JP2010088377A - Cell recovery system, cell recovery method, and fat-derived cell-containing liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To retrieve fat-derived cells in high efficiency through fully removing a digestive enzyme in a short time by a simple operation. <P>SOLUTION: A cell recovery system 1 includes: a filter 2 for catching fat-derived cells; on one side thereof, a cell source container 3 connected via a first changeover valve 6 switchable alternatively and intended for containing a cell suspension A containing an adipose tissue and a digestive enzyme liquid, a first cleansing liquid container 4 for containing a cleansing liquid B, and a retrieving container 5; and, on the other side of the filter 2, a waste liquid container 7 connected via a second changeover valve 10 switchable alternatively and intended for containing a waste liquid passed through the filter 2, a second cleansing liquid container 8 for containing the cleansing liquid, and an eluate feed means 9 for feeding an eluate C for retrieving cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell recovery apparatus and a cell recovery method, and a fat-derived cell-containing liquid recovered by these.

  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). In addition, a method for recovering adipose-derived cells at a high yield by using such a filter for adipose tissue digestive fluid containing an enzyme or the like that digests adipose tissue is disclosed (for example, see Patent Document 2). .)

Japanese Patent No. 39389973 JP 2007-289076 A

  However, when separating adipose tissue digestive liquid with a filter, the digestive enzyme contained in the adipose tissue digestive liquid is captured by the filter and collected together with fat-derived cells that are desired to be finally collected. There is. In order to remove the digestive enzyme trapped in the filter before recovery, it may be possible to wash the lactate Ringer solution etc. through the filter, but the fat-derived cells trapped in the filter penetrate deep into the filter, Conversely, there is a problem that the recovery rate is lowered.

  The present invention has been made in view of the above-described circumstances, and is capable of sufficiently removing digestive enzymes in a short time with a simple operation, and a cell recovery apparatus and a cell recovery method capable of recovering cells at a high yield And an adipose-derived cell-containing solution obtained thereby.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a cell capture filter that captures adipose-derived cells, and a fat tissue connected to the cell capture filter via a first switching valve that can be selectively switched on one side of the cell capture filter. Alternatively, on the other side of the cell capture filter, a cell source container for containing a cell suspension containing a digestive enzyme solution, a first washing solution container for containing a washing solution, and a collection vessel A waste liquid container for containing a waste liquid passing through the cell capture filter, a second wash liquid container for containing a wash liquid, and a cell connected to the cell capture filter via a switchable second switching valve The cell recovery device includes an eluate supply means for supplying an eluate for recovery.

Further, the present invention provides a cell capturing step for allowing a cell suspension containing an adipose tissue and a digestive enzyme solution to flow from one side to a cell capturing filter that captures fat-derived cells, and the cell capturing filter with respect to the cell capturing filter. A first washing step in which a washing solution is passed from the same side as the suspension is passed, and a washing solution is passed from a side opposite to the passage of the cell suspension through the cell capture filter. The second washing step and the eluate for collecting the fat-derived cells captured by the cell trapping filter are passed from the side opposite to the passage of the cell suspension through the cell trapping filter. And a recovery step of recovering the liquid that has passed through the cell capture filter in the elution step. Moreover, this invention is a cell collection | recovery method in which a fat origin cell passes a cell capture filter by gravity in the said cell capture step.
Furthermore, the present invention is a fat-derived cell-containing solution recovered using the cell recovery device.
Furthermore, this invention is the fat origin cell containing liquid obtained by the said cell collection | recovery method.

  According to the present invention, the cell source container is disposed above the cell trapping filter, and the first switching valve is operated to connect the inlet side (one side) of the cell trapping filter to the cell source container side. When the cell suspension in the cell source container is caused to flow into the cell capture filter by gravity or the like by connecting the waste container to the outlet side of the cell capture filter by operating the switching valve, the fat in the cell suspension The derived cells are captured by the cell trapping filter, and other contents flow through the cell trapping filter and flow into the waste container. At this time, some of the digestive enzymes contained in the cell suspension also remain in the cell capture filter together with the fat-derived cells.

  In this state, by switching the first switching valve to the first cleaning liquid container, the cleaning liquid in the first cleaning liquid container is caused to flow from the inlet side (one side) of the cell trapping filter by gravity or the like. Thereby, the digestive enzyme solution remaining in the cell trapping filter is pushed away from the outlet side (the other side) of the cell trapping filter and flows into the waste liquid container. At the same time, the fat-derived cells captured by the cell trapping filter are pushed deeply into the filter.

  After that, by inverting the cell recovery device itself of the present invention, the second washing liquid container is arranged above the cell trapping filter, and the second switching valve is operated so that the outlet side of the cell trapping filter ( The second cleaning liquid container is connected to the other side, and the first switching valve is operated to connect the first cleaning liquid container to the inlet side (one side) of the cell trapping filter. Then, the cleaning liquid in the second cleaning liquid container flows into the cell trapping filter from the outlet side (the other side) due to gravity or the like, and flows toward the inlet side. As a result, the fat-derived cells that have been deeply pushed into the cell trapping filter are pushed back toward the inlet side (one side) to a shallow position in the cell trapping filter and remain in the cell trapping filter. The digestive enzyme is also pushed out of the cell capture filter from the inlet side (one side).

  That is, adipose-derived cells remain in the cell capture filter at a shallow position similar to the state in which the cell suspension is simply passed through the cell capture filter, while the digestive enzymes remaining in the cell capture filter are sufficiently eliminated. can do. The liquid passed through the cell trapping filter flows into the first washing liquid container.

  In this state, the second switching valve is switched to the eluate supply means side, the first switch valve is switched to the collection container side, and the eluate supply means is operated to supply the eluate to the cell trapping filter. The adipose-derived cells captured by the eluate at a shallow position of the cell trapping filter are peeled off from the cell trapping filter and flow into the recovery container to be recovered. Thereby, an adipose-derived cell from which digestive enzymes are sufficiently eliminated and an adipose-derived cell-containing solution containing the adipose-derived cell can be efficiently obtained.

  In addition, the present invention provides a cell capturing step for passing a cell suspension containing an adipose tissue and a digestive enzyme solution from one side by gravity or the like through a cell capturing filter for capturing fat-derived cells, and the cell capturing filter. A first washing step in which a washing solution is passed from the same side as the passage of the cell suspension, and a washing solution from a side opposite to the passage of the cell suspension through the cell capture filter A second washing step for passing the solution and an eluate for collecting the fat-derived cells captured by the cell trapping filter from the side opposite to the cell suspension through the cell trapping filter. A cell recovery method comprising an elution step, and a recovery step of recovering the liquid that has passed through the cell capture filter in the elution step, and a fat-derived cell-containing liquid obtained thereby .

  According to the cell collection device and the cell collection method of the present invention, fat-derived cells from which digestive enzymes are sufficiently eliminated can be collected in a high yield within a short time with a simple operation. Moreover, in the fat-derived cell-containing liquid of the present invention containing the fat-derived cells obtained by the cell recovery apparatus of the present invention or obtained by the cell recovery method of the present invention, the digestive enzyme is sufficiently eliminated as described above. Therefore, for example, even when transplanted into a living body, the adverse effects on the living body due to enzymes are sufficiently reduced.

A cell-derived device 1, a cell collection method, and a fat-derived cell-containing solution obtained by 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, and a cell source container that is disposed on the inlet 2a side of the cell capture filter 2. 3. First switching for connecting the first washing liquid container 4 and the collection container 5 and the cell source container 3, the first washing liquid container 4 and the collection container 5 to the inlet 2a side of the cell trapping filter 2 alternatively. With respect to the valve 6 and the cell trapping filter 2, a waste liquid container 7, a second washing liquid container 8 and an elution liquid supply means 9 arranged on the outlet 2b side, and these waste liquid container 7, the second washing liquid container 8 and elution And a second switching valve 10 for selectively connecting the liquid supply means 9 to the outlet 2b side of the cell trapping filter 2. In the initial state, the first switching valve 6 and the second switching valve are fully closed.

As the cell trapping filter 2, a laminate of non-woven fabric filters having a 6-average fiber diameter of about 0.01 to 10 μm including polyester or polypropylene is employed.
The cell source container 3, the first cleaning liquid container 4, the recovery container 5, the waste liquid container 7, and the second cleaning liquid container 8 may all be containers made of a material such as flexible polyester such as a blood transfusion bag. These are aseptically connected to the cell capture filter 2.

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 first and second cleaning liquid containers 4 and 8 contain a cleaning liquid B such as physiological saline or lactated Ringer's solution, for example. Moreover, nothing is accommodated in the recovery container 5 and the waste liquid container 7 in the initial state.

The first switching valve 6 is composed of two three-way valves 6a and 6b. The first three-way valve 6 a is configured to selectively switch between the cell source container 3 and the first cleaning liquid container 4 with respect to the supply pipe 11. The second three-way valve 6 b is configured to selectively switch between the supply pipe 11 and the collection container 5 with respect to the cell trapping filter 2. Disposed between the first three-way valve 6a and the second three-way valve 6b is a mesh chamber 12 that captures a relatively large piece of adipose tissue and prevents it from flowing into the cell capture filter 2. is there.
The second switching valve 10 is constituted by a single four-way valve.

  The eluate supply means 9 is an eluent C that has little degeneration of adipose-derived cells, for example, physiological saline, buffer solution such as Dulbecco's phosphate buffer solution (D-PBS), Hanks solution, medium such as RPMI 1640 Is a syringe accommodated.

A cell recovery method using 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 three-way valve 6a constituting the first switching valve 6 is provided in the cell source container 3 and the second three-way valve 6b is provided in the supply pipe 11 so that the cell source container 3 is disposed above Switch. Further, the second switching valve 10 is switched to the waste liquid container 7 (in the drawing, the three-way valves 6a and 6b and the blackened portions in the second switching valve 10 indicate the closed direction).

  In this way, after the cell suspension A in the cell source container 3 flows into the supply pipe 11 through the first three-way valve 6a by gravity and is passed through the mesh chamber 12, The liquid is passed through the cell trapping filter 2 through the two three-way valve 6b. By passing through the mesh chamber 12, relatively large adipose tissue or the like in the cell suspension A is captured in the mesh chamber 12, and fat-derived cells and digestive enzyme solution isolated from the adipose tissue are stored in the cell capture filter 2. Pass the liquid.

  The cell capture filter 2 mainly captures fat-derived cells (cell capture step). The capture depth of the fat-derived cells in this state in the cell capture filter 2 is 0.4 to 4.0 cm (first depth position). Most of the liquid components such as the digestive enzyme solution pass through the capture filter 2, but a part of them adheres and remains. The liquid that has passed through the cell trapping filter 2 is collected in the waste liquid container 7 as the waste liquid D (see FIG. 3) via the second switching valve 10.

  When all the cell suspension A in the cell source container 3 is discharged, as shown in FIG. 3, the first three-way valve 6b of the first switching valve 6 is moved to the first washing liquid container 4 side. Switch. As a result, the cleaning liquid B in the first cleaning liquid container 4 flows into the cell trapping filter 2 from the inlet 2a side by gravity and flows through the cell trapping filter 2 (first cleaning step). The digestive enzyme remaining in the cell trapping filter 2 is washed away by the washing liquid B and flows into the waste liquid container 7 as the waste liquid D. On the other hand, since the fat-derived cells trapped in the cell trapping filter 2 are also pushed by the cleaning liquid B, they enter the deeper position (second depth position) in the cell trapping filter 2.

  When almost all of the washing liquid B is discharged from the first washing liquid container 4, the second switching valve 10 is switched to the second washing liquid container 8 side to invert the entire cell recovery apparatus 1 (FIG. 4). Thereby, the cleaning liquid B in the second cleaning liquid container 8 flows into the cell trapping filter 2 from the outlet 2b side through the second switching valve 10 due to the influence of gravity or the like, and passes through the cell trapping filter 2. (Second washing step). Accordingly, in the first washing step, the fat-derived cells that have entered the cell capture filter 2 up to the second depth position are pushed back to the first depth position by the washing liquid B, which is shallower.

In this state, as shown in FIG. 5, the second three-way valve 6b is switched to the collection container 5 side, the second switching valve 10 is switched to the eluate supply means 9 side, and the eluate supply means 9 is operated. Thus, the eluate C is passed through the cell trapping filter 2 from the outlet 2b side (elution step). As a result, the fat-derived cells that have been captured at the first depth position on the inlet 2a side of the cell capture filter 2 are peeled off from the cell capture filter 2.
Then, the fat-derived cells thus peeled together with the eluate C flow into the collection container 5 through the second three-way valve 6b and are collected (collection step).

  Thus, according to the cell collection device 1 and the cell collection method according to the present embodiment, the fat-derived cells and the like captured by the cell capture filter 2 are not collected as they are, but are washed once with the washing liquid B, and thus collected. The amount of the digestive enzyme solution mixed in the fat-derived cells can be reduced. As a result, fat-derived cells with little damage can be efficiently recovered, and the recovered fat-derived cells can be maintained in a healthy state.

In addition, when the washing liquid B is simply washed, the fat-derived cells enter deep into the cell trapping filter 2 and the recovery rate of the fat-derived cells is lowered. In the washing step 2, by supplying the washing liquid B from the opposite side to the cell trapping filter, the fat-derived cells that have entered deep into the cell trapping filter are once returned to a shallow position in the trapping filter and then further removed from the filter. Since it peels and collect | recovers with the eluate C, the fall of the collection | recovery rate of a fat origin cell can be prevented.
That is, according to the cell collection device 1 and the cell collection method according to the present embodiment, the advantage that fat-derived cells in which the content of the digestive enzyme solution is sufficiently reduced by a simple configuration can be collected at a high collection rate. There is.

  In the present embodiment, the first and second switching valves 6 and 10 have been described by exemplifying a three-way valve or a four-way valve that can be selectively switched to a route at any position, but the present invention is not limited thereto. Instead, the path may be alternatively 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 verista pump, a pressurized device, or the like.

In the adipose-derived cell-containing liquid containing the adipose-derived cells according to this embodiment that is collected at a high recovery rate by the cell collection device 1 according to this embodiment, the adipose-derived cells include vascular endothelial cells, fibroblasts, and hematopoiesis. Stem cells, blood cells, adipose-derived stem cells and the like are included. 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. In the cell collection | recovery apparatus of FIG. 1, it is a figure which shows the 1st washing | cleaning step which wash | cleans the fat origin cell capture | acquired by the cell capture filter in the forward direction. In the cell collection device of FIG. 1, it is a figure which shows the 2nd washing | cleaning step which wash | cleans the fat origin cell captured by the cell capture filter in the reverse direction. 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 1st washing | cleaning liquid container 5 Recovery container 6 1st switching valve 7 Waste liquid container 8 2nd washing | cleaning liquid container 9 Elution liquid supply means 10 2nd switching Valve A Cell suspension B Wash solution C Eluate D Waste solution

Claims (5)

A cell capture filter for capturing fat-derived cells;
To accommodate a cell suspension containing adipose tissue and digestive enzyme solution connected to the cell capture filter via a first switching valve that can be selectively switched on one side of the cell capture filter. A cell source container, a first cleaning liquid container for containing a cleaning liquid, and a recovery container;
A waste liquid container for storing waste liquid that has passed through the cell capture filter, connected to the cell capture filter via a second switching valve that can be selectively switched on the other side of the cell capture filter, A cell recovery apparatus comprising: a second cleaning liquid container for containing a cleaning liquid; and an eluate supply means for supplying an eluate for cell recovery. A cell capture step of passing a cell suspension containing adipose tissue and digestive enzyme solution from one side through a cell capture filter that captures adipose-derived cells;
A first washing step in which a washing solution is passed from the same side as the cell suspension is passed through the cell capture filter;
A second washing step in which a washing solution is passed from the opposite side of passing the cell suspension through the cell capture filter;
An elution step of allowing an eluate for recovering the fat-derived cells captured by the cell capture filter to flow from the opposite side of the cell suspension filter through the cell suspension; and
A cell recovery method comprising: a recovery step of recovering the liquid that has passed through the cell capture filter in the elution step.   The cell collection method according to claim 2, wherein in the cell capturing step, fat-derived cells are passed through a cell capturing filter by gravity.   A fat-derived cell-containing solution recovered using the cell recovery device according to claim 1.   A fat-derived cell-containing solution obtained by the cell recovery method according to claim 2 or 3.

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