WO2013085020A1

WO2013085020A1 - Cell culture method and culture apparatus

Info

Publication number: WO2013085020A1
Application number: PCT/JP2012/081750
Authority: WO
Inventors: 黒岩　保幸
Original assignee: Kuroiwa Yasuyuki
Priority date: 2011-12-09
Filing date: 2012-12-07
Publication date: 2013-06-13
Also published as: JP2013121334A; JP5596007B2

Abstract

The purpose of the present invention is to develop a culture method/system which enables the separation and culture of a cell in a completely closed system. The present inventors have achieved the above-mentioned purpose by providing a culture method, a culture system and a culture apparatus, in all of which a body fluid containing a blood cell is mixed with an erythrocyte-aggregating agent in a closed system in an aseptic manner, then allowing the resultant mixture to stand to thereby precipitate an erythrocyte, and then injecting a lymphocyte-containing buffy coat layer into a closed culture apparatus in a closed manner, thereby culturing the blood cell.

Description

Cell culture method and culture apparatus

The present invention relates to a cell culture field in which cells are isolated from a body fluid containing cells in a closed system, and the cells are closedly dispensed in a closed system incubator to perform everything from cell separation to culture in a closed system.

When blood and bone marrow fluid is collected and centrifuged with a coagulation inhibitor, red blood cells and plasma are separated, and a membrane-like layer (buffy coat) containing white blood cells is present between the red blood cell layer and the plasma layer. It is formed. When using hemocyte cells in regenerative medicine, etc., it is a very important process to collect and culture the buffy coat aseptically.

Conventionally, the obtained whole blood is subjected to specific gravity centrifugation to separate blood cells in an open system, and then cultured in an open or closed culture vessel. However, cells could not be treated under closed system conditions. This means that the process of separating and culturing blood cells is always exposed to the possibility of microbial contamination from the environment. However, when culturing cells in body fluids, especially blood, bone marrow fluid or umbilical cord blood, mononuclear cells are usually separated in an open system by Ficoll specific gravity centrifugation to remove red blood cells in the body fluid. Yes. Since this process cannot be performed in a closed system, it was difficult to completely carry out everything from cell separation to culture in a closed system.

As described above, a method for obtaining a buffy coat from blood or bone marrow fluid is widely known (Japanese Patent Laid-Open No. 2002-171965). However, a method of separating nucleated cells by centrifuging the buffy coat is common. In general, a method for separating and culturing a buffy coat in a closed system is not performed.

Also, when cells are cultured in regenerative medicine for clinical application to humans, it is necessary to confirm the safety of the cells for clinical application by sterility tests and mycoplasma tests. In general, it is necessary to conduct a sterility test for 2 weeks for a sterility test and a mycoplasma test for about 1 month for a mycoplasma. Therefore, when the cells are applied to humans immediately after culturing, the results of the sterility test or mycoplasma test must be obtained at the end of the culturing. However, in the case of performing separation / cultivation including a process in a non-closed system, it is impossible to completely rule out contamination of microorganisms from the environment, and a test on the final product is essential. After completion of the culture, a sterility test or a mycoplasma test must be performed, and as a result, the test result must be determined after administration.

In order to avoid such drawbacks, it is necessary to start a sterility test or mycoplasma test before the start of culturing. In order for such a sterility test or mycoplasma test to be performed before the start of the culture is effective, It is necessary to separate the buffy coat in a closed system, send it to the culture apparatus in a closed manner, and perform the steps from cell separation to culture in a completely closed system. However, separation and culture of cells in such a closed system have not been performed so far.

JP2002-171965

An object of the present invention is to develop a culture method and system capable of performing cell separation and culture in a completely closed system.

The inventors of the present invention settle the erythrocytes by aseptically mixing the body fluid containing the cells and the hemagglutinating agent in a closed system and allowing to stand in order to carry out the process from cell separation to culture in a completely closed system. And removing the buffy coat layer in a closed manner, including the liquid in the layer containing cells such as lymphocytes (buffy coat layer) being sent to the closed culture apparatus in a closed manner. And also providing a closed culture method for cells contained in body fluids, or carrying out these methods, including performing culture after feeding the buffy coat layer thus obtained The above problems have been solved by providing a culture system and a culture apparatus.

According to the method of the present invention, a body fluid containing cells can be processed from the collection consistently under conditions of a closed system, and as a result, the buffy coat layer is separated in the closed system, and the obtained By culturing the buffy coat, it is possible to provide a method for culturing cells contained in a body fluid in a closed system.

Thus, the ability to consistently collect and culture cells in a closed system not only provides sterile and mycoplasma-free, safer cells suitable for transplantation, but also prior to transplantation. The sterility test or mycoplasma test that must be carried out can be performed early before or simultaneously with the culture.

FIG. 1 is a diagram showing an example of an apparatus for culturing in a closed system of the present invention. In this figure, blood collected by a blood collection tube 11 is aseptically closed using a liquid feeding means to a container 14 into which a hemagglutinating agent is dispensed via a connecting tube 13 connected to an injection needle 12. Deliver liquid. When the container 14 is a vacuum blood collection tube, the liquid feeding pump may not be used. FIG. 2 is a diagram showing an example of an apparatus for culturing in a closed system of the present invention. In this figure, blood collected by the blood collection tube 21 is aseptically fed in a closed system using a liquid feeding means to a container 25 into which a hemagglutinating agent has been dispensed via a connecting tube 23. When the container 25 is a vacuum blood collection tube, the liquid feed pump need not be used. FIG. 3 is a diagram showing an example of an apparatus for culturing in a closed system of the present invention. In this figure, the collected blood in the blood transfusion bag 31 is aseptically sent in a closed system using the liquid feeding means to the container 35 into which the hemagglutinating agent has been dispensed via the connecting tube 33. When the container 35 is a vacuum blood collection tube, the liquid feed pump need not be used. FIG. 4 is a diagram showing a situation when the buffy coat is separated using the hemagglutinating agent in the closed system of the present invention. In this figure, after feeding blood into a container containing a hemagglutinating agent, mix by inverting and mixing the two liquids uniformly. After standing for 30 minutes to 1 hour, the agglomerated red blood cells settle, and the upper layer is buffed. It shows that the coat layer 42 has been formed. The buffy coat layer contains mononuclear cells, polynuclear cells and platelets suspended in plasma. FIG. 5 is a diagram showing an example of an apparatus for culturing in a closed system of the present invention. In this figure, the buffy coat layer 51 formed in FIG. 2 is aseptically sent in a closed system to the culture bag 55 in which OKT3 is solid-phased by using the liquid feeding pump 54 through the connecting tube 53. To do.

The inventors of the present invention aseptically mix body fluid containing cells and hemagglutinating agent in a closed system, and allow to settle and remove erythrocytes by allowing them to stand. A method of separating the buffy coat layer in a closed manner, including the liquid supply of the coat layer) to the closed culture apparatus in a closed manner, and the buffy thus obtained It shows that it is possible to provide a closed culture method for cells contained in a body fluid, including culturing after the coating layer is fed, and performing from completely separating cells to culturing in a completely closed system. Indicated.

In the prior art, there has been known a method of removing red blood cells by adding a hemagglutinating agent to a body fluid containing cells such as blood or bone marrow fluid, and allowing to stand (Japanese Patent Application Laid-Open No. 2002-171965). In the method described above, a method of centrifuging nucleated cells by centrifuging the buffy coat is generally used, and a method of culturing by adding the buffy coat as it is to the culture medium has not been generally performed. Further, the buffy coat was collected in a closed system, and the solution was closed and sent to a culture apparatus, and the process from cell separation to culture was not performed in a completely closed system.

In contrast, in the present invention, the buffy coat layer containing nucleated cells can be separated by simply mixing the body fluid containing the collected cells with the hemagglutinating agent and allowing to stand under closed conditions. Only the buffy coat was transferred to the culture solution while maintaining the desired conditions, and it was shown that the buffy coat cells obtained while maintaining the aseptic conditions in the closed system could be used for cultivation as they were.

In the present invention, a body fluid containing cells and a hemagglutinating agent are mixed under closed system conditions (that is, under aseptic conditions). The “body fluid containing cells” used in the present invention is selected from blood, umbilical cord blood, bone marrow fluid, ascites fluid or pleural fluid. Examples of “cells” contained in these body fluids include nucleated cells of the immune system, such as leukocytes such as neutrophils, basophils, and eosinophils, T cells, B cells, NK There are lymphocytes such as cells and monocytes.

Any hemagglutinating agent to be mixed with a body fluid containing cells as described above can precipitate red blood cells but not other nucleated cells when mixed with a body fluid containing cells. However, it is possible to separate and collect nucleated cells by simply leaving them after mixing the body fluid containing cells and the hemagglutinating agent, without performing centrifugation. It is preferable to have it. Examples of hemagglutinating agents that can be used in the present invention include dextrans, Ficolls, hydroxyethylcellulose (HES), and the like. As the hemagglutinating agent, dextrans and hydroxyethyl cellulose (HES) are more preferable.

When used in the present invention, the hemagglutinating agent is particularly efficient for nucleated cells contained in the buffy coat when the final concentration of hemagglutinating agent is 0.0005% to 10.0% when mixed with a body fluid containing cells. It can be recovered well. In the present invention, nucleated cells contained in the buffy coat can be collected more efficiently when the final concentration of the hemagglutinating agent is 0.1% to 3.75%.

The method of the present invention includes, for example, feeding blood collected by a syringe, feeding blood collected by a vacuum blood collection tube, feeding blood collected by a blood transfusion bag, and bone marrow perforation. It is possible to send bone marrow fluid collected by a syringe equipped with a needle, or to feed cord blood collected in a collected cord blood collection bag as specific embodiments. In the above, the above-mentioned body fluid is sent to the container containing the hemagglutinating agent through a sterilized tube in a closed system.

In order to send the body fluid sample to the container containing the hemagglutinating agent, a pressure difference, a pump, gravity, or the like can be used. When a pressure difference is used for liquid feeding, for example, by reducing the internal pressure of the container containing the hemagglutinating agent, the body fluid can be moved to the container containing the hemagglutinating agent via the tube. Alternatively, pressure can be applied from the outside to the container itself containing the body fluid containing cells, and the body fluid can be moved to the container containing the hemagglutinating agent via the tube. Alternatively, when a pump is used for liquid feeding, the body fluid can be moved through the tube from the container that contains the body fluid to the container containing the hemagglutinating agent using the liquid feeding pump.

The present invention can also provide an apparatus for realizing the above-described method. Specifically, a first container for containing a body fluid containing collected cells, a second container for containing a hemagglutinating agent and a culture solution, a first container for connecting the first container and the second container. It is possible to provide a device equipped with the first communication means and the first liquid feeding means for moving the blood in the first container to the second container via the communication means. This device can be used to separate a buffy coat layer from a body fluid containing cells in a closed system.

As the first liquid feeding means, a pressure difference, a pump, gravity, or the like can be used. When using a pressure difference, it is necessary to reduce the internal pressure of the second container. In addition, the first container, the second container, and the first communication means used in this apparatus need to be sterilized.

Specific examples of this device are shown in Figs. As an example, in FIG. 1, blood is used as the body fluid, and the pump 15 is used as the liquid feeding means. A connecting tube 13 (corresponding to a first communication means) including an injection needle 12 is connected to a container 11 (corresponding to a first container) containing blood, and a liquid feeding pump 15 (first liquid feeding means) The blood was mixed with the hemagglutinating agent by moving the blood in the container 11 to the container 14 (corresponding to the second container) into which the hemagglutinating agent was dispensed.

As another example, in FIG. 2, blood is used as the body fluid, and gravity is used as the liquid feeding means. Connect the connecting tube 23 (corresponding to the first communication means) to the container 21 (corresponding to the first container) containing blood, and using gravity (corresponding to the first liquid feeding means), The blood was mixed with the hemagglutinating agent by moving the blood in the container 21 to the container 25 (corresponding to the second container) into which the hemagglutinating agent was dispensed.

As yet another example, in FIG. 3, blood is used as the body fluid, and the compressive force from the outside with respect to the first container is used as the liquid feeding means. A connecting tube 33 (corresponding to the first communication means) is connected to a container 31 (corresponding to the first container, for example, a blood transfusion bag) containing blood, and a compressive force (first to the first container) The blood was mixed with the hemagglutinating agent by moving the blood in the container 31 to the container 35 (corresponding to the second container) into which the hemagglutinating agent was dispensed. .

When the body fluid containing cells and the hemagglutinating agent are mixed in the second container and allowed to stand using the apparatus of FIGS. 1 to 3, as shown in FIG. 4, an aggregate of erythrocytes (41 ), And the buffy coat (42) was separated in the upper layer.

The present invention also provides a second container that houses a buffy coat layer separated from blood in a closed system, a third container for culturing the collected buffy coat, and the second container and the third container are connected. And a second liquid feeding means for moving the buffy coat layer of the second container to the third container via the second communication means. . This device can be used to sort a buffy coat layer separated from a body fluid containing cells in a closed system.

As the second liquid feeding means, a pressure difference, a pump, gravity, or the like can be used. When using a pressure difference, it is necessary to reduce the internal pressure of the third container. Further, the second container, the third container, and the second communication means used in this apparatus need to be sterilized. And as the third container, as a closed system culture apparatus, the closed system culture flask in JP-A-2005-58103 and the culture bag in JP-A-2007-175028 can be used, and the inside thereof, A cell culture solution for culturing the moved buffy coat layer is included.

It is also possible to solidify CD3 in the third container and stimulate the buffy coat layer moved from the second container directly with CD3 in the third container. By using a container having such a structure, lymphocytes can be directly activated at the timing of culturing as well as culturing.

Figure 5 shows a specific example of this device. In FIG. 5, the buffy coat formed in the container 14, the container 25, or the container 35 of FIGS. 1 to 3 is used, and a pump 54 is used as the liquid feeding means. A connecting tube 53 (corresponding to the second connecting means) is connected to the container 53 (corresponding to the second container) containing the separated buffy coat, and the liquid feeding pump 54 (corresponding to the second liquid feeding means) Was used to move the buffy coat in the container 51 to the container 55 (corresponding to the third container) into which the cell culture solution had been dispensed.

In this way, cells such as activated lymphocytes can be obtained under closed system conditions. This cell is characterized in that it has no contamination such as bacteria or mycoplasma.

Hereinafter, in order to describe the present invention in more detail, examples of the present invention will be shown.

Example 1 Examination of Hemagglutinating Agent The purpose of this example was to investigate hemagglutinating agents that can be used in the method and apparatus of the present invention.

2 g of dextran T500 (Sigma Aldrich) and 2 g of dextran 70 (Tokyo Kasei) were dissolved in physiological saline (Otsuka Pharmaceutical) and autoclaved at 120 ° C. for 20 minutes. 6% HES40 solution (Nipro) and 6% Sarinhes solution (hydroxyethyl starch injection solution, Fresenius Kirby) were diluted 3 times with physiological saline to prepare 2% solutions each.

Equal volumes of 3% of peripheral blood collected with heparin blood collection tube (Nipro) and 2% of each prepared solution were mixed (final concentration 1%) and allowed to stand for 30 minutes. In dextran T500 and HES40, erythrocytes settled, and a buffy coat containing yellow leukocytes and lymphocytes was formed in the upper layer. 3 mL of the buffy coat layer was collected so that erythrocytes were not mixed.

Dextran 70 and sarinhes were left for a further 30 minutes for a total of 1 hour. From the dextran 70 on which the buffy coat layer was formed, 3 μmL of the buffy coat was separated so as not to contaminate red blood cells. Sarinhes was unable to separate the buffy coat because of the poor formation of the buffy coat layer.

Further, 4 μmL of physiological saline was added to 2 μmL of peripheral blood to dilute it 3 times, and then it was layered on a 15 μmL centrifuge tube into which 3 μmL of Ficoll solution (Lymphosepar I, Immunobiological Laboratories) was dispensed. The centrifuge tube containing the peripheral blood layered on Ficoll solution was centrifuged at 400 × g for 20 minutes with brake off. The lymphocyte fraction formed in the intermediate layer (boundary with Ficoll) was collected so that erythrocytes would not be mixed. The sorted lymphocyte layer was transferred to a new 15 μmL centrifuge, and physiological saline was added to make 14 μmL, followed by centrifugation at 430 × g for 10 minutes. The supernatant was discarded and suspended in 4 ml of serum-free medium Artemis-1 (Nippon Techno Service) (former product name: SKY-2 (Skycell Science)). The number of nucleated cells in the sorted buffy coat, the number of nucleated cells separated by Ficoll and suspended in Artemis-1 medium, and the number of nucleated cells in whole blood are shown in Table 1.

Serum-free medium (5 mL) was dispensed into a 25 cm ² activation flask (prepared by the method of Sekine et al.) On which OKT-3 was immobilized, and 2 mL of each buffy coat or 1 mL of whole blood was added. On the other hand, 2 mL of nucleated cells separated by Ficoll and suspended in Artemis-1 medium were dispensed into an activated flask, and 3 mL of Artemis-1 medium was added to make a total of 5 mL.

The culture was started in a carbon dioxide incubator (37 ° C., 5% CO ₂ ). On the fourth day of culture, 5 mL of Artemis-1 medium was added. On the fifth day, 15 mL of Artemis-1 medium was added, and the culture was continued. On day 7 of culture, all cells in the 25 cm ² activation flask were added to a 225 cm ² flask containing 75 mL of Artemis-1 medium, and the culture was continued. On the 11th day, 100 mL of Artemis-1 medium was added, and the culture was continued. On day 14, all cells were collected and the number of cells was counted (Table 2).

With dextran T500 and HES40, more than 9 × 10 ⁸ nucleated cells were obtained. In dextran 70, 8 × 10 ⁸ or more nucleated cells were obtained, and 7 × 10 ⁸ or more nucleated cells were obtained from cells separated by Ficoll, whereas in culture with whole blood, 5 × Only about 10 ⁸ nucleated cells were obtained, and cell proliferation ability was reduced in whole blood.

Example 2: Examination of Hemagglutinating Agent Concentration This example was carried out for the purpose of examining preferred concentrations of Destran T500 and HES40, which were satisfactory as hemagglutinating agents as a result of examination in Example 1.

6% Destran T500 and 6% HES40 prepared in the same manner as in Example 1 were diluted with physiological saline to prepare a 1% to 3% concentration solution. 2 mL of peripheral blood collected with a heparin blood collection tube (Nipro) was added to 2 mL of each concentration solution, mixed by inversion, allowed to stand for 30 minutes, and 2 mL of the buffy coat layer was collected. In the same manner as in Example 1, 2 mL of each buffy coat was added to a 25 cm ² activation flask containing 5 mL of Artemis-1 medium and cultured for 14 days. After completion of the culture, the number of nucleated cells was counted (Table 3). With dextran T500 and HES40, 7 × 10 ⁸ or more nucleated cells were recovered in the range of 1% to 3% (final concentration 0.5% to 1.5%).

Example 3: Examination of the relationship between the hemagglutinating agent concentration and the number of cells The purpose of this example was to examine the relationship between the hemagglutinating agent concentration and the number of cells used.

20% Destran T500 prepared in the same manner as in Example 1 was diluted with physiological saline to prepare a 2.5% to 20% concentration solution. Further, 2% dextran T500 was diluted with physiological saline to prepare 0.0001%, 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% concentration solutions. 2 μmL of peripheral blood collected with a heparin blood collection tube (Nipro) was added to 2 μmL of each concentration solution, mixed by inversion, and allowed to stand for 30 minutes. The 0.0001% to 0.2% concentration solution was allowed to stand for another 30 minutes for a total of 1 hour. The buffy coat layer was collected. Table 4 shows the amount of the buffy coat collected and the number of nucleated cells.

Same as in Example 1 using 0.2%, 0.3%, 5.0%, 7.5% concentration solution (initial concentration) and cells separated by Ficoll out of the separation conditions in which the number of recovered cells was 3.0 × 10 ⁶ or more. Each buffy coat 2 mL (0.2 mL condition was 1.8 mL) or cells collected with Ficoll were added to a 25 cm ² activation flask containing 5 mL of Artemis-1 medium and cultured for 14 days. After completion of the culture, the number of nucleated cells was counted (Table 5). Over 6 × 10 ⁸ nucleated cells were recovered in the range of 0.2% to 5.0% (final concentration 0.1% to 2.5%) of dextran T500.

Example 4: Culture of the collected buffy coat The purpose of this example was to examine the conditions for further culturing the buffy coat obtained by the method of the present invention.

In a clean bench, 5 mL of blood collected using a vacuum blood collection tube containing heparin (Terumo VP-H100) was added to 5 mL of 2% dextran physiological saline. Terumo's VP-P100) and the injection needle (Terumo's NN-2070C), extension tube (Terumo's SF-ET2022) and lock connector (Terumo's TS-LC11) A 1: 1 mixture of 2% dextran saline was prepared.

When left for 30 minutes, erythrocytes settled and a pale yellow layer was formed on top of the blood collection tube. A syringe needle (NN-2070C manufactured by Terumo Corporation) connected to an activated bag of 300 μmL was inserted from the top, and a 5 μmL buffy coat layer was fed into an activation bag containing 50 μmL of serum-free culture solution by a peristaltic pump. Using a tube sealer (AC-155 manufactured by Terumo Corporation), the tube connected to the activation bag is sealed and cut off.

¡Repeat this operation and send a total of 20mL buffy coat to the activated bag. After mixing the buffy coat and the culture well, start the culture in a 37 ° C carbon dioxide incubator.

4 days later, the bag containing the serum-free culture solution and the activation bag are combined with a sterile connector (Terumo, TSCD-202), and 50 mL of culture solution is added. After sealing with a tube sealer, separate the two bags. Return the activated bag to which the culture solution has been added to the 37 ° C carbon dioxide incubator and continue the culture. On the next day, connect the activation bag and the bag containing serum-free medium, and add 150 mL of serum-free medium. Return the activated bag to which the culture solution has been added to the 37 ° C carbon dioxide incubator and continue the culture.

Two days later, the culture bag containing 750 ml of serum-free culture solution and the activation bag were connected, and the entire amount of the culture solution in the activation bag was injected into the culture bag containing 750 ml. A culture bag of a total volume of 1000 mL was cultured for 4 days in a 37 ° C. carbon dioxide incubator. A 1000-mL culture bag was joined to a culture bag containing 1000 mL of fresh culture solution, and the culture solutions in the two bags were uniformly mixed. After the volume of the culture solution in each of the two bags was 1000 mL, each was sealed with a tube sealer and cut into two bags.

The two bags were further cultured for 3 days in a 37 ° C carbon dioxide incubator. After stirring the culture solution so that the cells were evenly distributed, 10 mL of cell suspension was collected from each sampling port.

The cell density of bag 1 was 3.36 × 10 ⁶ cells / mL, the cell density of bag 2 was 3.20 × 10 ⁶ cells / mL, and the total cell number was 6.56 × 10 ⁹ cells.

In addition, the activation bag for 300 mL used in this Example was attached with a 21-G injection needle from a sampling port with 10 mL of physiological saline (Otsuka Pharmaceutical) supplemented with 50 μg of anti-CD3 antibody (OKT-3). The solution was injected into a 300 mL culture bag (TAZETTA-F manufactured by Kojin Bio Inc.) using a syringe. Sterile air was injected into the bag with a syringe, and the bag was inflated and shaken at room temperature for 2 hours to immobilize OKT3 on one side of the culture bag.

The 1 L separation bag (Nipro) dispensed with 1 L of physiological saline and the OKT solid phase immobilization bag were joined with an aseptic bonding machine, and 300 mL of physiological saline was added to the OKT3 solid phase culture bag. The solution in the bag was agitated to be uniform and allowed to stand for 5 minutes. After 5 minutes, the other tube of the activation bag was joined to an empty separation bag, and the liquid in the activation bag was transferred to a separation bag for waste liquid. The middle part of the tube was pinched with forceps to stop the liquid. This operation was repeated two more times.

After sealing with a tube sealer, the three bags were separated. An activation bag for activation culture was prepared by injecting 50 mL of serum-free culture solution into the activation bag.

Example 5: Culture of collected buffy coat The purpose of this example was to examine other conditions for further culturing the buffy coat obtained by the method of the present invention.

5 mL of blood collected with a 30 mL syringe was aseptically injected into five 10 mL serum vacuum blood collection tubes (Terumo VP-P100) supplemented with 5 mL of 2% dextran saline. After inversion mixing, the mixture was allowed to stand for 30 minutes. A total of 20 mL of the buffy coat layer was injected in the same manner as in Example 4 with 50 mL of Artemis-1 medium in an OKT3 solid phase 300 mL culture bag, and the culture was continued to 2 L. The number of cells collected after 2 weeks was 7.64 × 10 ⁹ cells.

11 Container with blood 12 Injection needle 13 Connection tube 14 Container with erythrocyte agglutinating agent 15 Fluid pump 21 Blood collection tube 22 Injection needle 23 Connection tube 24 Sterilization filter 25 Container with erythrocyte agglutinating agent 31 Blood transfusion bag 32 Injection needle 33 Connection tube 34 Sterilization filter 35 Container in which hemagglutinating agent is dispensed 41 Precipitated red blood cell layer 42 Buffy coat layer 51 Buffy coat layer 52 Injection needle 53 Connection tube 54 Feed pump 55 OKT3 solid phase into which the culture solution has been dispensed Bag 56 Buffy coat delivered

Claims

Mix body fluid containing cells and hemagglutinating agent,
The erythrocytes are settled and removed by standing,
The solution of the layer containing the cells is sent to the closed culture device in a closed manner and cultured.
A closed culture method of cells contained in a body fluid.
Mix body fluid containing cells and hemagglutinating agent,
The erythrocytes are settled and removed by standing, and the fluid of the layer containing the cells is closedly sent to the closed culture device.
A method for separating the buffy coat layer in a closed manner.
The body fluid containing cells and the hemagglutinating agent are mixed by feeding the body fluid containing cells in a closed system through a tube to the container containing the hemagglutinating agent. Method.
The method according to any one of claims 1 to 3, wherein the body fluid containing cells is selected from blood, umbilical cord blood, bone marrow fluid, ascites fluid, or pleural effusion fluid.
The method according to any one of claims 1 to 4, wherein the hemagglutinating agent is selected from dextran and hydroxyethyl cellulose (HES).
The method according to any one of claims 1 to 5, wherein the final concentration of the hemagglutinating agent is 0.0005% to 10.0%.
The method according to claim 6, wherein the final concentration of the hemagglutinating agent is 0.1% to 3.75%.
The apparatus according to any one of claims 3 to 7, wherein the feeding of the body fluid to the container containing the hemagglutinating agent is performed by a pressure difference, a pump, and gravity.
9. The method according to any one of claims 3 to 8, wherein blood collected by a syringe is sent in a closed system through a sterilized tube to a container containing a hemagglutinating agent.
9. The bone marrow fluid collected by a syringe equipped with a bone marrow perforation needle is sent to a container containing a hemagglutinating agent through a sterilized tube in a closed system. the method of.
9. The umbilical cord blood collected in a umbilical cord blood collection bag is sent in a closed system through a sterilized tube to a container containing a hemagglutinating agent, according to any one of claims 3 to 8. The method described.
A first container for containing a body fluid containing the collected cells;
A second container containing a hemagglutinating agent and a culture solution;
A first communication means for connecting the first container and the second container, and a first liquid feeding means for moving the blood in the first container to the second container via the communication means,
A device for separating a buffy coat layer from a body fluid containing cells in a closed system.
The apparatus according to claim 12, wherein the first liquid feeding means includes a pressure difference, a pump, and gravity.
The apparatus according to claim 12 or 13, wherein the first container, the second container, and the first communication means are sterilized.
The device according to any one of claims 12 to 14, wherein the first container is selected from a cord blood collection bag, a vacuum blood collection tube, and a blood collection bag.
A second container containing a buffy coat layer separated from blood in a closed system,
A third container for culturing the collected buffy coat,
Second communication means for connecting the second container and the third container, and a second liquid feed for moving the buffy coat layer of the second container to the third container via the second communication means means,
A device that sorts out the buffy coat layer.
The apparatus according to claim 16, wherein the second liquid feeding means includes a pressure difference, a pump, and gravity.
The apparatus according to claim 16 or 17, wherein the second container, the third container, and the second communication means are sterilized.
The apparatus according to any one of claims 16 to 18, wherein the third container contains a cell culture medium.
Activated lymphocytes obtained by the method according to any one of claims 1 to 11.