pore diameter of about 20 μm to 100 μm, preferably 25 μm to 40 μm may be employed.

In the adipose-derived cell-containing liquid containing the adipose-derived cells according to the present embodiment collected by the cell collection apparatus 1 according to the present embodiment, the adipose-derived cells include vascular endothelial cells, fibroblasts, Examples include hematopoietic stem cells, hematopoietic cells, and adipose-derived stem cells. Here, the stem cell is a cell collected from adipose tissue for the purpose of cell therapy or the like, and refers to a cell having multipotency and self-renewal ability. In addition, the adipose tissue may be human-derived subcutaneous adipose tissue, visceral adipose tissue, white adipose tissue, brown adipose tissue, etc., and a fat portion of a human or the like collected with a sharp instrument such as scissors, liposuction, etc. It may be collected by a method.
In the fat-derived cell-containing solution according to the present embodiment, the digestive enzyme is sufficiently eliminated as described above, and therefore, the adverse effect of the enzyme on the living body is sufficiently reduced even when transplanted into the living body.

It is a whole lineblock diagram showing the cell recovery device concerning one embodiment of the present invention. In the cell collection | recovery apparatus of FIG. 1, it is a figure which shows the capture | acquisition step which makes a cell capture filter capture | acquire fat-derived cells. FIG. 2 is a diagram showing a washing step of washing the fat-derived cells captured by the cell trapping filter in the forward direction in the cell collection device of FIG. 1. It is a figure which shows the step which elutes and collect | recovers the fat origin cells capture | acquired by the cell capture filter in the cell collection | recovery apparatus of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell collection | recovery apparatus 2 Cell capture filter 2a Inlet 2b Outlet 3 Cell source container 4 Washing liquid container 6 Fat content capture filter 7 Recovery container 8,10 Piping 9 1st switching valve 11 2nd switching valve 12 Waste liquid container 13 Eluate supply Means 14 Third switching valve A Cell suspension B Wash solution C Eluate D Waste solution

Claims (6)

A cell capture filter for capturing fat-derived cells;
To accommodate a cell suspension containing adipose tissue and digestive enzyme solution connected to a pipe connected to one side of the cell trapping filter via a first switching valve that can be selectively switched A washing solution container for containing a cell source vessel and a washing solution;
A fat-capturing filter that is provided in the pipe and captures the fat contained in the cell suspension supplied from the cell source container;
A recovery container connected to the pipe between the fat trapping filter and the cell trapping filter via a second switching valve that can be alternatively switched;
A waste liquid container for storing waste liquid that has passed through the cell capture filter, connected to the cell capture filter via a third switching valve that can be selectively switched, on the other side of the cell capture filter; A cell recovery apparatus comprising an eluate supply means for supplying an eluate for cell recovery.   The cell collection device according to claim 1, wherein the fat trapping filter is a nonwoven fabric and has an average fiber diameter of 20 µm to 100 µm.   The cell collection device according to claim 2, wherein an average fiber diameter of the fat capturing filter is 30 µm or more and 50 µm or less.   The cell collection device according to claim 1, wherein the fat trapping filter is a porous material having a large number of through pores, and an average pore diameter thereof is 20 µm to 100 µm.   The cell collection device according to claim 4, wherein the fat trapping filter has an average pore diameter of 25 µm or more and 40 µm or less. An adipose-derived cell-containing solution recovered by the cell recovery device according to any one of claims 1 to 5.

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