WO2020196347A1

WO2020196347A1 - Container for concentrating cells

Info

Publication number: WO2020196347A1
Application number: PCT/JP2020/012533
Authority: WO
Inventors: 義久鈴木; 薫尾前; 博史井川
Original assignee: 公益財団法人神戸医療産業都市推進機構; 株式会社フコク
Priority date: 2019-03-28
Filing date: 2020-03-19
Publication date: 2020-10-01
Also published as: JP2020162447A; JP7226738B2

Abstract

This container 10 for concentrating cells has a discharge part 12 at one end portion of a flexible container body 11, and accommodates a cell suspension containing cells of interest in a sealed state, centrifuges the cell suspension, and precipitates the cells of interest at the one end side to produce a cell concentration, wherein: the container body 11 has a first accommodation part 17 and a second accommodation part 21 which is provided on a one end-side bottom section 19 of the first accommodation part 17 to communicate with the first accommodation part 17 and stores the cell concentration; the bottom section 19 of the first accommodation part 17 is tilted downward toward the second accommodation part 21; and the discharge part 12 is provided on a one end portion of the second accommodation part 21 and is closed such that the cell concentration can be extracted from a tip 29. This container can concentrate cells while maintaining a closed system with a simple structure and can also easily extract the cell concentration without waste.

Description

Cell concentration container

The present invention relates to a cell concentration container that contains a cell suspension and concentrates by centrifugation.

Conventionally, since it is required to contain a cell suspension and centrifuge, a hard container having easy to obtain strength has been often used, but a technique for centrifuging using a flexible container is also known. ..
For example, in Patent Document 1 below, a container body having some or all flexibility is provided with a first storage portion having a large volume and a second storage portion having a small volume for storing centrifugal precipitates. A container for centrifugation has been proposed. In this centrifuge container, a port portion is provided above the first accommodating portion, and after centrifugation, the container wall at the boundary between the first accommodating portion and the second accommodating portion is brought into close contact with the container wall from the outside. Then, the port portion and the second accommodating portion were put into a non-communication state, and the cells in the second accommodating portion were separated and washed. In such a container, when the target cells after separation contained in the second container are collected from the second container, if the container is deformed or the like, it takes time and effort.

In Patent Document 2 below, unnecessary cells were separated from a cell-containing solution containing nucleated cells and unnecessary cells, and the nucleated cells were collected or concentrated in a collection bag. In this document, mononucleosis was recovered by centrifuging with high centrifugal force, separating by filtration with a filter, transferring to a conical tube or the like, and further centrifuging. Therefore, it took time and effort to transfer the container.

In Patent Document 3 below, in a blood bag to be centrifuged in order to improve the separation accuracy of blood plasma components, leukocytes, red blood cells, etc., each component is separated by connecting a tube to the upper end, the lower end, and the middle abdomen of the container. And it was drained from each tube. Therefore, in the case of a small amount of components, waste is likely to occur because they remain in the container or in the route until collection.

Further, in Patent Document 4 below, it is composed of a capacity part A and a capacity part B, a capacity part B is provided below the capacity part A, the capacity is A> B, and the capacity part A and the capacity part B are easily separated or divided. Possible centrifuge containers have been proposed. However, since the cells in the volume B separated from the bag are taken out from the centrifuge container by a syringe or the like, it takes time and effort to collect the separated cells.

Japanese Unexamined Patent Publication No. 2011-125813 International Publication WO2005 / 035737 Japanese Unexamined Patent Publication No. 08-82621 Japanese Unexamined Patent Publication No. 2008-220319

As described above, in any of the above techniques, in the conventional system for collecting or concentrating the cells that settle by centrifugation using a flexible container, when the container is transferred or the like, the cells are transferred. It took time and effort, and wasted.

Therefore, an object of the present invention is to provide a cell concentration container capable of concentrating cells while maintaining a closed system with a simple structure and easily extracting the cell concentrate without waste.

In order to achieve the above object, the cell concentration container of the present invention has a discharge portion at one end of a flexible container body, and a cell suspension containing the target cells is contained in a sealed state and centrifuged. This is a cell concentration container in which the target cells settle on one end side to generate a cell concentrate, and the main body of the container is located on the first storage portion and the bottom of the first storage portion on the one end side. It has a second accommodating portion that is provided in communication with the first accommodating portion and stores the cell concentrate, and the bottom of the first accommodating portion is formed in a downward gradient toward the second accommodating portion and discharged. The portion is provided at one end of the second accommodating portion and is characterized in that the cell concentrate can be withdrawn from the tip and closed so as to be able to be drawn out.
The present invention can be applied to a cell concentration container in which the target cell is a mononucleosis and the mononucleosis is concentrated from a cell suspension containing the mononucleosis.

In the cell concentration container of the present invention, the second container may be formed of a flexible tube. In this case, it is preferable that the flexible tube can be closed by being pressurized from the outside on the side of the second accommodating portion of the discharging portion.
The cell concentration container of the present invention may include a hard member having a flow path at least at the tip of the discharge portion, and the flow path may be blocked by an elastic member capable of penetrating the extraction tool. In this case, it is preferable that the elastic member is provided with a slit.
It is preferable that the discharge portion is configured so that the insertion position of the extraction tool can be regulated so that the tip opening of the extraction tool penetrating the elastic member is arranged at a position adjacent to the elastic member in the container body.

According to the cell concentration container of the present invention, since the container body has flexibility, the volume can be increased or decreased in a sealed state, and each operation for cell concentration can be performed while maintaining a closed system with a simple structure. ..
The container body is provided with a second accommodating portion communicating with the bottom portion on one end side of the first accommodating portion, and the bottom portion on one end side of the first accommodating portion is formed with a downward gradient toward the second accommodating portion. 2 A discharge portion is provided at one end of the accommodating portion so that the cell concentrate can be extracted from the tip and closed. Therefore, by centrifuging, the target cells settle to one end and collect, so that the cell concentrate is generated at a position where it can be easily extracted from the tip of the discharge portion in the second accommodating portion. Therefore, this cell concentrate can be directly withdrawn without waste. Moreover, since the cell concentrate is extracted from the second accommodating portion, that is, the tip of the accumulation site of the cell concentrate, it is easy to suppress the deformation and perform the extraction operation even in the flexible and easily deformable container body.
Therefore, according to the present invention, it is possible to provide a cell concentration container capable of performing cell concentration while maintaining a closed system with a simple structure and easily and reliably withdrawing the cell concentrate without waste. ..

In the cell concentration container of the present invention, if the target cell is a mononucleosis and the mononucleosis is concentrated from the cell suspension containing the mononucleosis, the cell concentrate can be extracted without waste and used effectively. be able to.
In the cell concentration container of the present invention, if the second storage portion is formed of a flexible tube, various operability is good. In particular, if the flexible tube can be closed by applying pressure from the outside on the second accommodating portion side of the discharge portion, the second accommodating portion can be easily opened and closed from the outside to drain the cell concentrate, and the operation can be performed. Good sex.

In the cell concentration container of the present invention, if the discharge portion is provided with a hard member having a flow path at the tip and the flow path is blocked by an elastic member capable of penetrating the extraction tool, the extraction tool can be penetrated. The cell concentrate can be easily extracted, and the hard member can be supported and operated when the extraction tool is penetrated, so that the operability is good. Further, since the elastic member is mounted on the inner diameter of the hard member, it becomes easy to control the fitting allowance, that is, the sealing force of the elastic member with respect to the inner diameter of the hard member, and reliable sealing can be performed. If the elastic member is provided with a slit, the elastic member can be penetrated even if the extraction tool is thick, and the cell concentrate can be extracted from a larger diameter channel and damage the cells when the extraction is performed. Is prevented. Therefore, the cell concentrate can be easily withdrawn while obtaining a reliable sealing force.
Further, if the discharge portion is configured so that the insertion position of the extraction tool can be regulated so that the tip opening of the extraction tool penetrating the elastic member is arranged at a position adjacent to the elastic member, the cell concentrate is extracted by the extraction tool. When withdrawing, the cell concentrate does not easily remain inside and can be withdrawn without waste.

It is a front view of the container for cell concentration which concerns on embodiment of this invention. It is a figure explaining the structure of the discharge part of the cell concentration container which concerns on embodiment of this invention. It is sectional drawing of the adapter used when the container for cell concentration which concerns on embodiment of this invention is attached to a centrifuge. It is a bottom view of the adapter shown in FIG. 3A.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the centrifuge container of the present embodiment, the cell suspension containing the target cells is contained in a sealed state and centrifuged, so that the target cells settle to one end side and are concentrated to generate a cell concentrate. It is a container.

The cell suspension of the present embodiment is a suspension collected from a living body and containing target cells, and various kinds of solutions such as centrifugation and filtration of a solution collected from a living body or a solution collected from a living body are used. A liquid consisting of a part of the components obtained by separation by the method or a diluted liquid thereof. In the present embodiment, it is a liquid in which red blood cells are separated from cerebrospinal fluid, peripheral blood, etc. and contains monocytes as target cells.

As shown in FIG. 1, the cell concentration container 10 of the present embodiment has a flexible container body 11, a cell concentrate discharge portion 12 provided at one end of the container body 11, and the other end. It includes a connected connecting tube 13 and a suspension portion 15 provided at the other end.

The container body 11 is provided at the first accommodating portion 17 and the bottom portion 19 on one end side of the first accommodating portion 17 in communication with the first accommodating portion 17, and a cell concentrate is generated and accumulated during centrifugation. It has two accommodating portions 21 and. In the present embodiment, the first accommodating portion 17 and the second accommodating portion 21 are joined via the joining portion 23.

The first accommodating portion 17 is formed in a bag shape by facing the flexible films and sealing them around each other. On one end side of the first accommodating portion 17, a reducing portion 25 whose width is reduced along the axis L of the first accommodating portion 17 is provided. A second accommodating portion 21 is joined to the end of the reduced accommodating portion 25 via a joint portion 23, and the bottom portion 19 of the first accommodating portion 17 is formed with a downward slope toward the second accommodating portion 21 side. ..

On the other end side of the first accommodating portion 17, a plurality of devices that can be connected to other instruments for supplying a cell suspension into the first accommodating portion 17 and discharging a separation solution, a washing solution, or the like. The connecting tube 13 is connected, and a suspension portion 15 for stably suspending the cell concentration container 10 from another member is provided and sealed.

The second accommodating portion 21 is formed of a flexible tube, is joined to the first accommodating portion 17 via the joint portion 23, and extends from the first accommodating portion 17 to one end side along the axis L.
The joint portion 23 is made of a molded body, and the outer peripheral surface is accommodated inside the reduced portion 25 in the first accommodating portion 17 and airtightly joined, and the second accommodating portion 21 is outside the tubular portion 27 protruding in the axial L direction. It is fitted and airtightly joined. Inside the joint portion 23, a through hole forming a part of the second accommodating portion 21 is provided in the axial L direction, and the second accommodating portion 21 communicates with the first accommodating portion 17. That is, it can be regarded as the second accommodating portion 21 including the internal volumes of the joint portion 23 and the tubular portion 27.

The discharge portion 12 is provided at one end of the second accommodating portion 21, and is configured so that one end of the second accommodating portion 21 is airtightly closed and the cell concentrate can be extracted from the tip 29. As shown in FIG. 2, the discharge portion 12 has a flexible tube-shaped second accommodating portion 21 that is externally fitted and airtightly joined, and a flow path that communicates with the inside of the second accommodating portion 21 inside. A hard member 33 made of a molded body having 31, an elastic member 35 that airtightly closes the flow path 31, and a cap 37 that can be closed by surrounding the tips of the elastic member 35 and the hard member 33 from the outside and applying pressure. A clip 39 such as a clamp that presses and seals the second accommodating portion 21 from the outside at a position close to the hard member 33. The hard member 33 is formed to be at least harder than the first accommodating portion 17 and the second accommodating portion 21.

In the present embodiment, the elastic member 35 is formed so that various extraction tools 41 such as the needle tube and the tip of the syringe can penetrate. The elastic member 35 is provided with a slit 43 in the L direction of the axis, so that a thicker extraction tool 41 can easily penetrate the elastic member 35. The elastic member 35 is configured to be sealed by being pressure-compressed from the outer peripheral side via the hard member 33 by tightening the cap 37 while being arranged in the flow path 31 of the hard member 33. There is.

In order to maintain the closed state at the time of centrifugation, the discharge portion 12 of the present embodiment is closed by pressing the lower end of the second accommodating portion 21 from the outside by the clip 39, and is capped at the end of the second accommodating portion 21. By tightening 37, pressure is applied from the outside to seal the flow path 31.

When the extraction tool 41 is passed through the discharge unit 12, the ejection tool 41 comes into contact with the hard member 33, the cap 37, or the like, and the insertion position into the second accommodating portion 21 is restricted. The tip opening 41a of the extraction tool 41 is located adjacent to the elastic member 35 inside the second accommodating portion 21, that is, in the present embodiment, the second accommodating portion 21 of the elastic member 35 constituting the lower inner wall of the second accommodating portion 21. It can be placed near the exposed surface to the inside.

In the cell concentration container 10 of the present embodiment, the first accommodating portion 17, the joining portion 23, the second accommodating portion 21, and the hard member 33 are each accommodated as the same resin or a resin that can be heat-welded to each other. It is composed of a resin that is safer for cell suspensions and cell concentrates, and each joint is heat-welded.

More specifically, in the present embodiment, the first accommodating portion 17 is formed from an ethylene-vinyl acetate copolymer resin film in a bag shape, and the joint portion 23 is formed from an ethylene-vinyl acetate copolymer resin molded body or the like. The second accommodating portion 21 is mainly formed of an ethylene-vinyl acetate copolymer resin tube or the like in a substantially tubular shape, and the hard member 33 is composed of an ethylene-vinyl acetate copolymer resin molded body or the like. In addition to the above, polyethylene resin, polypropylene resin, ethylene-propylene copolymer resin, polybutadiene resin, styrene-butadiene copolymer resin and their hydrogenated resin, polyurethane resin, and a mixture of these resins can be used according to the application. However, it is applied in consideration of adhesion and heat-weldability. Considering airtightness, maintenance of aseptic condition, generation of volatile gas, etc., bonding by heat welding is preferable, and it is preferable to form the same material having good heat welding property.

In order to concentrate the target cells in the cell suspension using the cell concentration container 10 to obtain a cell concentrate, the cell concentration container 10 is sterilized in advance with the connecting tube 13 and the discharge portion 12 closed. Then, the cell concentrate and the components and liquids to be appropriately mixed are housed and sealed from the connecting tube 13 using a drawing tool 41 such as a syringe.

The cell concentration container 10 is attached to the centrifuge using, for example, an adapter 45 as shown in FIG. As shown in the cross-sectional view of FIG. 3A, the adapter 45 stably arranges the cell concentration container 10 on the jig 46 provided at the accommodation position of the centrifuge, and corresponds to the inner surface of the jig 46. It has an outer peripheral surface 47, and has an inner peripheral surface 48a that supports a side peripheral portion of the first accommodating portion 17 and an inclined surface 48b that supports a reduced portion 25 on one end side of the first accommodating portion 17 inside. An internal arrangement portion 49 is provided on one end side of the inclined surface 48b so that the second accommodating portion 21 and the discharge portion 12 can be stably arranged so as to penetrate in the axial direction. As shown in the plan view of FIG. 3B, the internal arrangement portion 49 is wound, for example, so that the small diameter portion 49a in which the tubular portion 27 of the first accommodating portion 19 is arranged and the second accommodating portion 21 are not blocked. It is provided with a large diameter portion 49b that is bent and accommodated.

Centrifugation is performed with the cell concentration container 10 attached to the centrifuge using this adapter 45. In the present embodiment, it is possible to apply a high centrifugal force of 650 G or more within the range of the centrifugal force that can be used by the target cells after concentration.
As a result, the target cells settle and are sequentially accumulated on the discharge portion 12 side of the second accommodating portion 21, and a cell concentrate is generated. When the amount of the cell concentrate produced is larger than the volume of the second accommodating portion 21, the cell concentrate may be accommodated in the reduced portion 25 or the like of the first accommodating portion 17. When partitioning the first accommodating portion 17 and the second accommodating portion 21 with a clamp 51 or the like described later, a second accommodating portion 21 having a size that accommodates the entire amount of the generated cell concentrate is prepared and the second accommodating portion 21 is provided. It is desirable to accommodate in 21.

After the centrifugation for a predetermined time is completed, care is taken not to disperse the precipitated cells, and the cell concentration container 10 is hung using the suspension portion 15, and the first accommodation is performed as shown by a virtual line in FIG. By sandwiching a position crossed by the clamp 51 from the outside of the portion 17 or the second accommodating portion 21, the inside of the first accommodating portion 17 and the inside of the second accommodating portion 21 are liquid-tightly partitioned. The supernatant liquid contained in the other end of the first accommodating portion 17 is extracted from the connecting tube 13 from the clamp 51 and discharged. At this time, if a scale for measuring the position of the liquid level when suspended by the suspension portion 15 is provided in advance on the side surface of the container main body 11, the amount of liquid discharged by partitioning with the clamp 51 based on this scale. It is possible to adjust. Then, if necessary, physiological saline may be added from another connecting tube 13, suspended and centrifuged in the same manner, partitioned by a clamp 51, and the supernatant liquid may be withdrawn to wash the cells.

After the final centrifugation, the clamp 51 is removed and the concentrated cells are loosened from the outside by tapping or the like and suspended. In this state, the extraction tool 41 such as a syringe is inserted from the tip of the discharge portion 12 to penetrate the elastic member 35, and the tip opening 41a of the extraction tool 41 is positioned adjacent to the elastic member 35 inside the second accommodating portion 21. A cell concentrate can be obtained by arranging and draining the liquid in the second containing portion 21.

According to the cell concentration container 10 of the present embodiment, since the container body 11 is flexible, the volume can be increased or decreased in a sealed state, and each operation for cell concentration is performed while maintaining a closed system with a simple structure. be able to.

In the cell concentration container 10 of the present embodiment, the container body 11 is provided with the second storage portion 21 communicating with the bottom 19 on the one end side of the first storage portion 17, and is provided on the one end side of the first storage portion 17. The bottom portion 19 is provided on the side of the second accommodating portion 21 with a downward slope, and a discharge portion 12 is provided at one end of the second accommodating portion 21 so that the cell concentrate can be extracted from the tip 29 and closed.

By centrifuging, the target cells settle on one end side of the first accommodating portion 17 and gather, so that the cell concentrate is generated at a position in the second accommodating portion 21 that can be easily removed from the tip 29 of the discharge portion 12. Therefore, this cell concentrate can be easily and without waste in a short distance. Since the cell concentrate is directly extracted from the tip 29, even the flexible and easily deformable container body 11 can be extracted without being affected by this deformation. According to the cell concentration container 10 of the present embodiment, cell concentration can be performed while maintaining a closed system with a simple structure, and the cell concentrate can be easily and reliably withdrawn without waste.

In the present embodiment, since a small amount of cell concentrate is produced from a large amount of cell suspension in the cell concentration container 10, various deformations are likely to occur when the flexible container body 11 is large and handled. The second accommodating portion 21 is formed small. By extracting the cell concentrate from the tip 29 of the discharge portion 12 provided at one end of the second accommodating portion 21 with the extraction tool 41, the cell concentrate is extracted regardless of the first accommodating portion 17, which is easily deformed greatly. It can be manipulated and the cell concentrate can be easily and without waste. Here, the target cell is a mononucleosis, and the cell concentrate in which the mononucleosis is concentrated can be extracted without waste and used effectively.

According to the cell concentration container 10 of the present embodiment, if the second accommodating portion 21 is formed of a flexible tube and the discharge portion 12 pressurizes the second accommodating portion 21 from the outside so as to be occluded. The second container 21 can be easily opened and closed from the outside to drain the cell concentrate, and the operability is good.

According to the cell concentration container 10 of the present embodiment, the discharge portion 12 includes a hard member 33 having a flow path 31 at the tip 29, and the flow path 31 is blocked by an elastic member 35 through which the extraction tool 41 can penetrate. .. Therefore, the cell concentrate can be easily extracted by passing the extraction tool 41 through the elastic member 35, and at that time, the hard member 33 can be supported and operated, so that the operability can be improved.
Since the elastic member 35 is mounted on the flow path 31 formed of the inner diameter of the hard member 33, it is easy and reliable to control the fitting allowance, that is, the sealing force of the elastic member 35 with respect to the inner diameter of the hard member 33. Can be sealed. As a result, even when a high centrifugal force is applied to the discharge unit 12, reliable sealing is possible.

In the cell concentration container 10 of the present embodiment, since the elastic member 35 is provided with the slit 43, the elastic member 35 can be penetrated even if the extraction tool 41 is thick, and the cell concentrate can be flowed in a larger diameter. It can be extracted from the road 31 and can prevent damage to cells during extraction.

According to the cell concentration container 10 of the present embodiment, the tip opening 41a of the extraction tool 41 penetrating the elastic member 35 can be arranged at a position adjacent to the elastic member 35. Therefore, when the cell concentrate is extracted by the extraction tool 41, the cell concentrate of the second accommodating portion 21 can be extracted to the bottom, and the cell concentrate is unlikely to remain inside and can be extracted without waste.

The above embodiment can be appropriately modified within the scope of the present invention.
In the above embodiment, the example of mononucleosis has been described as an example of the target cell, but the cell is not particularly limited as long as it is a cell that can be precipitated and concentrated by centrifuging the cell suspension. For example, it may be a floating cell, an adhesive cell, a cultured cell cultured by various methods, or various cell spheroids.
In the above embodiment, as the container body 11 of the cell concentration container 10, the first storage portion 17 is composed of a bag made of a flexible film, the second storage portion 21 is composed of a tube, and both are joined. However, the container may be a container in which the first accommodating portion 17 and the second accommodating portion 21 are integrally formed by a continuous flexible film. For example, a container body 11 made of a continuous film in which a reduction portion 25 is provided on one end side of the first accommodation portion 17 and a second accommodation portion 21 narrower than the first accommodation portion 17 is provided at the lower end of the reduction portion 25. It is also possible to configure. In that case, for example, a hard member 33 may be airtightly joined to one end of the second accommodating portion 21 so that the through flow path 31 communicates with the inside of the second accommodating portion 21.

In the above embodiment, the discharge portion 12 has a structure in which the flow path 31 of the hard member 33 is closed by the elastic member 35 and sealed by the cap 37, and the end portion of the second accommodating portion 21 is pressed by a clip 39 such as a clamp. Although an example in which both the closed structure and the closed structure are provided has been described, it is also possible to provide only one of the structures. The discharge unit 12 may be configured to be openable and closable. For example, it is possible to use a screw cap opening / closing mechanism, a needle plug, a connector with a plug, etc., so that the discharge unit 12 and the second accommodating unit 21 can be closed from the outside to prevent leakage from the discharge unit. It can be reliably prevented and is effective when a high centrifugal force is applied to the discharge unit 12.
In the above embodiment, the length of the second accommodating portion 21 is relatively short, but when the second accommodating portion 21 is mainly composed of a flexible tube, the second accommodating portion 21 has a longer flexibility. It is also possible to adopt a sex tube. When setting in the centrifuge, the second accommodating portion 21 made of a flexible tube is bent so as not to be blocked, and the discharging portion 12 is held above the container or at a position on the rotation center side during centrifugation, and is in that state. You may centrifuge with. In this case, since the discharge unit 12 is located above the container or in the direction of the center of rotation during centrifugation, the centrifugal force applied to the discharge unit 12 can be relaxed.

Next, examples of the present invention will be described.
Mononucleosis was concentrated and recovered from peripheral blood using the cell concentration container 10 shown in FIG. In the cell concentration container 10, the first container 17 is made of a film of an ethylene-vinyl acetate copolymer, the second container 21 is made of a polyvinyl chloride tube, and the joint 23 is made of a polyethylene molded product. The cell concentration container 10 was sterilized in advance by gamma ray irradiation.

120 mL peripheral blood was collected in a blood bag containing CPDA solution using a needle 21G Safe Touch PSV Set ™. The blood bag containing the CPDA solution contained 16.8 mL of the CPDA solution. Blood components were measured by sampling 5 mL of peripheral blood, and the white blood cell count, lymphocyte count, and monocyte count were measured. White blood cell count, the calculated total number of mononuclear cells in peripheral blood 110mL from lymphocyte count, number of nucleated cells in 6700cells / μL, is in 120mL was 804cells × ¹⁰ 6 / 120mL. The mononuclear cell count is 2770cells / μL, was 332.4cells × ¹⁰ 6 / 120mL in 120 mL.

From 136.8 mL of CPDA solution-containing peripheral blood in a blood bag containing CPDA solution, 120 mL is used for separation of the centrifuge (SEPAX2, trademark) to 110 mL of peripheral blood before separation by the centrifuge (SEPAX2, trademark). the number of cells contained, the number of nucleated cells, respectively 646.5cells × ¹⁰ 6 / 110mL, mononuclear cell counts became 267.3 cells × ¹⁰ 6 / 110mL.

Mononucleosis was separated and recovered by a centrifuge (SEPAX2, trademark). Centrifuge using 120 mL of CPDA solution-containing peripheral blood (peripheral blood volume is 110 mL), 100 mL of human lymphocyte separation medium (Ficoll-Paque PREMIUM 1.073, GE Healthcare, trademark), and 1000 mL of physiological saline. (SETAX2, trademark) was set and a mononuclear cell separation process program was performed to recover 45 mL of mononuclear cell suspension in a collection bag.

Specifically, 91 mL of a medium for separating human lymphocytes was introduced into a centrifugal chamber, and peripheral blood containing CPDA solution was introduced into the centrifugal chamber to make a total volume of 211 mL, and a predetermined centrifugal force was applied for 15 minutes to perform centrifugation. .. The buffy coat and plasma were temporarily evacuated to a evacuation bag, and the red blood cells were discarded.

After washing the centrifugal chamber with physiological saline, the buffy coat and plasma were returned from the evacuation bag to the centrifugal chamber, physiological saline was added, and the mixture was centrifuged, and the supernatant was discarded. The operation of washing the centrifuge chamber and centrifuging with saline was repeated twice. Physiological saline was added to the product to make 45 mL and placed in a collection bag. 45 mL of the cell suspension was collected from the collection bag with a syringe, and 2 mL of the cell suspension was sampled in a sterility test container and subjected to the sterility test. As a result, no bacteria were detected.

1 mL of the cell suspension was sampled, blood components were measured using a multi-item automatic blood cell analyzer (XN-Series, manufactured by Simex, trademark), and the number of lymphocytes and monocytes were measured. The cell recovery rate was calculated using the above data, and the cell viability was measured using the trypan blue reagent. As a result, number of nucleated cells in 4208cells / μL, is in 45mL was 189.4cells × ¹⁰ 6 / 45mL. Mononuclear cell counts in 3970cells / μL, is in 45mL 178.7cells × ¹⁰ 6 / 45mL, is in 42mL was 166.7cells × ¹⁰ 6 / 42mL.

The cell recovery rate was calculated to be (178.7 cells × 10 ⁶ ) / (267.3 cells × 10 ⁶ ) × 100, which was 66.8%. The survival rate was 99.5%.
When a part of 45 mL of the cell suspension was sampled in a sterility test container and subjected to the sterility test, no bacteria were detected.

Next, cell concentration was performed using the cell concentration container 10 as shown in FIG.
83 mL of physiological saline was injected into the cell concentration container 10 containing 42 mL of the cell suspension to make a total volume of 125 mL. The cell concentration container 10 is attached to the shape-retaining adapter shown in FIG. 3, set in a tabletop cooling centrifuge 5500 (manufactured by Kubota Seisakusho Co., Ltd.), set to 20 degrees, and centrifuged at 650 G for 10 minutes. Cell concentration was performed. It was confirmed that there was no liquid leakage after cell concentration.

After concentrating the cells, be careful not to disperse the precipitated cells, suspend the cell concentration container 10 using the suspension portion 15, and use the clamp 51 from the outside of the first storage portion 17 as shown by a virtual line in FIG. By sandwiching a crossing position, the inside of the first storage portion 17 and the inside of the second storage portion 21 were liquid-tightly partitioned, and the cell concentrate was stored in the second storage portion 21. At this time, the amount of the cell concentrate contained in the second accommodating portion 21 from the clamp 51 to the discharging portion 12 was adjusted to 4 mL or less.

The supernatant on the upper part of the clamp 51 was extracted from the connecting tube 13 provided at the other end of the first accommodating portion 17. A sample for endotoxin measurement was collected from this supernatant, and the amount of endotoxin was measured by an endotoxin measurement system (Endosafe (R) -PTS (PTS100), registered trademark, manufactured by Waken Yakuhin Co., Ltd.). 05 It was less than EU / mL and satisfied the standard endotoxin value of the drug to be administered to the medullary cavity of the Japanese Pharmacy. The Sample RT CV was 0%, the Spike RT CV was 10.6%, and the Spike Recovery was 92%.

Subsequently, the clamp 51 was removed, and the concentrated cells were loosened from the outside by tapping or the like and suspended. A syringe is inserted from the tip of the discharge portion 12 to penetrate the elastic member 35, the tip opening 41a of the syringe is arranged at a position adjacent to the elastic member 35 inside the second accommodating portion 21, and the cell suspension is extracted with the syringe. It was. When the volume of the cell suspension in the syringe was measured, the collected amount was 4 mL.

The mononucleosis count of the obtained cell concentrate was measured.
A part of the cell suspension in the syringe was sampled, blood components were measured using a multi-item automatic blood cell analyzer (XN-Series, manufactured by Simex, trademark), and the number of lymphocytes and monocytes was measured. .. The cell recovery rate of mononucleosis was calculated using the above data, and the cell viability was measured using the trypan blue reagent. As a result, number of nucleated cells in 40304cells / μL, is in 4mL was 161.2cells × ¹⁰ 6 / 4mL. Mononuclear cell counts in 37851cells / μL, was 151.4cells × ¹⁰ 6 / 4mL is in 4 mL.

The cell recovery rate was calculated to be (151.4 cells × 10 ⁶ ) / (166.7 cells × 10 ⁶ ) × 100 = 90.8%. The survival rate was 99%. When a part of the cell concentrate in the syringe was sampled in a sterility test container and subjected to the sterility test, no bacteria were detected.

Table 1 summarizes the number of cells, the cell recovery rate, and the cell survival rate in each step in the above.

Table 2 summarizes the endotoxin measurement amount and the sterility test results.

As is clear from Table 1, the mononucleosis suspension was recovered from the peripheral blood by a centrifuge (SEPAX2, trademark), and the mononucleosis concentrate was recovered by the cell concentration container 10 and found to be 4 mL or less. The cell recovery rate was as high as 90.8%. After collecting peripheral blood, endotoxin measurement is used to confirm the safety of each stage of cell separation with a centrifuge (SEPAX2, trademark), cell concentration in a cell concentration container 10, and filling into a syringe, which is the final form. A sterility test was performed, but no particular problematic results were found.

10 ... Cell concentration container; 11 ... Container body; 12 ... Discharge part; 13 ... Connecting tube; 15 ... Suspension part; 17 ... First storage part; 19 ... Bottom;
21 ... 2nd accommodating part; 23 ... Joint part; 25 ... Reduced part; 27 ... Cylinder part; 29 ... Tip;
31 ... Flow path; 33 ... Hard member; 35 ... Elastic member; 37 ... Cap; 39 ... Clip;
41 ... Extractor; 41a ... Tip opening; 43 ... Slit; 45 ... Adapter; 47 ... Outer surface; 48 ... Inclined surface; 49 ... Internal arrangement;
51 ... Clamp

Claims

A cell suspension containing target cells is contained in a sealed state and centrifuged at one end of a flexible container body, so that the target cells settle on one end and the cells A cell concentration container from which a concentrate is produced.
The container body includes a first accommodating portion and a second accommodating portion provided at the bottom of the first accommodating portion on one end side in communication with the first accommodating portion to store the cell concentrate. Have and
The bottom portion of the first accommodating portion is formed in a downward slope toward the second accommodating portion side.
The cell concentration container is provided at one end of the second storage unit and is closed so that the cell concentrate can be extracted from the tip thereof.
The cell concentration container according to claim 1, wherein the target cell is a mononucleosis.
The cell concentration container according to claim 1 or 2, wherein the second storage portion is formed of a flexible tube.
The cell concentration container according to claim 3, wherein the flexible tube is pressurized from the outside and can be closed on the side of the second accommodating portion of the discharging portion.
The cell concentration container according to any one of claims 1 to 4, wherein the discharge portion includes a hard member having a flow path, and the flow path is blocked by an elastic member capable of penetrating the extraction tool.
The cell concentration container according to claim 5, wherein the elastic member is provided with a slit.
The discharge unit is configured to regulate the insertion position of the extraction tool so that the tip opening of the extraction tool penetrating the elastic member is arranged at a position adjacent to the elastic member. 6. The cell concentration container according to 6.
A cell concentration container for producing the cell concentrate by precipitating the target cells with a centrifugal force of 650 G or more.
The second accommodating portion is formed of a flexible tube.
The discharge portion includes a hard member having a flow path, the flow path is closed by an elastic member provided with a slit and capable of penetrating the extraction tool, and the flexibility is provided at a position close to the discharge portion. The cell concentration container according to claim 1 or 2, wherein the tube is pressurized from the outside and can be occluded.