JP2008189594A - Device for producing biological tissue prosthetic material and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a biological tissue prosthetic material efficiently by utilizing cell sources such as peripheral blood, cord blood, marrow fluid, fat tissue or placenta tissue by a simple operation in a short time. <P>SOLUTION: This device 1 for producing the biological tissue prosthetic material is provided by equipping a cell-capturing filter 2 for capturing nucleus cells effective for vascularization without capturing erythrocytes and platelets, a cell source container 3 housing the cell source A and a recovering container 4 housing the biological prosthetic material B and connected with the entrance port 2a side of the cell-capturing filter 2 through a first switching valve 7, a drain bag 5 connected to the exit port 2b side of the cell-capturing filter 2 through a second switching valve 8 and housing the erythrocytes and platelets permeating through the cell-capturing filter 2 and a recovering liquid-supplying means 6 for supplying the recovering liquid C. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an apparatus and a method for manufacturing a biological tissue filling body.

  Conventionally, blood cells that do not substantially capture red blood cells but that are effective for angiogenesis flow into a cell capture filter that captures the cells, capture the cells effective for angiogenesis by the cell capture filter, and collect the captured cells A method for producing a cell composition for neonatal therapy is known (for example, see Patent Document 1).

JP 2005-82567 A

  However, when producing a biological tissue complement using collected cells that are effective for angiogenesis, it is necessary to take out the seed from the collection container and seed it on the biological tissue supplement. At the time of taking out from the plant and sowing it on the living tissue supplement material, there is a possibility that bacteria etc. may be mixed in, and it is necessary to pay a high degree of care so that such a problem does not occur. In addition, body fluids after removal of cells that are effective for angiogenesis also contain a lot of substances that are effective for the production of biological tissue complements, which can be used effectively instead of simply returning them to the patient's body. Conceivable.

  The present invention has been made in view of the above-described circumstances, and is a biological tissue filling body that can efficiently produce a biological tissue filling body using peripheral blood in a short time with a simple operation. An object of the present invention is to provide a manufacturing apparatus and a manufacturing method.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a cell capture filter that captures nucleated cells effective for angiogenesis without capturing red blood cells and platelets;
A cell source container containing a cell source and a collection container containing a biological tissue filling material connected to the inlet side of the cell trapping filter via a first switching valve; And a drain bag for storing red blood cells and platelets permeated through the cell-capturing filter and a recovery liquid supply means for supplying a recovery liquid.

  According to the present invention, the first switching valve connected to the inlet side of the cell trapping filter is connected to the cell source container side containing the cell source, and the second switching valve connected to the outlet side of the cell trapping filter. When the cell source is allowed to flow into the cell trapping filter by gravity or other means while being connected to the drain bag side, nucleated cells effective for angiogenesis, for example, mononuclear cells are trapped by the cell trapping filter. The On the other hand, red blood cells and platelets pass through without being captured by the cell trapping filter and flow into the drain bag.

  Thereafter, the first switching valve is switched from the cell source container side to the recovery container side, the second switching valve is switched from the drain bag side to the recovery liquid supply means side, and the recovery liquid is supplied by the operation of the recovery liquid supply means. By supplying, the cell trapping filter is backwashed with the collected liquid. Thereby, the nucleated cells effective for angiogenesis captured by the cell trapping filter flow into the recovery container together with the recovery liquid, and are seeded on the biological tissue filling material accommodated in the recovery container. In other words, the nucleated cells effective for angiogenesis recovered from the cell source can be aseptically seeded on the body tissue filling material with a simple operation without touching the outside air, and the body tissue filling material can be efficiently produced. Can do.

In the said invention, it is good also as the said collection container being pressure-reduced.
In this way, when the first switching valve is switched from the cell source container side to the recovery container side, the nucleated cells captured by the cell trapping filter are easily sucked into the recovery container together with the recovery liquid. It becomes possible to manufacture the tissue complement more efficiently.

In the above invention, the recovery container may be provided with injection ports for calcium ions and cytokines.
Change from mononuclear cells to fibroblast-like cells by acting calcium ions on nucleated cells that are trapped by the cell trapping filter and collected in the collection container and effective for angiogenesis, especially mononuclear cells. Can do. Fibroblast-like cells have the same properties as mesenchymal stem cells, can be differentiated under predetermined conditions, and can produce a biological tissue filling body capable of repairing biological tissue at an early stage after patient compensation. In addition, the growth of fibroblast-like cells can be promoted by the action of cytokines.
In this case, the cytokine may be b-FGF.
The cell source may be peripheral blood, umbilical cord blood, bone marrow fluid, adipose tissue, or placental tissue.

  The present invention also includes a step of allowing a cell source to pass through a cell capture filter that captures nucleated cells effective for angiogenesis without capturing red blood cells and platelets, and supplying calcium ions to the red blood cells that have passed through the cell capture filter. A step of allowing the liquid obtained by supplying calcium ions to pass through the cell trapping filter from the reverse direction; a step of collecting the liquid that has passed through the cell permeation filter in the reverse direction; and A method for producing a body tissue filling body comprising a step of supplying the filling material.

  According to the present invention, by allowing a cell source to permeate through the cell trapping filter, nucleated cells effective for angiogenesis contained in the cell source are trapped by the cell trapping filter, and erythrocytes and platelets are permeated. By supplying calcium ions to the erythrocytes that have passed through the cell trapping filter, the erythrocytes are hemolyzed and destroyed, and a body fluid containing plasma contained therein can be obtained. Then, by allowing the obtained body fluid to permeate through the cell trapping filter from the reverse direction, nucleated cells effective for angiogenesis captured by the cell trapping filter are backwashed and recovered by the body fluid. Then, by supplying the collected body fluid to the biological tissue filling material, nucleated cells effective for angiogenesis can be seeded on the biological tissue filling material, and a biological tissue filling material can be easily obtained.

In this case, since the plasma obtained by supplying calcium ions to erythrocytes has an action of improving cell engraftment, it is possible to promote cell growth in the body tissue complement. Furthermore, since calcium ions contained in the body fluid change mononuclear cells to fibroblast-like cells, it is possible to produce a biological tissue supplement capable of repairing biological tissue at an early stage after implantation in the living body. .
In this case, the cell source may be peripheral blood, umbilical cord blood, bone marrow fluid, adipose tissue, or placental tissue.

  According to the present invention, a biological tissue complement is efficiently produced using a cell source such as peripheral blood, umbilical cord blood, bone marrow fluid, adipose tissue or placental tissue in a short time with a simple operation. There is an effect that can be.

A manufacturing apparatus 1 and a manufacturing method of a biological tissue filling body according to the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the manufacturing apparatus 1 for living body tissue complement according to this embodiment captures cells effective for angiogenesis without capturing red blood cells and platelets by causing peripheral blood A to flow in one direction. Cell trapping filter 2, blood container (cell source container) 3 containing peripheral blood A connected to the inlet side of cell trapping filter 2, recovery container 4 containing biological tissue filling material B, and cell trapping filter And a recovery liquid supply means including a drain bag 5 that stores red blood cells and platelets that have passed through the cell trapping filter 2 connected to the outlet side 2 and a syringe 6 that supplies the recovery liquid C.

A first three-way valve (switching valve) 7 is disposed between the cell trapping filter 2 and the blood container 3 and the collection container 4, and the blood container 3 or the collection container 4 is switched by switching the first three-way valve 7. Any one of these is alternatively connected to the cell trapping filter 2. In the figure, reference numeral 8 denotes a pinch valve.
In addition, a second three-way valve (switching valve) 9 is disposed between the cell trapping filter 2 and the drain bag 5 and the syringe 6. By switching the second three-way valve 9, the drain bag 5 or One of the recovery liquid supply means including the syringe 6 is selectively connected to the cell trapping filter 2.

As the cell trapping filter 2, a laminate of nonwoven fabric filters having an average fiber diameter of about 0.01 to 10 μm made of polyester or polypropylene is employed.
The blood container 3, the collection container 4 and the drain bag 5 are all connected to the cell capture filter 2 aseptically.

The biological tissue filling material B accommodated in the collection container 4 is made of, for example, β tricalcium phosphate (βTCP). Further, the inside of the collection container 4 is in a reduced pressure state.
Further, the collection container 4 is provided with a connection portion 4a to which supply means such as a syringe 10 (see FIG. 4) can be connected aseptically, and calcium ions D or b-FGF can be supplied through the connection portion 4a. It can be supplied by the syringe 10.

Further, in the syringe 6, there is little degeneration of cells effective for angiogenesis as the recovery solution C, for example, physiological saline, buffer solution such as Dulbecco's phosphate buffer solution (D-PBS), Hanks solution, RPMI 1640, etc. The medium etc. are accommodated.
Moreover, in order to collect | recover efficiently, it is better to employ | adopt the collection | recovery liquid C which has a fixed blood osmotic pressure, and dextran etc. may be added.

  In addition to peripheral blood, umbilical cord blood, bone marrow fluid, adipose tissue or placental tissue may be used as the cell source. Of these, those having high viscosity are preferably used after enzymatic treatment or the like to lower the viscosity and liquefy it.

The operation of the manufacturing apparatus 1 for a biological tissue filling body according to this embodiment configured as described above will be described below.
In order to manufacture a biological tissue filling body using the biological tissue filling body manufacturing apparatus 1 according to the present embodiment, the first three-way valve 7 is connected to the blood container 3 side, and the second three-way valve 9 is connected to the drain bag. 5 is connected, and the blood container 3 is disposed higher than the cell trapping filter 2.

  As a result, as shown in FIG. 2, the peripheral blood A stored in the blood container 3 descends in the pipe due to gravity. As the peripheral blood A, for example, 40 cc to 200 cc is preferably lowered at a dropping rate of 20 cc / min. The descending peripheral blood A passes through the first three-way valve 7 and is supplied to the cell trapping filter 2 from the inlet 2a side. When flowing through the cell trapping filter 2, nucleated cells that are effective in angiogenesis, for example, mononuclear cells, contained in the peripheral blood A are captured. On the other hand, red blood cells and platelets E pass without being captured by the cell trapping filter 2, pass through the second three-way valve 9, and flow into the drain bag 5 through the piping.

  When the peripheral blood A in the blood container 3 has sufficiently passed through the cell trapping filter 2, the first three-way valve 7 is switched to the collection container 4 side and the second three-way valve 9 is switched to the syringe 6 side. The syringe 6 is pressurized. Thereby, the collection liquid C in the syringe 6 is supplied from the outlet 2b side of the cell trapping filter 2.

Since the inside of the collection container 4 is set in a reduced pressure state, the liquid C in the cell trapping filter 2 is connected via a pipe as shown in FIG. 3 by switching the first three-way valve 7 to the collection container 4 side. Is sucked to the inlet 2a side.
That is, the recovery liquid C in the syringe 6 smoothly flows into the cell trapping filter 2 from the outlet 2b side by the pressurization on the outlet 2b side by the operation of the syringe 6 and the pressure reduction on the inlet 2a side by the recovery container 4. The capture filter 2 flows from the outlet 2b side toward the inlet 2a side. Thereby, the cell trapping filter 2 is back-washed, and the nucleated cells F effective for angiogenesis captured by the cell trapping filter 2 are recovered from the inlet 2a side into the recovery container 4 via the first three-way valve 7. It flows with liquid C.

  Since the biological tissue filling material B is accommodated in the collection container 4, the nucleated cells F effective for angiogenesis that have flowed in are seeded and adhered to the biological tissue filling material B. Thereby, a biological tissue complement is manufactured.

  In this case, according to the manufacturing apparatus 1 according to the present embodiment, it is possible to manufacture a biological tissue filling body by simply switching the two three-way valves 7 and 9 and operating the syringe 6 as the recovery liquid supply means. Therefore, it is possible to manufacture a living tissue complement very easily. Furthermore, since the peripheral blood A and the biological tissue filling material B are all maintained in the containers 3 and 4 and the piping without touching the outside air in the manufacturing process, there is no risk of infection, and the biological tissue filling material is manufactured aseptically. There is an advantage that you can.

  Moreover, with respect to the biological tissue filling body manufactured in this way, the syringe 10 is inserted into the connecting portion 4a provided in the collection container 4, and calcium ions D and b-FGF are supplied, whereby the biological tissue filling body. Nucleated cells F, particularly mononuclear cells that are effective for angiogenesis attached to the cells, can be changed to fibroblast cells. Since fibroblast cells have the same properties as mesenchymal stem cells, they can engraft and grow on biological tissue filling material B and can differentiate into biological tissue precursor cells such as osteoblasts. A biological tissue filling body capable of repairing a defect in a biological tissue at an early stage after being implanted can be produced.

Next, the manufacturing method of the biological tissue filling body which concerns on the 2nd Embodiment of this invention is demonstrated below with reference to FIGS.
Since the manufacturing method of the biological tissue filling body according to the present embodiment is performed using the same manufacturing apparatus 20 as the manufacturing apparatus 1 of the biological tissue filling body according to the first embodiment shown in FIG. Common parts are denoted by the same reference numerals and description thereof is omitted. Note that the second three-way valve 9 and the syringe 6 as the recovered liquid supply means in the manufacturing apparatus 1 according to the first embodiment are unnecessary.

  In the manufacturing method according to the present embodiment, the peripheral blood A stored in the blood container 3 is supplied to the cell capture filter 2 by opening the first three-way valve 7, and the nucleated cells F effective for angiogenesis are supplied to the cell capture filter 2. Step S1 for capturing the cells, Step S2 for supplying calcium ions G or 0.3-0.5% physiological saline to the red blood cells E that have passed through the cell trapping filter 2 and are stored in the drain bag 5, and a first step Step S3 for switching the three-way valve 7 to allow the liquid H in the drain bag 5 to pass through the cell trapping filter 2 in the reverse direction from the outlet 2b side of the cell trapping filter 2, and the liquid F passing through the cell trapping filter 2 in the reverse direction , H is collected in the direction of the collection container 4, and step S 5 is provided to supply the collected liquids F and H to the biological tissue filling material B in the collection container 4.

  When calcium ions G are supplied to peripheral blood containing a large amount of remaining red blood cells E from which nucleated cells F effective for angiogenesis are separated in step S1, red blood cells E are destroyed and internal plasma components H are drained. It remains in the bag 5. In step S3, the inside of the drain bag 5 is pressurized, and the body fluid containing the extracted plasma component H is caused to flow back toward the cell capture filter 2 side.

  Alternatively, the drain bag 5 is disposed at a higher position than the cell trapping filter 2, and the body fluid containing the plasma component H is caused to flow from the outlet 2b side to the inlet 2a side of the cell trapping filter 2 by gravity. Thus, the nucleated cells F effective for angiogenesis that have been captured by the cell capture filter 2 are separated from the cell capture filter 2 by the flow of the body fluid containing the plasma component H, and the inlet 2a side together with the body fluid containing the plasma component H Spill from. Since the first three-way valve 7 is switched so as to be connected to the collection container 4 side, a body fluid containing the plasma component H and the nucleated cells F effective for angiogenesis is placed in the collection container 4 in step S4. Collected.

  Since the biological tissue filling material B is accommodated in the collection container 4, nucleated cells F effective for angiogenesis are seeded on the biological tissue filling material B together with the plasma component H. Since the plasma component H improves the engraftment property of the nucleated cell F, the growth of the nucleated cell F on the biological tissue filling material B is promoted. Further, by setting the concentration of calcium ions G added to the drain bag 5 so that the calcium ions G remain in the liquids F and H that have flowed into the collection container 4, the calcium ions G are converted into mononuclear cells. Can be changed into fibroblast-like cells.

  In addition, according to the manufacturing method of the biological tissue filling body which concerns on this embodiment, although it decided to backwash the cell capture filter 2 with the liquid containing the plasma component H, it replaces with this and is obtained in the drain bag 5. The liquid containing a large amount of plasma component H may be centrifuged to obtain platelet-rich plasma (PRP) or poor platelet plasma (PPP), which may be added to the cells F.

  In the present embodiment, the cells F such as mononuclear cells effective for angiogenesis are captured by the cell capture filter 2 and then separated by backwashing and seeded on the biological tissue filling material B. The use of the mononuclear cell F is used not only for bone and cartilage treatment but also for cancer treatment using dendritic cells.

  Mononuclear cells obtained from peripheral blood can be differentiated into dendritic cells when cultured in a medium supplemented with GM-CSF and IL-4, which are cytokines that enhance phagocytic leukocyte function. When the protein extracted from the cancer cell is phagocytosed, the incorporated cancer antigen protein is fragmented in the cell to produce an abnormal peptide, which is presented as an antigen on the surface of the dendritic cell.

By returning the dendritic cells to the patient, a specific CTL (cytotoxic T lymphocyte) for cancer cells is induced in the body, and a therapeutic effect is obtained.
By applying this, the collection container 4 is not filled with the biological tissue filling material B but a medium supplemented with GM-CSF and IL-4 is placed, and the nucleated cells F captured by the cell capture filter 2 are allowed to flow. By culturing this, dendritic cells can be obtained aseptically and returned to the patient for cancer treatment.

It is a whole lineblock diagram showing the manufacturing device of the living body tissue filling object concerning one embodiment of the present invention. It is a figure explaining the effect | action of the manufacturing apparatus of FIG. 1, and is a figure which shows the step which makes a cell capture filter capture | acquire a nucleated cell. It is a figure explaining the effect | action of the manufacturing apparatus of FIG. 1, and is a figure explaining the step which seed | inoculates the nucleated cell capture | acquired by the cell capture filter on the biological tissue filling material in a collection container. It is a figure explaining the effect | action of the manufacturing apparatus of FIG. 1, and is a figure explaining the step which supplies a calcium ion and cytokine to the biological tissue supplementary material in a collection container. It is a flowchart which shows the manufacturing method of the biological tissue complement body which concerns on the 2nd Embodiment of this invention. In the manufacturing method of FIG. 5, it is a figure which shows the step which makes a cell capture filter capture | acquire a nucleated cell. In the manufacturing method of FIG. 5, it is a figure which shows the step which adds a calcium ion to the bodily fluid which contains many red blood cells accommodated in the drain bag. It is a figure which shows the step which pressurizes the drain bag of FIG. 7 and makes body fluid flow backward to a cell capture filter. It is a figure which shows the step which supplies the cell and body fluid which flowed back the cell capture filter of FIG. 8 to a biological tissue filling material.

Explanation of symbols

A Peripheral blood B Biological tissue filling material C Collection liquid 1,20 Production device 2 Cell capture filter 3 Blood container 4 Collection container 5 Drain bag 6 Syringe (collection liquid supply means)
7 First switching valve 8 Second switching valve

Claims (7)

A cell capture filter that captures nucleated cells that are effective in angiogenesis without capturing red blood cells and platelets;
A cell source container containing a cell source and a collection container containing a biological tissue filling material connected to the inlet side of the cell capture filter via a first switching valve;
A biological tissue provided on the outlet side of the cell trapping filter via a second switching valve and having a drain bag for storing red blood cells and platelets that have passed through the cell trapping filter and a recovery fluid supply means for supplying a recovery fluid Complement body manufacturing equipment.   The apparatus for manufacturing a biological tissue filling body according to claim 1, wherein the recovery container is decompressed.   The apparatus for producing a body tissue complement according to claim 1, wherein the recovery container is provided with injection ports for calcium ions and cytokines.   The apparatus for producing a body tissue complement according to claim 3, wherein the cytokine is b-FGF.   The apparatus for producing a biological tissue complement according to any one of claims 1 to 4, wherein the cell source is peripheral blood, umbilical cord blood, bone marrow fluid, adipose tissue, or placental tissue. Passing the cell source through a cell capture filter that captures nucleated cells that are effective in angiogenesis without capturing red blood cells and platelets;
Supplying calcium ions to red blood cells that have passed through the cell capture filter;
Allowing the liquid obtained by supplying calcium ions to pass through the cell capture filter from the opposite direction;
Recovering the liquid that has passed through the cell permeation filter in the reverse direction;
And a step of supplying the recovered liquid to the biological tissue filling material.   The method for producing a biological tissue complement according to claim 6, wherein the cell source is any one of peripheral blood, umbilical cord blood, bone marrow fluid, adipose tissue, and placental tissue.

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