JP2009136168A - Cell separation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell separation device whereby a required amount of normal cells can be transplanted immediately, after concentrating. <P>SOLUTION: This cell separation device 1 comprises a decomposition section 2 for decomposing a living body tissue for producing a cell suspension having isolated cells; a cell concentration section 3 for centrifuging the produced cell suspension to concentrate the cells; and a cell transfer path 6 for transferring the cell suspension from the decomposition section 2 to the cell concentration section, wherein a sample port 17 from which a portion of the cell suspension under transfer can be removed is disposed at the middle position of the cell transfer path 6. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell separation device.

  Conventionally, for example, a technique is known in which a collected cell suspension is decomposed with a digestive enzyme, and the obtained cell suspension is concentrated by a centrifuge (see, for example, Patent Document 1). It is necessary to examine the state of the cells, such as whether they contain a predetermined number of cells or whether they are infected with bacteria or the like, before transplantation.

International Publication No. 2005/01248 Pamphlet

However, it is desirable that the concentrated cells are transplanted immediately after the concentration, and the requirement cannot be satisfied if examined after the concentration.
This invention is made | formed in view of the situation mentioned above, Comprising: It aims at providing the cell separation apparatus which makes it possible to transplant the required quantity of healthy cells immediately after concentrating.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a decomposition processing unit that generates a cell suspension in which a biological tissue is decomposed to isolate cells, a cell concentration unit that concentrates cells by centrifuging the generated cell suspension, A cell transport path for transporting the cell suspension from the decomposition processing section to the cell concentration section, and a sample port capable of taking out a part of the transported cell suspension at a midpoint of the cell transport path A cell separation device is provided.

  According to the present invention, the biological tissue introduced into the decomposition processing unit is decomposed in the decomposition processing unit, thereby generating a cell suspension in which cells are isolated. The cells are transported to the cell concentration section through the transport path, and concentrated by being centrifuged in the cell concentration section, so that transplantable cells are separated. In this case, by removing a part of the cell suspension from the sample port provided in the middle of the cell conveyance path, it is possible to collect and inspect the cells immediately before being conveyed to the cell concentration unit It becomes. That is, since the centrifugation process in the cell concentration unit requires a predetermined time, the state of the cells contained in the collected cell suspension is examined during the centrifugation process, and when the concentration is completed, transplantation is performed. It has been determined whether or not it has a possible soundness and cell number, and transplantation can be performed quickly after concentration. In addition, as a result of the examination, when the soundness of the cells is not good or the number of cells is considered insufficient, the transplanting operation can be stopped.

In the above invention, the cell transport path is provided with a pump that feeds the cell suspension and a valve that opens and closes the cell transport path, and the sample port is located between the valve and the cell concentrator. And it is good also as arrange | positioning between the said separation process part and the said pump.
In this way, by opening the valve and driving the pump in the forward direction, the cell suspension generated in the separation processing unit is conveyed to the cell concentration unit via the cell conveyance path, and concentrated in the cell concentration unit. can do. In addition, by closing the valve in the middle of transportation and driving the pump in reverse, the pressure of the cell suspension placed in the cell transportation path between the valve and the pump can be increased and easily guided to the sample port. .

Moreover, in the said invention, the syringe for taking out a cell suspension from this sample port is good also as attaching to the said sample port so that attachment or detachment is possible.
By doing in this way, the cell suspension led to the sample port can be sucked out by a syringe and transported to an external cell inspection device.

Moreover, in the said invention, the cell test | inspection part which test | inspects the state of the taken-out cell suspension is good also as connecting to the said sample port.
By doing in this way, a part of cell suspension taken out before concentration by a cell concentration part can be led to the cell inspection part connected to the sample port, and the state can be inspected.

Moreover, in the said invention, it is good also as providing the display part which displays the continuation or cancellation of a process according to the test result by the said cell test | inspection part.
By doing in this way, when the cell state is normal as a result of the inspection by the cell inspection unit, the continuation of the process is displayed on the display unit, and the processing process is continued after confirmation by the operator. On the other hand, as a result of the examination, if there is any abnormality in the state of the cell, for example, if the number of cells is small, or if some bacteria are found, the continuation of the process is displayed on the display unit, The operator can stop the subsequent processing steps, omit the waste of concentration of cells having an abnormality, and can start the next concentration operation at an early stage.

  According to the present invention, there is an effect that a necessary amount of healthy cells can be transplanted immediately after concentration.

A cell separation device 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the cell separation device 1 according to this embodiment includes a decomposition processing unit 2 that contains and stirs a biological tissue, for example, adipose tissue, together with digestive enzymes, and a decomposition process performed by the decomposition processing unit 2. As a result, a cell concentration unit 3 that concentrates a cell suspension obtained by isolating adipose-derived cells from adipose tissue, and a cleaning solution that stores a cleaning solution for cleaning the adipose-derived cells concentrated in the cell concentration unit 3 A storage unit 4, a waste liquid recovery unit 5 that stores waste liquid discharged in the concentration process (including the washing process) in the cell concentration unit 3, and a transport path (cell transport path) 6 that connects them are provided. .

The degradation processing unit 2 includes an introduction unit 7 for introducing adipose tissue collected from a human into the degradation processing unit 2 and a filter that allows fat-derived cells isolated from the adipose tissue to pass therethrough and prohibits passage of the adipose tissue. 8 is provided.
In the example shown in the figure, the cell concentrating unit 3 includes two centrifuge containers 10 and a centrifuge 11 that rotates the centrifuge container 10 with its bottom surface radially outward. Display).

Reference numeral 12 denotes a bearing, reference numeral 13 denotes a seal member, and reference numeral 21 denotes a supernatant suction part for sucking the supernatant. The centrifuge container 10 is integrally provided with a syringe 10a for collecting the finally concentrated fat-derived cells.
Each of the cleaning liquid storage unit 4 and the waste liquid recovery unit 5 includes containers 4a and 5a for storing the cleaning liquid or the waste liquid.

The cell conveyance path 6 includes a plurality of tubes 14 connecting the decomposition processing unit 2, the cell concentration unit 3, the washing liquid storage unit 4 and the waste liquid collection unit 5, and a pump that pushes the tubes 14 from the outside to feed the liquid inside. 15, a joint 16 that branches the tube 14, a plurality of valves V _{1 to} V ₁₂ that open and close the tube 14 at each position, and a part of the cell suspension that can be taken out in the middle of the tube 14. And a sample port 17.

Sample port 17 is provided so as to branch a portion of the tube 14 between the valve V ₁ and the pump 15, are provided detachably syringe (not shown). Thereby, after introducing a part of the cell suspension heading from the decomposition processing unit 2 to the cell concentration unit 3 into the sample port 17 and then sucking it with the syringe attached to the sample port 17, the syringe is removed and the inspection device is removed. It can be transported.

The operation of the cell separation device 1 according to this embodiment configured as described above will be described below.
In order to decompose a living tissue, for example, adipose tissue and collect cell-derived cells using the cell separation device 1 according to the present embodiment, the adipose tissue and digestive enzyme solution are introduced into the decomposition processing unit 2 via the introduction unit 7. And agitation while warming, the digestion enzyme decomposes the adipose tissue to obtain a cell suspension in which adipose-derived cells are isolated from the adipose tissue.

In a state where a predetermined amount of the cell suspension is obtained, as shown by the black circles in FIG. 2, the valves V ₂ , V ₃ , V _{5 to} V ₇ , V _{10 to} V ₁₂ of the cell transport unit 6 are used. Is closed, the valves V ₁ , V ₄ , V ₈ , V ₉ are opened, and the pump 15 is rotated forward to suck the cell suspension in the decomposition treatment unit 2 by the pump 15, Supply.

Here, before all of the cell suspension is sent to the cell concentration section 3, as shown in FIG. 3, by reversing the pump 15 by closing the valve V _1, the cell suspension in the tube 14 The liquid can be reversed and sent to the sample port 17. The cell suspension in the sample port 17 can be collected in the syringe by attaching a syringe to the sample port 17 and sucking it. The cell suspension collected in the syringe is sent to an inspection device (not shown), and, for example, ATP inspection and LDH inspection are performed.

  In ATP test, ATP measurement reagent (Kikkoman) is added to the obtained cell suspension to measure the amount of ATP. Since the amount of ATP is proportional to the number of cells, the number of cells in the cell suspension is measured. can do. Moreover, since the amount of ATP increases extremely when microorganisms and fungi mix, it becomes possible to evaluate contamination of bacteria.

  In addition, the LDH test measures the amount of LDH by adding an LDH measurement reagent (manufactured by Wako Pure Chemical Industries) to the obtained cell suspension, and the LDH amount increases according to stress such as cell damage. Can measure the stress state of cells. LDH is known to show 100 IU / L / 37 ° C. in normal cell suspensions. In addition, the number of cells (viability determination) can be measured using a cytotoxicity measuring reagent based on LDH activity (for example, LDH-Cytotoxic Test manufactured by Wako Pure Chemical Industries, Ltd.).

After this, once again, as shown in FIG. 2, it is rotated forward pump 15 by opening the valve V _1, and sends the cell suspension in an amount sufficient cell concentration section 3. In the cell concentration unit 3, the cell suspension is accommodated in the centrifuge container 10, and the centrifuge 11 is operated to perform centrifugation, whereby fat-derived cells having a large specific gravity in the cell suspension are centrifuged. Collected at the bottom of the ten.

In this state, as shown in FIG. 4, by closing the valves V _{1 to} V ₅ and V _{8 to} V ₁₀ of the cell transport unit 6, opening the valves V ₆ and V ₇ , and reversing the pump 15, The supernatant produced in the centrifuge container 10 is sucked and sent to the container 5a of the waste liquid collection unit 5. By sufficiently sending the supernatant to the container 5a, a pellet of fat-derived cells and a slight supernatant remain in the centrifuge container 10. After this, as shown in FIG. 5, the valves V _{1 to} V ₄ , V ₆ , V ₇ , V ₁₀ are closed, the valves V ₅ , V ₈ , V ₉ , V ₁₁ , V ₁₂ are opened, and the pump 15 , The cleaning liquid stored in the cleaning liquid storage unit 4 is supplied into the centrifuge container 10, mixed with the fat-derived cells and the supernatant stored in the centrifuge container 10 and resuspended. .

  Then, the step of supplying and resuspending the washing liquid, the step of collecting fat-derived cells at the bottom of the centrifuge container 10 by the operation of the centrifuge 11, and the step of sucking and discarding the supernatant into the container 5 a of the waste liquid collection unit 5 Is repeated until the concentration of the digestive enzyme in the cell suspension is diluted so as not to damage fat-derived cells. Then, the adipose-derived cells accumulated at the bottom of the centrifuge container 10 are aspirated by the syringe 10a provided integrally with the centrifuge container 10, and the syringe 10a is separated from the centrifuge container 10 to be directly conveyed to the affected part. It becomes possible to transplant.

  In this case, according to the cell separation device 1 according to the present embodiment, the fat-derived cells that are finally collected are not inspected, but are arranged in the middle of conveyance from the decomposition processing unit 2 to the cell concentration unit 3. Since the sample collected from the sample port 17 is inspected, it is possible to inspect the state of the cells in the cell concentration unit 3 until the fat-derived cells are concentrated and collected by the syringe 10a. As a result, there is an advantage that the concentrated adipose-derived cells can be rapidly transplanted after being concentrated.

  When the cell suspension sampled from the sample port 17 is inspected, and the number of cells is small, the inspection result is displayed on a display unit (not shown), such as re-decomposing the cell in the decomposition processing unit 2. Feedback to the operator. In addition, if the cell suspension sampled from the sample port 17 is inspected and it is found that the cell is in a high stress state or infected with bacteria, the cell concentration process that is being performed is performed. Thus, it is possible to prevent unnecessary processing from being performed.

In the present embodiment, since the position of the sample port 17 is arranged between the valve V ₁ and the pump 15, but the cell suspension into the sample port 17 was able to supply the reverse rotation of the pump 15, to Instead, it may be arranged at another position.
In addition, the cell suspension supplied to the sample port 17 is taken out by a detachable syringe. Instead, an inspection device (cell inspection unit) is connected to the sample port 17 and supplied to the sample port 17. The cell suspension may be supplied as it is to the inspection apparatus to inspect the state of the cells. By doing in this way, the state of a cell can be inspected without going through an operator, and re-decomposition processing in the decomposition processing unit, suspension of concentration processing, etc. can be automatically performed according to the test result.

  In addition, the cell suspension taken out by the syringe is transported to the inspection apparatus and inspected. Instead of this, the measuring reagent of each item is stored in the syringe, or the reagent component is provided on the inner wall. It may be possible to immediately inspect the cell suspension by simply sucking the cell suspension into the syringe using a syringe coated with.

  Moreover, in this embodiment, although what provided integrally the syringe 10a for extract | collecting the concentrated fat origin cell in the centrifuge container 10 of the cell concentration part 3 was illustrated, it replaced with this and FIG. As shown in FIG. 4, a centrifuge container 10 having a cell aspiration tube 20 for aspirating concentrated fat-derived cells may be employed. The sucked adipose-derived cells may be sent to a cell collection container (not shown) by driving the pump 15.

  Moreover, although the fat tissue was illustrated as a biological tissue which isolate | separates a cell, it is not limited to this, It is good also as using when isolate | separating this biological tissue origin cell from another biological tissue.

It is a whole lineblock diagram showing the cell separation device concerning one embodiment of the present invention. In the cell separation device of FIG. 1, it is a whole block diagram which shows the flow of the cell suspension from a decomposition process part to a cell concentration part. FIG. 2 is an overall configuration diagram for explaining the supply of a cell suspension to a sample port in the cell separation device of FIG. 1. FIG. 2 is an overall configuration diagram for explaining collection of a supernatant separated in a cell concentration unit into a waste liquid container in the cell separation device of FIG. 1. FIG. 2 is an overall configuration diagram illustrating the supply of a cleaning liquid to fat-derived cells concentrated in a cell concentration unit in the cell separation device of FIG. 1. It is a figure which shows the modification of the centrifuge container in the cell separation apparatus of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell separator 2 Decomposition processing part 3 Cell concentration part 6 Conveyance path (cell conveyance path)
15 pump 17 sample port V ₁ ~V ₁₂ valve

Claims (5)

A decomposition processing unit for generating a cell suspension in which a biological tissue is decomposed to isolate cells;
A cell concentration section for concentrating cells by centrifuging the generated cell suspension;
A cell conveyance path for conveying a cell suspension from the decomposition treatment unit to the cell concentration unit,
A cell separation apparatus comprising a sample port capable of taking out a part of a cell suspension to be transported at a position midway along the cell transport path. A pump for feeding the cell suspension to the cell transport path, and a valve for opening and closing the cell transport path;
The cell separation device according to claim 1, wherein the sample port is disposed between the valve and the cell concentration unit and between the separation processing unit and the pump.   The cell separation device according to claim 1 or 2, wherein a syringe for taking out a cell suspension from the sample port is detachably attached to the sample port.   The cell separation device according to claim 1 or 2, wherein a cell inspection unit for inspecting a state of the extracted cell suspension is connected to the sample port.   The cell separation device according to claim 4, further comprising a display unit that displays whether the process is continued or stopped according to a test result by the cell test unit.

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