WO2013085020A1 - Procédé de culture cellulaire et appareil de culture - Google Patents

Procédé de culture cellulaire et appareil de culture Download PDF

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Publication number
WO2013085020A1
WO2013085020A1 PCT/JP2012/081750 JP2012081750W WO2013085020A1 WO 2013085020 A1 WO2013085020 A1 WO 2013085020A1 JP 2012081750 W JP2012081750 W JP 2012081750W WO 2013085020 A1 WO2013085020 A1 WO 2013085020A1
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Prior art keywords
container
cells
culture
buffy coat
hemagglutinating agent
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PCT/JP2012/081750
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English (en)
Japanese (ja)
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黒岩　保幸
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Kuroiwa Yasuyuki
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Publication of WO2013085020A1 publication Critical patent/WO2013085020A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • a method for obtaining a buffy coat from blood or bone marrow fluid is widely known (Japanese Patent Laid-Open No. 2002-171965).
  • a method of separating nucleated cells by centrifuging the buffy coat is common.
  • a method for separating and culturing a buffy coat in a closed system is not performed.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 3 is a diagram showing an example of an apparatus for culturing in a closed system of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pressure difference 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.
  • 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.
  • 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.
  • a pump 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.
  • the first liquid feeding means a pressure difference, a pump, gravity, or the like can be used.
  • a pressure difference it is necessary to reduce the internal pressure of the second container.
  • the first container, the second container, and the first communication means used in this apparatus need to be sterilized.
  • 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.
  • blood is used as the body fluid, and gravity is used as the liquid feeding means.
  • 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.
  • 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. .
  • 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.
  • the second liquid feeding means a pressure difference, a pump, gravity, or the like can be used.
  • a pressure difference it is necessary to reduce the internal pressure of the third container.
  • the second container, the third container, and the second communication means used in this apparatus need to be sterilized.
  • 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.
  • lymphocytes can be directly activated at the timing of culturing as well as culturing.
  • Figure 5 shows a specific example of this device.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ mL 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.
  • the sorted lymphocyte layer was transferred to a new 15 ⁇ mL centrifuge, and physiological saline was added to make 14 ⁇ mL, 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)).
  • Artemis-1 Nippon Techno Service
  • SKY-2 Stecell Science
  • 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).
  • dextran T500 and HES40 more than 9 ⁇ 10 8 nucleated cells were obtained.
  • 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.
  • 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.
  • 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 ⁇ mL of peripheral blood collected with a heparin blood collection tube (Nipro) was added to 2 ⁇ mL 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.
  • Example 2 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.
  • 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 ⁇ mL was inserted from the top, and a 5 ⁇ mL buffy coat layer was fed into an activation bag containing 50 ⁇ mL of serum-free culture solution by a peristaltic pump.
  • a tube sealer AC-155 manufactured by Terumo Corporation
  • 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.
  • a sterile connector Teumo, TSCD-202
  • 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
  • the total cell number was 6.56 ⁇ 10 9 cells.
  • 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 ⁇ g 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'objectif de la présente invention est de développer un procédé/système de culture qui permet la séparation et la culture d'une cellule dans un système complètement clos. Les présents inventeurs ont réalisés l'objectif mentionné ci-dessus en fournissant un procédé de culture, un système de culture et un appareil de culture, dans chacun desquels un liquide organique contenant une cellule sanguine est mélangé avec un agent d'agrégation d'érythrocytes dans un système clos d'une manière aseptique, puis en laissant reposer le mélange ainsi obtenu pour précipiter ainsi un érythrocyte, puis par l'injection d'une couche leucoplaquettaire contenant des lymphocytes dans un appareil de culture clos d'une manière fermée, mettant ainsi en culture la cellule sanguine.
PCT/JP2012/081750 2011-12-09 2012-12-07 Procédé de culture cellulaire et appareil de culture WO2013085020A1 (fr)

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JP2011270699A JP5596007B2 (ja) 2011-12-09 2011-12-09 細胞培養方法および培養装置
JP2011-270699 2011-12-09

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020003504A1 (fr) * 2018-06-29 2020-01-02 株式会社サンプラテック Système de culture par perfusion
CN113073077A (zh) * 2021-04-07 2021-07-06 德泉生物医学技术(深圳)有限公司 一种使用封闭式系统培养临床级脐带血间充质干细胞的方法
WO2022076660A1 (fr) * 2020-10-09 2022-04-14 Vbc Holdings Llc Procédé en système fermé et kit d'ensembles jetables pour isoler des cellules stromales mésenchymateuses à partir d'un lipoaspirat

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JP2009284860A (ja) * 2008-05-30 2009-12-10 Asahi Kasei Kuraray Medical Co Ltd 単核細胞と血小板の濃縮方法

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Publication number Priority date Publication date Assignee Title
JP2002171966A (ja) * 2000-12-04 2002-06-18 Human Tekku:Kk 臍帯血由来活性化リンパ球及び該リンパ球を主成分とする製剤ならびに該製剤の製造方法、該製剤調整用キット。
JP2005058103A (ja) * 2003-08-13 2005-03-10 Lymphotec:Kk 閉鎖系細胞培養用容器及び該容器を用いた細胞の増殖培養方法、ならびに該培養方法を用いた免疫治療剤、細胞増殖培養用キット
JP2009284860A (ja) * 2008-05-30 2009-12-10 Asahi Kasei Kuraray Medical Co Ltd 単核細胞と血小板の濃縮方法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020003504A1 (fr) * 2018-06-29 2020-01-02 株式会社サンプラテック Système de culture par perfusion
JPWO2020003504A1 (ja) * 2018-06-29 2021-07-08 株式会社サンプラテック 灌流培養システム
JP7162226B2 (ja) 2018-06-29 2022-10-28 株式会社サンプラテック 灌流培養システム
WO2022076660A1 (fr) * 2020-10-09 2022-04-14 Vbc Holdings Llc Procédé en système fermé et kit d'ensembles jetables pour isoler des cellules stromales mésenchymateuses à partir d'un lipoaspirat
CN113073077A (zh) * 2021-04-07 2021-07-06 德泉生物医学技术(深圳)有限公司 一种使用封闭式系统培养临床级脐带血间充质干细胞的方法

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