JP2008220319A - Vessel for centrifugal separation, and supporting instrument for the vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vessel for centrifugal separation for efficiently conducting operations including centrifuging a cell-containing solution, counting sedimented cells in a closed system, removing a surplus liquid and ejecting sedimented cells. <P>SOLUTION: The vessel for centrifugal separation, which is to be used in concentrating cells, is provided, essentially comprising volumetric parts (A) and (B) and having the following characteristics: (a) There is the volumetric part (B) under the volumetric part (A), wherein the volumetric part (A) is larger in volume than the volumetric part (B). (b) The volumetric parts (A) and (B) are easily separable from each other. (c) The volumetric part (B) is equipped with a scale for counting cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a centrifuge container for cell concentration suitable for cell medicine.

  In recent years, cell therapy using cells prepared in vitro for treatment, repair, and formation of living tissue has attracted attention.

  For example, Nagatani et al. Have reported that when mesenchymal stem cells collected from bone marrow are cultured and amplified and injected into mice with heart failure, the cardiac function is improved (Non-patent Document 1).

  Moreover, Yoshimura et al. Reported that it is very useful for surgical tissue formation when a fresh stem cell fraction contained in human adipose tissue is collected and reinjected together with adipose tissue (Non-patent Document 2).

  In general, when preparing cells for transplantation purposes, the cells are concentrated using a centrifuge installed in a place with special hygiene management called a Cell Processing Center (CPC), and a blood cell counter is used. Cell suspension solution is prepared based on the data and transported from the CPC to the operating room. However, such a process immediately before the implementation of cell therapy requires a lot of labor and time and is very complicated.

To date, a large volume part with an internal volume of 5 to 100 ml and a small volume part with an internal volume of 0.5 to 5 ml can be separated by making the boundary thin, or connected by screws, or flanges There is known a centrifuge container in which a large-capacity part and a small-capacity part are formed so as to be separable by being connected to each other by a locking tool (Patent Document 1). This centrifuge container does not require the transfer of the treatment liquid from the centrifuge container to the microtube as in the prior art, and has the advantage that the labor of the transfer is reduced and cell loss and damage are eliminated. However, in cell medicine, a process in which cells must be prepared in a non-open system so that bacteria and the like are not mixed, specifically, measurement of the number of cells, operation of removing only the supernatant so as not to destroy the precipitated cells, The above-mentioned CPC is necessary for the operation of refilling cells into a syringe or the like for administration to a patient, and it has not been possible with such a centrifuge container alone.
Nagatani et al., Circulation, 23; 112 (8); 1128-35 (2005) Yoshimura et al., Oral abstracts, IFATS, V-3 (2006) Japanese Patent Laid-Open No.8-108096

  An object of the present invention is to determine the amount of cells contained only by centrifuging a solution containing cells in a non-open system, and further, by separating or dividing the small container in which the cells have settled, For centrifugation suitable for cell medicine, which can easily remove unnecessary supernatant without using a station, and can easily prepare a cell suspension from the settled cells (refill into a syringe). To provide a container.

  The inventors have intensively studied to solve this problem.

  As a result, at least a) there is a capacitive part (B) below the capacitive part (A), and these capacities are (A)> (B), b) the capacitive parts (A) and (B) are easily separated, Alternatively, c) When a centrifuge container that satisfies the condition that the volume part (B) has a scale for measuring the amount of cells is used, the amount of cells can be easily determined by reading the amount of precipitated cells. In addition, by separating or dividing the volume part (B) containing the precipitated cells, unnecessary supernatant can be removed, and cell medicine can be performed from the volume part (B) containing the precipitated cells. The surprising discovery that the cell suspension used in the above can be easily prepared has led to the completion of the present invention.

Therefore, the present invention provides the following.
[1]
A centrifuge container used for concentrating cells having at least capacity parts (A) and (B) and having the following characteristics.
a) There is a capacitor part (B) at the bottom of the capacitor part (A), and their capacity is (A)> (B)
b) Capacitance part (A) and (B) can be easily separated or divided
c) The volume part (B) has a scale for measuring the amount of cells [2]
Furthermore, the container for centrifugation according to [1] having the following characteristics
d) Capacitors (A) and (B) have ports that can be connected to the outside [3]
The means for separating or dividing the capacity parts (A) and (B) is selected from a screw type, a cutting type, a folding type, a cock type, and a valve type. 4]
The container for centrifugation according to [1] to [3], wherein the capacity parts (A) and (B) have a shape selected from a bag, a tube, a bottle, and a tube.
[5]
The centrifuge container [6] according to [1] to [4], wherein the scale for measuring the amount of cells can measure 1 x 10 ⁵ cells or more, or 0.001 cm ³ or more.
[1] to [5] support for installing the centrifuge container in the centrifuge

  When the centrifuge container of the present invention is used, when the solution containing cells is centrifuged, the amount of cells can be easily obtained by simply reading the scale under a non-open system without using a blood cell counter or the like. Furthermore, by separating or dividing a small container in which cells have settled, the supernatant can be removed without using a suction device, and cells are prepared from the separated container using a syringe or the like while maintaining a non-open system. can do. It is very convenient because a cell suspension suitable for cell therapy can be efficiently prepared.

  Hereinafter, the present invention will be described in detail.

  The cell as used in the present invention is not particularly limited as long as it is a cell in biology.

  For example, stem cells, leukocytes, bone marrow derived cells, bone marrow derived mesenchymal stem cells, adipose derived cells, adipose derived mesenchymal stem cells, fibroblasts, epithelial cells, endothelial cells, various cultured cells, erythrocytes, platelets, bacteria, fungi Yeast, plant cells, etc., or a mixture thereof.

  Concentration as used in the present invention means that a solution containing cells is centrifuged by a centrifuge or the like, and the cells are accumulated in the lower part of the centrifuge container, so that the concentration of the cells is increased compared with that before the operation. It means to increase.

  There is no particular limitation on the centrifugal separator for concentrating and the centrifugal conditions thereof, and any rotational speed, time, temperature, and rotor size suitable for the sedimentation of each cell may be used. For example, when the cultured bone marrow-derived mesenchymal stem cells are concentrated, the solution is centrifuged in a centrifuge at 1200 rpm at room temperature for 5 minutes.

  The capacity section referred to in the present invention is a volume constituting the centrifuge container and its container, and is a part of the centrifuge container necessary for holding the solution without leaking. The material of the container here is not particularly limited, and examples thereof include commonly used plastic, glass, and metal.

  The term “consisting of the capacity parts (A) and (B)” as used in the present invention refers to an embodiment in which at least the capacity parts (A) and (B) are connected to form an integral centrifuge container.

  In the present invention, easy separation or separation is not particularly limited as long as the connected capacitor parts (A) and (B) can be physically easily separated or divided, for example, the capacitor part Connect (A) and (B) with screws and separate them by removing the screws, and design the joint of the capacitor (A) to be a female screw and the joint of (B) to a male screw (Or vice versa), the connection and separation can be made, the capacities (A) and (B) can be made of a soft material, and the joint can be cut with scissors etc. during separation, the capacities Folding part is made at the joint of (A) and (B), and when separating, a load is applied to the folding part as a fulcrum, and the capacitive parts (A) and (B) are folded, and the capacitive parts (A) and (B ) Is fixed with a seal, and when separating, the seal is removed and separated, and a cock or valve is provided between the capacity parts (A) and (B), and the parts are separated by them. Ku, the capacity portion (A) and between (B) pressed by a finger, or a manner such that to divide the contents easily. An example of a preferred mode is that the centrifugation operation is performed in a centrifuge container consisting of a transparent soft plastic bag, and the supernatant is in the volume part (A) and the cells are sedimented in the volume part (B). It may be closed by pinching between A) and (B) with a finger or an instrument, and cutting between (A) and (B) with scissors so that the sedimented cell mass does not collapse. Alternatively, the container may be a centrifuge container made of a transparent hard tube, and the contents may be divided by providing a cock, a valve, or the like between the capacity parts (A) and (B).

The volume (mL or cm ³ ) of the volume part (A) is not particularly limited, but is preferably a volume exceeding 10 ml from the viewpoint of the amount of treatment necessary for cell medicine. The volume of the volume part (B) is not particularly limited as long as it is smaller than the volume part (A), but is preferably 10 μl to 10 ml, more preferably 100 μl to 5 ml.

The amount of cells referred to in the present invention means the number of cells or the volume thereof. The scale for measuring the amount of cells means a scale marked on the container (B) in order to measure the number of cells or the volume thereof. For example, when measuring the amount of bone marrow mononuclear cells or adipose tissue-derived adherent cells, place multiple scales on the container (B) every 1 x 10 ⁷ cells or every 0.05 cm ³ and visually check them. It is a mode of measuring. In general cell populations such as leukocytes, erythrocytes, platelets, cultured cells, and adherent cells, the number and volume of cells are approximately proportional to each other, so that they can be converted.

The range of the scale for measuring the amount of cells is preferably 1 × 10 ⁵ cells or more, or 0.001 cm ³ or more. Measurement of a cell amount smaller than these is not preferable because it is difficult to measure visually. The upper limit may be arbitrary as required and there is no particular limitation.

  In order to obtain the amount of cells from the scale, it is necessary to determine the relationship between the amount of cells that settles and the scale of the container (B) in advance. For example, a certain amount of cells may be precipitated in advance under appropriate centrifugation conditions (constant rotation speed, time, temperature, rotor size), and the relationship between the amount and the position of the scale may be obtained. Thereafter, the amount of cells is determined based on the relationship.

  The mouth that can be connected to the outside in the present invention means a mouth or a lid necessary for connecting the inside and the outside of the capacity parts (A) and (B) and transferring the solution and cells to the inside and outside. For example, it may be a screw type, a connector type, a luer lock type, a rubber insertion port for a syringe needle, a cock, or the like. Preferably, a connector type, a luer lock type, a rubber insertion port for a syringe needle, a cock or the like that can connect the inside and the outside of the capacity part while maintaining a non-open system. To illustrate the usage of the mouth of the volume part (B), when transferring the cells settled in the volume part (B) to a separately prepared syringe, the needle is inserted from the syringe needle insertion port provided in the volume part (B). The cells inserted and settled in the volume part (B) are loosened with a syringe and sucked.

  As used in the present invention, a support for installing in a centrifuge means an inner cup, a rotor, an adjustment tool, an adapter, a stage, a binder, etc. necessary for installing and holding a cell separation container in a cell separation apparatus Point to. For example, in order to maintain the shape of the capacity part (B) during the centrifugal separation operation, the capacity part (B) can be inserted into the recess of the support designed to be concave.

  Although the specific example of the centrifuge container of this invention is illustrated in FIGS. 1-3 and the support tool of the centrifuge container is illustrated in FIG. 4, this invention is not limited to these.

  FIG. 1 is an example of a centrifuge container (folding type) that takes the form of a centrifuge tube. Put the cell-containing solution in the volume parts (A) and (B) and cover them. After centrifugation, the amount of cells settled in the volume part (B) is measured on a scale. Then, the flow of the liquid is blocked by strongly pressing the lower part of the folding part, and the capacity parts (A) and (B) are separated by applying a load with the folding part as a fulcrum. Next, the surplus liquid in the separated volume part (B) is removed with a syringe, and then the necessary cell suspension is prepared by loosening the precipitated cells.

  FIG. 2 is an example of a centrifuge container (cock type) that takes the form of a centrifuge tube. Put the cell-containing solution in the volume parts (A) and (B) and cover them. After centrifugation, the amount of cells settled in the volume part (B) is measured on a scale. And by closing the cock between the volume parts (A) and (B), the flow of the inner solution is divided, and the syringe needle is inserted from the syringe needle insertion port of the volume part (B), and the necessary cell suspension is removed. Prepare.

  FIG. 3 is an example of a centrifuge container that takes the form of a bag. Put the cell-containing solution in the volume parts (A) and (B) and cover them. After centrifugation, the amount of cells settled in the volume part (B) is measured on a scale. Then, the liquid flow is blocked by strongly pressing the scissors cutting line slightly, and the capacities (A) and (B) are separated by inserting scissors in the scissors cutting line. And in the state which maintained the non-opening type | system | group of the capacity | capacitance part (B), by inserting a syringe needle from a syringe connection port and raising and lowering the pusher of a syringe, the settled cell is loosened and they are extracted to a syringe.

  FIG. 4 is an example of a support used for installing a centrifuge container in the form of a bag in a centrifuge. The capacity part (B) is brought into the recess of the support tool to protect the deformation of the capacity part (B) from the centrifugation operation.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1]
<Preparation of bone marrow-derived mesenchymal stem cells>
Human bone marrow (Fresh whole bone marrow) was purchased from Veritas and cultured (37 ° C., 5% CO ₂ ) in a serum-containing medium (DMEM + 10% FBS).

  Medium exchange was performed every 2 to 3 days, and mesenchymal stem cells that reached subconfluence were subcultured by trypsin treatment.

  The mesenchymal stem cells thus obtained were collected by trypsin treatment and used as a test solution.

<Bag-type centrifuge container>
A centrifuge container of the present invention was prepared, and a test solution containing the above mesenchymal stem cells was introduced.

  It was centrifuged with a centrifuge (1500 rpm, 5 minutes), and the cells were sedimented in the volume part (B).

The amount of precipitated cells was found to be 3 × 10 ⁷ cells by reading the scale marked on the volume part (B).

  Next, the supernatant was removed from the liquid inlet of the capacity part (A) while sealing the top of the precipitated cells with a finger (FIG. 5).

  And the capacity | capacitance part (B) was cut off with scissors (FIG. 6), and the sedimented cell was sucked up with the syringe.

  The total time taken from centrifugation to filling the syringe with the cultured mesenchymal stem cells derived from human bone marrow was 7 minutes.

  FIG. 7 shows the relationship between the volume and number of human bone marrow-derived cultured mesenchymal stem cells that have been spun down under the above conditions.

[Example 2]
<Preparation of adipose tissue-derived cells>
A rabbit weighing 3.5 kg (Japanese white breed) was euthanized by an overdose of Ketalar / Celactal, and the dorsal subcutaneous fat was collected.

  The white fat was finely chopped with surgical scissors and shaken at 37 ° C. with 100 mL of 0.075 w / v% collagenase / phosphate buffer (pH = 7.4) (one hour).

  By this operation, the subcutaneous fat was decomposed into a viscous liquid having fluidity.

  The enzyme-treated solution was allowed to stand to separate the solution into two layers, and a lower aqueous solution layer (enzyme-treated solution) was collected.

  This aqueous solution layer was passed through a cell separation column described in JP-A-2006-121260, and contained adipose-derived stem cells were separated and recovered.

  The cell recovery solution of nucleated cells obtained in this way (most of red blood cells and platelets were removed) was used as a test solution.

<Bag-type centrifuge container>
A container for centrifugation according to the present invention was prepared, and the cell recovery solution for the fat-derived nucleated cells was introduced therein. It was centrifuged with a centrifuge (1500 rpm, 5 minutes), and the cells were sedimented in the volume part (B).

The amount of precipitated cells was found to be 1 × 10 ⁷ by reading the scale marked on the volume part (B).

  Next, the supernatant was removed from the liquid inlet of the volume part (A) while the top of the precipitated cells was sealed with a finger.

  Then, the volume part (B) was cut with scissors, and the precipitated cells were sucked up with a syringe.

  The total time required for filling the syringe with nucleated cells collected from the adipose tissue was 7 minutes.

It is a figure which shows an example of the centrifuge tube type container for centrifugation (folding type). It is a figure which shows an example of the centrifuge tube type centrifuge container (cock type). It is a figure which shows an example of the container for bag type centrifugation. It is a figure which shows an example of the supporter of the container for centrifugation. It is a figure which shows the bag-type centrifuge container made as a prototype. It is a figure which shows the small container (capacity part (B)) isolate | separated from the container for centrifugation. The relationship between the volume and the number of human bone marrow-derived cultured mesenchymal stem cells that have been centrifuged is shown.

Explanation of symbols

2 Opening and closing cock 3 Scissor cutting line 4 Supporter 5 Supernatant liquid in the volume part (A) 6 Sedimented cells 11, 21, 31, 51 Lids 12, 22, 32, 42, 52 Volume part (A)
13, 23, 33, 43, 53, 63 Capacity (B)
14, 24, 34, 54 Cell measurement scale 15, 45 Folding part 16, 26, 36 Syringe needle insertion slot

Claims (6)

A centrifuge container used for concentrating cells having at least capacity parts (A) and (B) and having the following characteristics.
a) There is a capacity part (B) at the bottom of the capacity part (A), and the capacity is (A)> (B),
b) Capacitance part (A) and (B) can be easily separated or divided
c) The volume part (B) has a scale for measuring the amount of cells. Furthermore, the container for centrifugation of Claim 1 which has the following characteristics.
d) Capacitors (A) and (B) have ports that can be connected to the outside The centrifuge container according to claim 1 or 2, wherein the means for separating or dividing the capacity parts (A) and (B) is selected from a screw type, a cutting type, a folding type, a cock type, and a valve type. The container for centrifugation according to any one of claims 1 to 3, wherein the capacity parts (A) and (B) have a shape selected from a bag, a tube, a bottle, and a tube. The centrifuge container according to any one of claims 1 to 4, wherein the scale for measuring the amount of cells can measure 1 x 10 ⁵ cells or more, or 0.001 cm ³ or more. A support for installing the centrifuge container according to claim 1 in a centrifuge.

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