JP2012055227A - Cell treatment device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell treatment device for acquiring a necessary amount of cells without increasing the risk of foreign objects or bacteria existing in an external air mixed therein.SOLUTION: The cell treatment apparatus 1 includes: a tissue digestion part 11 where biological tissues are digested in a digestion container, and cell suspension is produced; a centrifugal separation containers 2a, 2b for accommodating and turning the cell suspension produced in the tissue digestion part 11, and centrifugally separating the cell suspension into supernatant and cell group; and a sheet heater 22 for keeping warm the cell group in the centrifugation containers 2a, 2b.

Description

  The present invention relates to a cell processing apparatus.

  Conventionally, cell processing apparatuses that separate and concentrate adipose tissue-derived cells present in adipose tissue aspirated from a patient are known (see, for example, Patent Documents 1 and 2). Cell therapy can be carried out by transplanting the cells separated using this cell treatment device to a diseased site of a patient.

JP 2005-519883 Gazette JP-A-9-187269

  By the way, when the amount of adipose tissue sucked from the patient is small, the number of cells necessary for cell therapy may not be reached. In addition, when cells are used for immunotherapy, a large amount of cells are required and may be administered in several divided doses. However, since cell separation involves aspiration surgery, fewer patients reduce the burden on patients. This is preferable because it can be performed.

However, according to the cell processing devices disclosed in Patent Documents 1 and 2 described above, since the amount of cells as a final product depends on the amount of adipose tissue sucked from a patient, the number of suction operations increases. , There was a disadvantage of increasing the burden on the patient.
In addition, when cells are grown in a culture apparatus, the cells that are the final product must be taken out of the centrifuge container and replaced with a culture apparatus. There was a disadvantage that the risk of contamination increased.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a cell processing apparatus capable of obtaining a necessary amount of cells without increasing the risk of contamination by foreign substances and bacteria in the outside air. It is said.

In order to achieve the above object, the present invention employs the following means.
The present invention includes a digestion container that digests a living tissue to generate a cell suspension, and stores and rotates the cell suspension generated in the digestion container, and the cell suspension is divided into a supernatant and a group of cells. A cell processing apparatus including a centrifuge container for centrifugal separation and a heat retaining means for retaining a cell group in the centrifugal container is employed.

  According to the present invention, the cell suspension produced by digesting the living tissue in the digestion vessel is accommodated in the centrifuge container and rotated, so that each component of the cell suspension is changed due to the difference in specific gravity. Centrifuge. Specifically, a cell group having a large specific gravity is collected in the bottom lowermost layer of the centrifuge container, and a supernatant such as lipid or plasma is collected in the upper layer. And the cell group isolate | separated in this way can be culture | cultivated by heat-retaining in a centrifuge container with a heat-retaining means.

  By doing in this way, the cell group taken out from the living tissue can be cultured and increased, the amount of living tissue collected from the patient can be reduced, and the burden on the patient at the time of collecting the tissue can be reduced. Furthermore, since the cell group can be cultured in the centrifuge container, a series of treatments can be performed in a closed system, reducing the risk of contamination and reducing the labor of the operator.

  In the above invention, the cleaning liquid container for storing the cleaning water, the medium container for storing the culture medium, the liquid supply means for supplying the liquid into the centrifuge container, the cleaning liquid container, the medium container, and the liquid supply means. It is good also as providing the piping to connect and the liquid supply switching means which is provided in this piping and switches the liquid supply to the said centrifuge container with a wash water and a culture medium.

  By doing in this way, the liquid supply to a centrifuge container can be switched with a wash water and a culture medium by a liquid supply switching means. Thereby, a cell group can be wash | cleaned by supplying washing water in a centrifuge container. Moreover, culture of a cell group can be accelerated | stimulated by supplying a culture medium in a centrifuge container.

In the above-mentioned invention, a drainage means for draining the liquid in the centrifuge container and a drainage container for storing the liquid drained from the drainage means may be provided.
By doing in this way, the liquid in a centrifuge container can be discharged | emitted to a drainage container from a drainage means. Thereby, while washing | cleaning property of a cell group is improved by changing the washing | cleaning liquid in a centrifuge container, culture | cultivation of a cell group can be promoted by changing the culture medium in a centrifuge container.

The said invention WHEREIN: It is good also as providing the scaffold supply means which supplies a scaffold in the said centrifuge container.
By supplying the scaffold into the centrifuge container, cells can be cultured on the surface of the scaffold instead of the inner wall of the centrifuge container. Thereby, it is possible to eliminate the necessity of peeling the cell group from the inner wall of the centrifuge container, and it is possible to easily collect the cell group. The scaffold may be mixed into the medium and supplied to the centrifuge container, or may be supplied to the centrifuge container by a system different from the medium.

  According to the present invention, there is an effect that a necessary amount of cells can be obtained without increasing the risk of contamination by foreign substances or bacteria in the outside air.

It is a whole lineblock diagram showing the cell treatment device concerning one embodiment of the present invention. It is a flowchart which shows the process performed by the cell processing apparatus of FIG. It is detail drawing of the centrifuge processing container of FIG.

Hereinafter, a cell treatment apparatus according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the cell processing apparatus 1 according to this embodiment includes a tissue digestion unit (digestion container) 11 that digests a living tissue to generate a cell suspension, and centrifuges the cell suspension. A centrifuge 5, a cleaning solution bag (cleaning solution container) 6a, 6b for storing a cleaning solution, a draining bag (draining vessel) 25 for storing a draining solution, a medium bag (medium container) 20 for storing a medium, Tubes (piping) 7 and 15 for connecting them, tube pumps (liquid supply means and drainage means) 8 provided in the tubes 7 and 15, valves V1 to V7 provided in the tube 7, and control thereof And a control unit (liquid supply switching means) 10 for performing the above operation.

  As shown in FIG. 1, the tissue digestion unit 11 is a device for digesting biological tissue such as adipose tissue, and includes a processing container 12 that contains the adipose tissue A, and a filter disposed in the processing container 12. 13, a shaker 14 on which the processing container 12 is placed, a tube 15 connected to the processing container 12, and a valve V <b> 6 provided at an intermediate position of the tube 15.

  The processing container 12 is a cylindrical container and accommodates a mixed solution of adipose tissue A collected from a living body and a digestive enzyme solution B that digests the adipose tissue A. The processing container 12 has a bottom surface 12a inclined in one direction, and a discharge port 12b connected to the tube 15 is opened at the lowest position of the bottom surface 12a. The processing container 12 is closed at the top by a lid 12 c, and the lid 12 c is injected with an inlet 12 d for introducing a digestive enzyme into the processing container 12 and adipose tissue A is injected into the processing container 12. A fat injection port 12e is provided. In the processing container 12, with the progress of cell processing with the digestive enzyme solution B, fat-derived cells (cell group) constituting the adipose tissue A are isolated and floated, and a cell suspension C is generated.

  The filter 13 has a large number of holes having a diameter smaller than that of the fat tissue A, is disposed substantially horizontally above the bottom surface 12a, and covers the discharge port 12b. As a result, the adipose tissue A is restricted from being discharged from the discharge port 12b in a state where it is collected from the body, and is discharged through the pores of the filter 13 while the cell group is isolated by the digestive enzyme solution B. It is discharged from the outlet 12b.

  The shaker 14 rotates the processing container 12 horizontally along a predetermined circumferential orbit. Further, a sheet heater 16 is provided between the processing container 12 and the shaker 14 (the bottom surface of the processing container 12) so as to keep the inside of the processing container 12 at about 37 ° C.

  When the shaker 14 is operated, the processing container 12 placed on the shaker 14 is horizontally rotated along a predetermined circumferential orbit. Thereby, the adipose tissue A is agitated in the digestive enzyme solution B, and digestion is promoted. Then, when the shaker 14 is stopped and the processing container 12 is allowed to stand, the digestive adipose tissue A containing digested adipose tissue A containing cells separated from the adipose tissue A due to the difference in specific gravity, that is, Cell suspension C is separated into lower layers. The cell suspension C generated in this way is sent into the centrifuge containers 2a and 2b of the centrifuge 5 through the tubes 7 and 15 by operating the tube pump 8.

The centrifuge 5 includes two centrifuge containers 2a and 2b, a rotor 3 connected to the centrifuge containers 2a and 2b, and a motor 4 that rotationally drives the rotor 3.
In the centrifuge containers 2a and 2b, the cell suspension C generated by the tissue digestion unit 11 and sent via the tubes 7 and 15 by the tube pump 8 is accommodated. Further, one end of a tube 7 is inserted into each of the centrifuge containers 2a and 2b.

The rotor 3 includes a column 3a that is driven to rotate about an axis in the vertical direction by a motor 4, and two arms 3b that extend radially outward from the column 3a.
Between the both ends of the arm 3b, one bucket 3c for storing the centrifuge containers 2a, 2b is provided one by one, and is supported so as to be swingable around a horizontal axis.

  With the above configuration, when the rotor 3 is rotationally driven by the motor 4, the bucket 3c and the centrifuge containers 2a and 2b are integrally rotated around the vertical axis. Then, due to the centrifugal force, the bucket 3c and the bottoms of the centrifuge containers 2a and 2b are swung in a direction away from the support column 3a, and the bottoms are rotated outward in the radial direction. Thereby, the components of the cell suspension C in the centrifuge containers 2a and 2b are centrifuged due to the difference in specific gravity. Specifically, a cell group having a large specific gravity is collected in the bottom bottom layer of the centrifuge containers 2a and 2b, and a supernatant such as lipid and plasma is collected in the upper layer.

  The other end of the tube 7 connected to each of the centrifuge containers 2a and 2b is connected to the outside of the centrifuge 5 via a pipe line in the column 3a. For example, a rotary joint is employed for the tube 7 connecting portion of the support column 3a, and even when the support column 3a rotates, the tube 7 connected to the outside can be supplied and discharged without being twisted.

  The tube pump 8 feeds the liquid in the tube 7 by rotating the tube 7 while crushing it with a plurality of rollers (not shown). The tube pump 8 can be operated in forward and reverse directions, and can supply and drain liquid to the centrifuge containers 2a and 2b by changing the rotation direction.

  The centrifuge 5 is accommodated in a housing 21, and a seat heater (heat retaining means) 22 is provided around the housing 21 to keep the interior of the housing 21 at about 37 ° C. As a result, the cell suspension C (cell group) in the centrifuge containers 2a and 2b is incubated at a temperature of about 37 ° C.

The controller 10 controls the opening / closing operation of the valves V <b> 1 to V <b> 7 and the rotation direction of the tube pump 8.
Specifically, when sending the cell suspension C from the tissue digestion unit 11 into the centrifuge containers 2a and 2b, the control unit 10 opens the valves V1, V2, and V6 and rotates the tube pump 8 in the forward direction. Let

  When supplying the cleaning liquid from the cleaning liquid bag 6a into the centrifuge containers 2a and 2b, the control unit 10 opens the valves V1, V2, and V3 and rotates the tube pump 8 in the normal direction. When supplying the cleaning liquid from the cleaning liquid bag 6b into the centrifuge containers 2a and 2b, the control unit 10 opens the valves V1, V2, and V4 and rotates the tube pump 8 in the normal direction.

  When supplying the culture medium from the culture medium bag 20 into the centrifuge containers 2a and 2b, the control unit 10 opens the valves V1, V2, and V5 and rotates the tube pump 8 in the normal direction. When the liquid in the centrifuge containers 2a and 2b is discharged to the drainage bag 25, the control unit 10 opens the valves V1, V2, and V7 and rotates the tube pump 8 in the reverse direction.

The operation of the cell processing apparatus 1 configured as described above will be described below using the flowchart shown in FIG.
First, a cannula or the like is inserted into a patient's body, and adipose tissue A in the living body is aspirated (step S1). Hereinafter, description will be divided into a cell treatment step (step S10) in which the adipose tissue A thus sucked is digested to take out a cell group, and a culture step (step S20) in which the cell group obtained by the cell treatment step is cultured. To do.

  In the cell processing step shown in step S10, first, adipose tissue A (with a tumenic solution) is injected into the processing container 12 from the fat injection port 12e provided in the lid 12c of the processing container 12 (step S2).

Next, the adipose tissue A in the processing container 12 is washed (step S3).
Specifically, after discharging the tumescent liquid in the processing container 12, the cleaning liquid is supplied into the processing container 12, the red blood cells in the adipose tissue A are suspended in the cleaning liquid, and the cleaning liquid is discharged from the processing container 12. To do. The adipose tissue A is washed by repeating this operation several times.

Next, digestion of the adipose tissue A in the processing container 12 is performed (step S4).
Specifically, the digestive enzyme solution B is put into the processing container 12, the shaker 14 is operated, the adipose tissue A and the digestive enzyme solution B are stirred in the processing container 12, and the fat from the digestive enzyme is stirred. Promotes tissue A digestion. Then, by leaving the shaker 14 to stand, the adipose tissue A in the processing container 12 is separated into two layers (the upper layer is the mature adipose tissue A and the lower layer is the digested cell suspension C).

  Next, the cell suspension C is transferred from the tissue digestion unit 11 into the centrifuge containers 2a and 2b by opening the valves V1, V2 and V6 and rotating the tube pump 8 forward (step S5).

Next, the cell suspension C in the centrifuge containers 2a and 2b is washed (step S6).
Specifically, first, the motor 4 is operated to rotate the centrifuge containers 2a and 2b so that the bottoms are directed outward in the radial direction. Thereby, the cell suspension C in the centrifuge containers 2a and 2b is centrifuged into a cell group and a supernatant liquid by the difference in specific gravity of the components.

Next, after the motor 4 is stopped and the centrifugal rotation is stopped, the valves V1, V2, and V7 are opened, and the tube pump 8 is rotated in the reverse direction so that the supernatant liquid in the centrifugal containers 2a and 2b is drained. To 25.
Then, the valves V1, V2, V3 (V4) are opened, and the tube pump 8 is rotated forward so that the cleaning liquid is supplied from the cleaning liquid bag 6a (cleaning liquid bag 6b) into the centrifuge containers 2a, 2b. Actuate and centrifuge.
By repeating the above operation a plurality of times, the cell suspension C in the centrifuge containers 2a and 2b is washed.

Next, the cell suspension C in the centrifuge containers 2a and 2b is concentrated (step S7).
Specifically, as in the cleaning step of step S6, first, the motor 4 is operated, and the centrifuge containers 2a and 2b are rotated so that the bottoms are directed outward in the radial direction. Thereby, the cell suspension C in the centrifuge containers 2a and 2b is centrifuged into a cell group and a supernatant liquid by the difference in specific gravity of the components.

Next, after the motor 4 is stopped and the centrifugal rotation is stopped, the valves V1, V2, and V7 are opened, and the tube pump 8 is rotated in the reverse direction so that the supernatant liquid in the centrifugal containers 2a and 2b is drained. To 25.
Thereby, concentration of the cell suspension C (cell group) of the centrifuge containers 2a and 2b is performed.

Next, the cell group (final product) centrifuged in the centrifuge containers 2a and 2b is taken out (step S8).
In this case, when the amount of the cell group obtained as the final product is smaller than the required amount, the cell group is cultured in the culturing step shown in Step S20.

  In the culture process shown in step S20, first, the valves V1, V2, and V5 are opened and the tube pump 8 is rotated forward to supply the culture medium from the culture medium bag 20 to the centrifuge containers 2a and 2b. The medium is changed from the washing solution to the culture medium (step S11).

Next, the motor 4 is operated to rotate the centrifuge containers 2a and 2b, whereby the cell group is resuspended (step S12).
Next, as shown in FIG. 3, the cell culture scaffold C is injected into the centrifuge containers 2a and 2b using, for example, a syringe (scaffold supply means) 26 (step S13). The scaffold C may be any microcarrier, collagen, alginate bead or the like that generally functions as a scaffold and may be changed depending on the purpose of treatment. Specifically, for regeneration of bone, granules of tricalcium phosphate or hydroxyapatite (both manufactured by Olympus Terumo Biomaterials), for regeneration of cartilage, collagen (for example, manufactured by Collagen), alginic acid (for example, Kyosei Pharmaceutical Co., Ltd.) Made in a gel or bead form and used as scaffolding material C.

Next, by operating the motor 4 and rotating the centrifuge containers 2a and 2b, the cell group is uniformly seeded on the scaffold C (step S14).
In addition, said step S13-step S14 are not an essential process, It is good also as culturing a cell group, without performing these processes.

Next, the seat heater 22 is operated, and the cell groups in the centrifuge containers 2a and 2b are incubated at about 37 ° C. (step S15).
At this time, in order to promote the culture of the cell group, it is preferable to exchange the medium in the centrifuge containers 2a and 2b.

  When exchanging the culture medium (step S21), first, the motor 4 is operated, and the centrifuge containers 2a and 2b are rotated so that the bottoms are directed radially outward. Thereby, the cell suspension C in the centrifuge containers 2a and 2b is centrifuged into a cell group (including the scaffold D) and a supernatant (including the medium) by the difference in specific gravity of the components.

By discharging the supernatant containing this medium, the old medium is removed from the centrifuge containers 2a and 2b (step S16).
And after removing an old culture medium, a new culture medium is again supplied in centrifuge container 2a, 2b, and culture | cultivation of a cell group can be performed effectively (step S17).

  As described above, according to the cell processing apparatus 1 according to the present embodiment, the cell suspension C generated by digesting the biological tissue in the tissue digestion unit 11 is accommodated in the centrifuge containers 2a and 2b. The components of the cell suspension C are centrifuged due to the difference in specific gravity. Specifically, a cell group having a large specific gravity is collected in the bottom bottom layer of the centrifuge containers 2a and 2b, and a supernatant such as lipid and plasma is collected in the upper layer. And the cell group isolate | separated in this way can be heat-retained and culture | cultivated by the sheet | seat heater 22 in centrifuge container 2a, 2b.

  By doing in this way, the cell group taken out from the living tissue can be cultured and increased, the amount of living tissue collected from the patient can be reduced, and the burden on the patient at the time of collecting the tissue can be reduced. Furthermore, since the cell group can be cultured in the centrifuge containers 2a and 2b, a series of treatments can be performed in a closed system, reducing the risk of contamination and saving the labor of the operator. Can do.

  Further, by controlling the opening / closing operation of the valves V1 to V7 and the direction of rotation of the tube pump 8 by the control unit 10, the liquid supply to the centrifuge containers 2a and 2b can be switched between the washing water and the culture medium. Thereby, a cell group can be wash | cleaned by supplying wash water in centrifuge container 2a, 2b. Moreover, culture of a cell group can be accelerated | stimulated by supplying a culture medium in centrifuge container 2a, 2b.

  Further, the liquid in the centrifuge containers 2a and 2b can be discharged from the tube pump 8 to the drainage container. Thereby, while washing | cleaning property of a cell group is improved by replacing | exchanging the washing | cleaning liquid in centrifuge containers 2a and 2b, culture | cultivation of a cell group can be promoted by replacing | exchanging the culture medium in centrifuge containers 2a and 2b.

  Moreover, by supplying the scaffold D into the centrifuge containers 2a and 2b, cells can be cultured on the surface of the scaffold D, not on the inner walls of the centrifuge containers 2a and 2b. Thereby, it is possible to eliminate the necessity of peeling the cell group from the inner walls of the centrifuge containers 2a and 2b, and the cell group can be easily recovered. The scaffold D may be mixed into the medium and supplied into the centrifuge containers 2a and 2b, or may be supplied into the centrifuge containers 2a and 2b by a system different from the medium.

As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.
For example, as a heat retaining means for culturing the cell groups in the centrifuge containers 2a and 2b, the seat heater 22 is provided in a housing that accommodates the entire centrifuge 5, but instead of this, a centrifuge container is provided. It is good also as providing a heat retention means in each of these.

  Moreover, in this embodiment, although the structure provided with two washing | cleaning liquid bags and one culture medium bag was illustrated, this invention is not limited to the number of these bags. For example, one or three or more cleaning solution bags may be provided, or two or more medium bags may be provided. Moreover, it is good also as culture | cultivating a cell group only by heat retention as a structure which does not provide the culture medium bag 20 without supplying a culture medium.

DESCRIPTION OF SYMBOLS 1 Cell processing apparatus 2a, 2b Centrifugal container 3 Rotor 3a Post 3b Arm 3c Bucket 4 Motor 5 Centrifugal machine 6a, 6b Washing liquid bag (cleaning liquid container)
7 Tube (Piping)
8 Tube pump (liquid supply means, drainage means)
10 Control unit (Liquid supply switching means)
11 Tissue digestion unit (digestion container)
12 Processing Container 13 Filter 14 Shaker 15 Tube (Piping)
16 Seat heater 20 Medium bag (medium container)
21 Housing 22 Seat heater (heat insulation means)
25 Drainage bag (drainage container)
26 Syringe (scaffold supply means)
A Adipose tissue B Digestive enzyme solution C Cell suspension D Scaffold
V1-V7 valve

Claims (4)

A digestion vessel that digests living tissue to produce a cell suspension;
A centrifuge container that contains and rotates the cell suspension produced in the digestion vessel, and centrifuges the cell suspension into a supernatant and a group of cells;
A cell treatment apparatus comprising a heat retaining means for retaining a cell group in the centrifuge container. A cleaning liquid container for storing cleaning water;
A medium container for storing the medium;
Liquid supply means for supplying a liquid into the centrifuge container;
A pipe connecting the washing liquid container, the medium container and the liquid supply means;
The cell processing apparatus according to claim 1, further comprising a liquid supply switching unit that is provided in the pipe and switches a liquid supply to the centrifuge container between washing water and a culture medium. Drainage means for draining the liquid in the centrifuge container;
The cell treatment apparatus according to claim 2, further comprising a drainage container that stores the liquid discharged from the drainage unit. The cell processing apparatus according to claim 1, further comprising scaffold supply means for supplying a scaffold into the centrifuge container.

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