WO2013047974A1 - Appareil de culture de cellules tridimensionnelle et procédé de culture tridimensionnelle de cellules l'utilisant - Google Patents

Appareil de culture de cellules tridimensionnelle et procédé de culture tridimensionnelle de cellules l'utilisant Download PDF

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WO2013047974A1
WO2013047974A1 PCT/KR2012/004010 KR2012004010W WO2013047974A1 WO 2013047974 A1 WO2013047974 A1 WO 2013047974A1 KR 2012004010 W KR2012004010 W KR 2012004010W WO 2013047974 A1 WO2013047974 A1 WO 2013047974A1
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Prior art keywords
culture
cells
cultured
dimensional
protrusion
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PCT/KR2012/004010
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English (en)
Korean (ko)
Inventor
박태현
김정아
박정극
Original Assignee
서울대학교 산학협력단
동국대학교 산학협력단
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Priority claimed from KR1020120050142A external-priority patent/KR101412155B1/ko
Application filed by 서울대학교 산학협력단, 동국대학교 산학협력단 filed Critical 서울대학교 산학협력단
Publication of WO2013047974A1 publication Critical patent/WO2013047974A1/fr

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    • 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
    • C12M23/10—Petri dish
    • 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
    • C12M23/12—Well or multiwell plates
    • 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
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/06—Magnetic means

Definitions

  • the present invention relates to a three-dimensional cell culture apparatus and a three-dimensional culture method of cells using the same.
  • Another method of three-dimensional culture of cells is a hanging drop culture.
  • FIG. 2 is a conceptual diagram showing a general method of three-dimensional culture of cells through a hanging drop culture (hanging drop culture).
  • the number of collected cells is counted as shown in FIG. 2 (a), and then about 300 to 500 cells are collected using a pipette or the like.
  • the cover 11 of the 100 is dispensed in the form of drops and inverted to incubate.
  • the hanging cells are collected at the end of the drop by gravity to form an embryonic cell mass, which is generally referred to as embryoid body.
  • Figure 3 A, B is another form of this cultivation method developed by 3d biomatrix, using a cover plate with a predetermined size of holes, the cell culture solution is accommodated in the cover plate, the cells from the hole by gravity Represents the form of three-dimensional culture.
  • Another object of the present invention is to provide a method for three-dimensional culture of cells using the three-dimensional cell culture tool.
  • the present invention to solve the above problems
  • a culture plate unit including a well for culturing cells
  • It provides a three-dimensional cell culture tool comprising a; protruding portion extending from the surface in contact with the culture plate portion of the cover portion into the well of the culture plate portion.
  • the protrusion is characterized in that the magnetic.
  • a method of causing the protrusion to show magnetic properties is not particularly limited, and in one embodiment of the present invention, the protrusion may be made of a permanent magnet material.
  • the protrusion includes an electromagnet, the control unit for controlling the size and supply time of the current supplied to the electromagnet and a power supply for supplying power to the electromagnet for a predetermined time by the control unit It is done.
  • the length of the protrusion is characterized in that 1/20 to 9/10 of the depth of the well.
  • the culture plate portion comprises a plurality of wells for culturing the cells, wherein the protrusions are characterized in that it is formed to include one per well for culturing the cells.
  • the protrusion is characterized in that it is formed to include a plurality per well for culturing the cells.
  • the end of the protruding portion is not particularly limited in shape, but is preferably formed sharply in order to concentrate the magnetism and efficiently culture the cells in three dimensions.
  • the present invention also provides
  • It provides a three-dimensional culture method of cells using the three-dimensional cell culture tool of the present invention comprising the step of three-dimensional culture of the cells by forming the spheroid by the magnetism of the protrusion.
  • the cells which are desired to be cultured in the spheroid form are first cultured in a culture solution containing nanoparticles, and are aggregated by external magnetism to be cultured in the spheroid form.
  • Cells cultured basally in a culture medium containing nanoparticles contain the nanoparticles inside the cells, nanoparticles adhere to the cell membrane, or magnetic nanoparticles adhere to the cells by other mechanisms, thereby moving the cells by external magnetism. Will be induced.
  • the nanoparticles typically refer to very small particles having a diameter ranging from 1 nanometer to a few hundred micrometers. Dyes and pigments because of their small size; Aesthetic or functional coatings; Tools for biological research, medical imaging and treatment; Magnetic recording media; Quantum dots; And even and uniform nanoscale semiconductors.
  • Magnetic nanoparticles have been proposed to be used in various biomedical applications, which include magnetic resonance imaging, hyperthermic treatment of malignant cells, and drug delivery. Recently, the nanoparticles were grown by culturing the cells in a mixture of nanoparticles. Techniques for introducing into cells and for immobilizing cells into which magnetic particles have been introduced have been developed.
  • the magnetic nanoparticles are characterized in that one or more selected from the group consisting of magnetite, Fe 3 O 4 , ⁇ -Fe 2 O 3 , manganese ferrite, cobalt ferrite and nickel ferrite.
  • the step of receiving the cultured cells and the culture solution in the well of the culture plate portion characterized in that the cultured cells and culture medium is accommodated so as not to contact the protrusion.
  • the step of receiving the culture solution containing the cultured cells in the well of the culture plate portion is characterized in that the end of the protrusion is accommodated so as to contact the cultured cells and the culture solution.
  • the end of the protrusion is accommodated so as to contact the cultured cells and the culture solution.
  • the step of basal culture of the cells to be cultured in the spheroid form in the culture medium containing the nanoparticles are a plurality of cells to be cultured in the spheroid form
  • the plurality of types of spheroid Cells desired to be cultured in the form is mixed in a culture solution containing the nanoparticles, characterized in that the basal culture at the same time.
  • Three-dimensional culture method of the cells using the three-dimensional culture tool of the present invention further comprising the step of basal culture of each of the first cell, the second cell to be cultured in the spheroid form separately in each culture solution containing nanoparticles;
  • Three-dimensional culture method of the cells using the three-dimensional culture tool of the present invention the step of culturing a plurality of types of cells to be cultured in the spheroid form separately in each culture medium containing nanoparticles;
  • Figure 4 shows a schematic diagram of the three-dimensional cell culture tool according to the present invention.
  • the three-dimensional cell culture apparatus includes a culture plate part 10 including a plurality of wells 30 for culturing cells; A cover part 20 covering the culture plate part; And one or more protrusions 50 extending from the surface in contact with the culture plate of the cover part toward the inside of the well of the culture plate.
  • the protrusion is characterized in that the magnetic, the method for this is not limited.
  • the protrusion may be made of a permanent magnet material, or may be made of an electromagnet material.
  • FIG. 4 illustrates a case in which the entire protrusion is made of a permanent magnet material (51) and a case in which the permanent magnet is mounted at the end (52).
  • FIG. 5 shows a schematic diagram when the protrusion is made of an electromagnet material.
  • the three-dimensional cell culture tool of the present invention includes an electromagnet protrusion, a culture plate 110, a plurality of wells 130 included in the culture plate, and a cover for covering the culture plate. 120 and an electromagnet protrusion extending from the surface in contact with the culture plate of the cover portion into each well of the culture plate, and connected to each tip to adjust the magnetism of the electromagnet material.
  • the external power supply unit 160 and the control unit 170 are included. In the present invention, whether the power applied to the external power source 160 is applied by the control unit 170, the intensity of the application, the application time, etc.
  • the overall shape and the well shape of the culture plate 10 are not particularly limited. Specifically, not only round bottom flasks, but also 6-well, 96-well plates and the like are generally used in the art for culturing cells. It can select suitably according to the culture purpose. As a preferred embodiment of the present invention, a case in which the culture plate has a multiwell shape is shown in FIG. 6, and a case in which the culture plate has a circular plate shape is shown in FIG. 7.
  • the shape and number of the tip may be appropriately selected by the capacity of the well for culturing the cells, and is not particularly limited, and the culture plate 10 as shown in FIG. ) Includes a plurality of wells 30 for cell culture, the protrusions may be formed to include one per well 30 for culturing the cells.
  • spheroids of the same trait may be simultaneously formed in each portion where a protrusion is located in the well for culturing a cell, and then three-dimensional. Further growth of the cultured cells is also effective when the culture medium is replaced or separated at once.
  • the length of the protrusion is not particularly limited, and is preferably 1/20 to 9/10 of the depth of the well. If the length of the protrusion is too long compared to the depth of the well, the number of cells in contact with the protrusion increases and the efficiency of the three-dimensional culture decreases. If the length of the protrusion is too short compared to the depth of the well, the magnetic influence range is small and the three-dimensional culture. The efficiency of will be reduced.
  • the tip is capable of three-dimensional culturing of the cells even without contact with the cell culture solution in the well, and three-dimensional culturing of the cells even in the contacted state.
  • culturing the cells in contact with the cell is preferable because the magnetic acts more directly, it is preferable to perform appropriate surface treatment on the surface of the protrusions because the cells adhere to the surface of the protrusions.
  • the surface of the protrusion may be coated with a polymer material (a high water-repellent material such as PEG) that interferes with cell adhesion, or by overlaying a material that prevents direct contact with the cell.
  • Three-dimensional cell culture method using the three-dimensional cell culture tool of the present invention comprises the steps of i) basal culture of the cells to be cultured in the spheroid form in the culture medium containing nanoparticles; ii) receiving a culture solution containing the cultured cells in a well of a culture plate; iii) covering the surface of the culture plate with a cover including the protrusion so that the protrusion is located inside the well; iv) culturing the cells three-dimensionally by forming the spheroids by the magnetism of the protrusions; It is configured to include.
  • the cell is desired to be cultured in spheroid form in the culture medium containing the nanoparticles.
  • the magnetic nanoparticles may be introduced into the cells by basal culture of the cells in the culture medium containing the nanoparticles, or attached to the cell membranes of the cells by surface modification of the nanoparticles.
  • a method of introducing magnetic nanoparticles into a cell may be a method generally used by those skilled in the art.
  • the magnetic nanoparticles may be included in the cell culture medium, preferably at a concentration of 10 pg to 1000 pg / cell,
  • the magnetic nanoparticles are introduced into the cells by culturing the cells in the included medium.
  • the magnetic nanoparticles superparamagnetic or ferromagnetic iron oxide nanoparticles are used.
  • the oxides included in the nanoparticles are ferro- or ferrimagnetic compounds such as magnetite, Fe 3 O 4 , ⁇ -Fe 2 O 3 , manganese ferrite, cobalt ferrite and nickel Ferrite.
  • the iron-oxide contained in the superparamagnetic nanoparticles used in the present invention is Fe 3 O 4 or ⁇ -Fe 2 O 3 , and most preferably Fe 3 O 4 .
  • the size of the well for culturing the cells of the three-dimensional cell culture tool of the present invention and the desired spheroid size for adjusting the volume and cell concentration of the culture medium containing the basal culture cells for culturing the cells It is accommodated in a well and covered with the cover portion so that the protrusion formed on the surface of the cover portion is located inside the well of the culture plate.
  • the cells containing the magnetic nanoparticles are gathered close to the tip by the magnetism of the protrusion of the cover part, and as a result, three-dimensional cell culture in which the cells aggregate with each other is possible.
  • the cells once aggregated can continue to be cultured while increasing the number of cells in the three-dimensional cell cultured spheroid, thereby further growing the size of the spheroid.
  • the present invention it is possible to control the cell type in the basal culture, or to culture the cells in various forms in the spheroid formation step after basal culture.
  • FIG. 1 a possible spheroid production method is schematically shown in FIG. 1
  • A) a method of randomly mixed spheroid form, ie, using a plurality of cell types in basal culture, forming spheroids by random mixing while agglomerating the plurality of types of cells
  • Core-shell spheroids i.e., after forming a spheroid with a first cell, a second cell forms a shell part using the first cell spheroid as a core part
  • C) fusion between spheroids fusion that is, it is possible to form various types of spheroids such as forming spheroids based on the first cell spheroid and the second cell spheroid.
  • 20 schematically shows the cases of A), B) and C), respectively.
  • the magnetic nanoparticles are introduced into the cells, and the cells are inoculated with the cells by culturing the magnetic force using the protrusions representing the magnetic, thereby inoculating the cells on the cover part. Even without a separate treatment such as the like, the cells have the effect of forming a spheroid in a suspended state in the basal culture.
  • Figure 1 shows the shape of the plate bottom used for three-dimensional culture of conventional cells.
  • 2 and 3 show the process and apparatus of the conventional culture method used for three-dimensional culture of conventional cells.
  • Figure 4 shows a cross-sectional view of a three-dimensional cell culture apparatus according to an embodiment of the present invention.
  • FIG. 5 and 6 show a three-dimensional cell culture apparatus using an electromagnet according to another embodiment of the present invention.
  • Figure 7 shows a cross-sectional view of a three-dimensional cell culture apparatus according to another embodiment of the present invention.
  • Figure 8 shows a method of three-dimensional culture of cells by the three-dimensional cell culture method of the present invention.
  • Figure 9 shows a cover picture of the three-dimensional cell culture apparatus prepared according to an embodiment of the present invention.
  • 10 to 12 show the results of culturing the cells using the three-dimensional cell culture tool of the present invention, introducing the fluorescent material into the cells and observed with a fluorescence microscope
  • Figure 13 shows the results of measuring the viability of cells cultured using the three-dimensional cell culture tool of the present invention.
  • FIG. 14 is a diagram illustrating a state of each cell by randomly extracting three-dimensional cultured cells formed in a cell culture tool in a case where the cell culture well includes a plurality of protrusions in one embodiment of the present invention.
  • Figure 15 shows the results of measuring the distribution of proteins involved in the skeleton within the cells in the case of cell culture using the three-dimensional culture tool of one embodiment of the present invention and in the two-dimensional culture comparative example.
  • Figure 16 shows the response of each cell to toxic substances in the case of culturing cells using the three-dimensional culture tool of one embodiment of the present invention and in the two-dimensional culture comparative example.
  • 17 to 19 show the results of measuring the process of forming a fusion spheroid using the three-dimensional culture tool of an embodiment of the present invention.
  • Fig. 20 schematically shows a possible spheroid production method in the present invention.
  • a 96-well plate was used as the culture plate, a tip-shaped protrusion was made of NdFeB, which is a permanent magnet material, and then attached to a cover of the 96-well plate to prepare a three-dimensional cell culture device.
  • the cover part of the prepared cell culture device is shown in FIG. 9.
  • HeLa cells as cell lines and Fe as magnetic nanoparticles 3 O 4 end The cells were cultivated in the medium contained so as to introduce 800 pg of particles into the cells.
  • Example 800 pg cells cultured in Example 1-2 were cultured to contain 500 cells per well of each of the three-dimensional cell culture apparatus prepared in Example 1. Cells were introduced with a substance that stains the nucleus of the cell (Hoechst) for easy observation.
  • Hoechst a substance that stains the nucleus of the cell
  • the cells aggregated and three-dimensional culture.
  • Example 11 Per well of the culture plate prepared in Example 1-1, the cells cultured in each of the basal cultures in Example 1-2 were cultured to contain 125 to 1000 cells, respectively, and after 5 minutes, z under a confocal microscope After reconstructing the 3D image after -axis scanning, the result of confirming whether the cells were cultured in 3D and the result of confirming in 3D images are shown in FIG. 11.
  • Example 1-2 the HeLa cells were basally cultured while changing the concentration of magnetic nanoparticles introduced into the cells during basal culture in the range of 200-800 pg / cell, and the culture plate prepared in Example 1-1 The wells were moved to 1000 cells / well, the covers were mounted, and the cells were incubated in three dimensions for 5 minutes.
  • Figure 12 it can be seen the degree of cell culture for each concentration. Depending on the concentration of the magnetic nanoparticles per well in the culture plate it can be seen that the difference in the size of the spheroid, which is a three-dimensional cells produced.
  • the cells cultured in 3D in Example 1-5 were subsequently cultured for 5 days, followed by fluorescence observation using the Live / DEAD viability kit, and the results are shown in FIG. 13.
  • Example 800 pg of the cells cultured in Example 1-2 were cultured to include 500 cells per well of each of the culture plates prepared in Example 2-1.
  • Example 2-1 After introducing the cells into which the magnetic particles prepared in Example 2-1 were introduced into the prepared three-dimensional cell culture device, the cover was mounted to allow the cells to be cultured in three dimensions, and the cultured three-dimensional cultured cells were randomized. The state of each cell was extracted as shown in FIG.
  • HeLa cells and mesenchymal stem cells were suspended in a 96 well plate with culture medium, and then allowed to settle for a certain period of time so that the cells settled at the bottom of the plate, and the cells were cultured by a general culture method in an incubator.
  • the three-dimensional cell cultured spheroid of Example 1 and the two-dimensional cultured cells of the comparative example have different protein distributions, and in the three-dimensional cell cultured spheroid of Example 1, between cells Organic influences tend to shift the distribution of proteins outward with the organic binding of actin.
  • Red fluorescence in FIG. 16 means dead cells.
  • Figure 16 in the case of the three-dimensional cell cultured spheroid of Example 1 of the present invention it can be seen that the response to the toxic drug appears from the outside of the spheroid, which is characterized by the characteristics of the three-dimensional cultured cells Shows.
  • the two cultured stem cell spheroids were mixed and cultured with a secondary spheroid in the cell culture apparatus prepared in Example 1 above.
  • FIG. 17 The process of forming the secondary spheroid according to the culture time is shown in FIG. 17.
  • the cells forming one of the two primary spheroids were shown in red by lipid membrane staining, and the cells forming the other spheroid were shown in blue by nuclear staining.
  • FIG. 17 it can be seen that over time, two primary spheroids are fused to form a new secondary fusion spheroid.
  • the spheroid culture was carried out first, and the cultured spheroids were spheroid cultured in a medium containing HeLa cells with GFP green color, and then core-shell type spores. Cultured with Lloyd, the results are shown in FIG. In Figure 18 it can be seen that the spheroid of the core shell structure is formed.

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Abstract

La présente invention concerne un appareil de culture de cellules tridimensionnelle et un procédé de culture tridimensionnelle de cellules l'utilisant. Dans l'appareil de culture de cellules tridimensionnelle et le procédé de culture de cellules tridimensionnelle l'utilisant de la présente invention, des cellules sont soumises à une culture basique dans un liquide de culture comportant des nanoparticules, des nanoparticules magnétiques étant introduites dans les cellules ou les membranes cellulaires, et un magnétisme est appliqué aux cellules dans lesquelles les nanoparticules magnétiques ont été introduites, de telle sorte que des sphéroïdes peuvent être formés dans un état de suspension dans des liquides de culture sans aucun traitement séparé de surface.
PCT/KR2012/004010 2011-09-27 2012-05-21 Appareil de culture de cellules tridimensionnelle et procédé de culture tridimensionnelle de cellules l'utilisant WO2013047974A1 (fr)

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KR20110097397 2011-09-27
KR10-2011-0097397 2011-09-27
KR1020120050142A KR101412155B1 (ko) 2011-09-27 2012-05-11 3차원 세포 배양 용구 및 이를 이용한 세포의 3차원 배양 방법
KR10-2012-0050142 2012-05-11

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WO2014183118A1 (fr) * 2013-05-10 2014-11-13 The Regents Of The University Of California Plateforme microfluidique numérique pour créer, conserver et analyser des spérules de cellule en trois dimensions
EP3112449A4 (fr) * 2014-02-25 2017-11-15 Kuraray Co., Ltd. Dispositif de fabrication de sphéroïdes, et procédé de récupération et procédé de fabrication de sphéroïdes
US11060065B2 (en) 2013-06-10 2021-07-13 Corning Incorporated Tissue structure and preparation method thereof
CN114774354A (zh) * 2022-05-23 2022-07-22 中山大学附属第三医院 一种细胞球的制备方法及应用
WO2022189615A1 (fr) * 2021-03-12 2022-09-15 Eceramix Gmbh Réseau de sources de plasma et procédé de traitement de matériau cellulaire

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Publication number Priority date Publication date Assignee Title
WO2014183118A1 (fr) * 2013-05-10 2014-11-13 The Regents Of The University Of California Plateforme microfluidique numérique pour créer, conserver et analyser des spérules de cellule en trois dimensions
US11060065B2 (en) 2013-06-10 2021-07-13 Corning Incorporated Tissue structure and preparation method thereof
EP3112449A4 (fr) * 2014-02-25 2017-11-15 Kuraray Co., Ltd. Dispositif de fabrication de sphéroïdes, et procédé de récupération et procédé de fabrication de sphéroïdes
AU2015221599B2 (en) * 2014-02-25 2019-11-28 Corning Incorporated Device for fabricating spheroid, and spheroid recovery method and manufacturing method
US10683477B2 (en) 2014-02-25 2020-06-16 Corning Incorporated Spheroid-producing device, method for recovering spheroids, and method for producing spheroids
CN113046237A (zh) * 2014-02-25 2021-06-29 康宁股份有限公司 球状体制造装置、球状体的回收方法及制造方法
US11208625B2 (en) 2014-02-25 2021-12-28 Corning Incorporated Spheroid-producing device, method for recovering spheroids, and method for producing spheroids
WO2022189615A1 (fr) * 2021-03-12 2022-09-15 Eceramix Gmbh Réseau de sources de plasma et procédé de traitement de matériau cellulaire
CN114774354A (zh) * 2022-05-23 2022-07-22 中山大学附属第三医院 一种细胞球的制备方法及应用
CN114774354B (zh) * 2022-05-23 2024-03-01 中山大学附属第三医院 一种细胞球的制备方法及应用

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