CN114736803A - Cell culture device and method for tumor microspheres - Google Patents
Cell culture device and method for tumor microspheres Download PDFInfo
- Publication number
- CN114736803A CN114736803A CN202210650823.3A CN202210650823A CN114736803A CN 114736803 A CN114736803 A CN 114736803A CN 202210650823 A CN202210650823 A CN 202210650823A CN 114736803 A CN114736803 A CN 114736803A
- Authority
- CN
- China
- Prior art keywords
- cell culture
- inner sleeve
- liquid
- cells
- sleeve structure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 57
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 26
- 239000004005 microsphere Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims description 14
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 239000011148 porous material Substances 0.000 claims abstract description 23
- 210000000078 claw Anatomy 0.000 claims abstract description 7
- 239000000463 material Substances 0.000 claims abstract description 7
- 230000002776 aggregation Effects 0.000 claims abstract description 4
- 238000004220 aggregation Methods 0.000 claims abstract description 4
- 230000009471 action Effects 0.000 claims abstract description 3
- 238000012258 culturing Methods 0.000 claims description 8
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 6
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 6
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 6
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 4
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 239000012930 cell culture fluid Substances 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 2
- 239000011806 microball Substances 0.000 claims 2
- 239000011805 ball Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 34
- 210000002220 organoid Anatomy 0.000 description 11
- 230000008859 change Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000001681 protective effect Effects 0.000 description 5
- 238000013461 design Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- -1 polydimethylsiloxane Polymers 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012604 3D cell culture Methods 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 238000002733 pharmacodynamic assay Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Images
Classifications
-
- 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
- C12M23/20—Material Coatings
-
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Immunology (AREA)
- Clinical Laboratory Science (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a cell culture device and a cell culture method for tumor microspheres, wherein the device is an inner sleeve structure for a cell culture pore plate, the bottom of the inner sleeve structure is provided with a plurality of identical inverted polygonal pyramid grooves which are connected together, and the bottom plane is fully paved with the inverted polygonal pyramid grooves; the lateral wall is fixed inside the cell culture pore plate single hole through three claws that along circumference equipartition, has the slit structure that the inside and outside liquid of a plurality of confession circulates and exchanges on the lateral wall between two adjacent claws. After the cells are inoculated, the cells can be uniformly accumulated in the inverted polygonal pyramid groove on the bottom surface of each inner sleeve under the centrifugal force action of a centrifugal machine; the sidewalls of the inverted pyramid can provide mechanical force to support the cells, forming cell spheres by cell aggregation without the need for exogenous support materials.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a cell culture device and method for tumor microspheres.
Background
Two-dimensional culture of tumor cells is not supported by a three-dimensional structure, and lacks of microenvironment for in vivo cell growth and differentiation to cause change of cell morphological structure, so that the difference between the established cell model and clinical experimental results is often large. The three-dimensional cell culture model is an experimental technology between animal experiments and monolayer cell culture and can reproduceThe interaction between cells in vivo has more physiological relevance and simultaneously does not influence the experimental flux. A large number of studies indicate that three-dimensional cell culture is more similar to in vivo survival in terms of cell proliferation and differentiation, cell-cell interaction, gene protein expression, and drug response. Therefore, the three-dimensional cell culture system has wide prospects in the aspects of drug discovery, disease simulation, targeted cancer treatment, regenerative medicine and the like, and the application of the three-dimensional cell culture system in verifying the preclinical results becomes possible to replace laboratory animal experiments[1-2]。
The three-dimensional cell culture techniques commonly used at present mainly include scaffold-based three-dimensional cell culture and scaffold-independent three-dimensional cell culture. Extracellular matrix is extracted from tissues as a main cell culture scaffold and is composed of components such as collagen, laminin, nidogen, growth factors and the like. Under certain conditions, the structure, composition and function of a cell basement membrane in vivo can be simulated, mechanical characteristics and physiological environment similar to the microenvironment of cells in vivo are constructed, and the growth and differentiation of the cells in vitro are supported[3]. And the pore plate with an ultralow adsorption surface is most applied in the three-dimensional cell culture technology independent of the bracket, and the method does not need special treatment and culture processes, and can directly culture a three-dimensional cell structure by inoculating digested cells into micropores by using a conventional cell culture method.
At present, scaffolds for three-dimensional cell culture are mostly derived from basement membrane matrixes of EHS mouse sarcomas, such as coring's Matrigel and Bio-techne's BME cultrex, and since most of tumor cells are of human origin, such Matrigel belongs to a heterogeneous component in a human tumor cell culture system. In addition, the components of the product are not clear, and the properties such as protein concentration, coagulation time and the like of each batch are different, so that the difference between the batches of the product is large, and the repeatability of the experiment is poor. In addition, the operation of using the matrigel is complicated, the matrigel needs to be carried out at low temperature, the storage condition is harsh (for example, BME cultrex needs to be stored at minus 80 ℃), the operation requirement on experimenters is high, and the cost is high.
The three-dimensional cell culture is carried out by utilizing the pore plate with the ultralow adsorption surface, although the method is simple and easy to implement, the obtained three-dimensional cell spheres are often poor in uniformity, are not beneficial to the replacement of a culture medium, and are limited by a cell culture vessel with low adhesion treatment, so that the application of the cell culture vessel in clinical experiments is limited.
[1] Duval K, Grover H, Han L H, et al. Modeling physiological events in 2D vs. 3D cell culture[J]. Physiology, 2017, 32(4): 266-277.
[2] Kapałczyńska M, Kolenda T, Przybyła W, et al. 2D and 3D cell cultures–a comparison of different types of cancer cell cultures[J]. Archives of medical science: AMS, 2018, 14(4): 910.
[3] Takebe T, Wells J M. Organoids by design[J]. Science, 2019, 364(6444): 956-959。
Disclosure of Invention
The invention aims to provide a cell culture device and a cell culture method for tumor microspheres aiming at the defects of the prior art, and on one hand, the problems of large batch-to-batch difference, poor experimental result repeatability, complex operation, high cost and the like caused by using exogenous cell matrix materials can be effectively avoided. On the other hand, the homogenization of the cultured tumor microspheres and the limitation of the size of the tumor microspheres can be realized, and the problems that the culture medium is inconvenient to replace in the process of culturing the tumor microspheres and the like are solved.
The purpose of the invention is realized by the following technical scheme: on one hand, the invention provides a cell culture device of tumor microspheres, which is an inner sleeve structure for a cell culture pore plate, wherein the bottom of the inner sleeve structure is provided with a plurality of identical inverted polygonal pyramid grooves which are connected together, and the bottom planes are fully paved with the inverted polygonal pyramid grooves; the lateral wall is installed inside the cell culture pore plate haplopore through three jack catchs along circumference equipartition, has the slit structure that the inside and outside liquid of a plurality of confession circulates and exchanges on the lateral wall between two adjacent jack catchs.
Further, the grooves in the shape of the inverted polygonal pyramids are arranged at the bottom of the inner sleeve structure in rows and columns.
Furthermore, the bottom of the inner sleeve structure is treated by polydimethylsiloxane PDMS, so that the adhesiveness is reduced.
Furthermore, the lower end of the slit structure has a certain distance from the bottom of the inner sleeve structure, and the distance is set according to requirements.
In another aspect, the present invention further provides a cell culture method for a cell culture device of tumor microspheres, the method comprising the following steps:
(1) in the inoculation stage, cell culture solution is injected into the inner sleeve structure in the single hole of the cell culture pore plate through a liquid transfer device, when the liquid level exceeds the lower end of the slit on the side wall of the inner sleeve, the liquid in the inner sleeve structure is communicated with the liquid in the cell culture pore plate, and therefore the cells cultured in the inner sleeve structure can exchange substances with all the cell culture solution;
after cells are inoculated, the cells are uniformly accumulated in the inverted polygonal pyramid groove at the bottom surface of each inner sleeve structure under the centrifugal force action of a centrifugal machine; each side wall of the inverted polygonal pyramid groove can provide mechanical force for supporting cells, and cell balls are formed through cell aggregation under the condition that exogenous supporting materials are not needed;
(2) and in the liquid replacement stage, a liquid transfer device is used for sucking liquid from the space between the inner sleeve structure and the cell culture pore plate, the liquid in the inner sleeve structure uniformly flows out from the slit, and the liquid feeding replacement of new culture liquid is completed between the inner sleeve and the pore plate.
The invention has the beneficial effects that: the invention provides a cell culture device of tumor microspheres, which breaks through the traditional three-dimensional cell culture mode, can form a three-dimensional cell structure with uniform size and controllable form by only utilizing the self physical supporting function of the device without an exogenous scaffold material, and avoids the problems of poor experimental repeatability, complicated operation process and the like caused by the exogenous scaffold material. The device can be matched with cell culture pore plates of various models, and in the aspect of application of pharmacodynamic assay, the groove design on the bottom surface of the inner sleeve can realize multi-hole assay in a single device, thereby simplifying the experimental process and improving the assay efficiency. In addition, the design of the culture device realizes the safe liquid change in the three-dimensional cell culture, and the disturbance to cells cultured at the bottom is small in the liquid change process, thereby greatly improving the stability and the construction efficiency in the three-dimensional cell culture process.
Drawings
FIG. 1 is a schematic view of an inner sleeve fixed inside a single hole of a hole plate;
FIG. 2 is a schematic view of the inner sleeve structure;
FIG. 3 is a schematic view of an inverted rectangular pyramid groove structure at the bottom of the inner sleeve;
FIG. 4 is a schematic view of the bottom of the inner sleeve being perforated;
FIG. 5 is a schematic view showing that cells are uniformly distributed in the micropores at the bottom of the inner sheath after plating;
FIG. 6 is a schematic comparison of the culture behavior of uniformly sized organoids cultured with a low adhesion, gelatin inner sleeve device;
FIG. 7 is a schematic illustration of a device for culturing uniform-sized tumor microspheres;
FIG. 8 is a schematic representation of the change in properties of organoids cultured in the device before and after administration;
in the figure, 110 is an inner sleeve structure; 120. a single hole of the orifice plate; 130. an inverted rectangular pyramid groove structure; 140. a slit structure; 150. the claw.
Detailed Description
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
The invention provides a cell culture device of tumor microspheres, which is shown in figure 1, figure 2 and figure 3. The device is an inner sleeve structure 110 used for a cell culture pore plate, inverted polygonal pyramid groove structures are arranged on the bottom surface of the inner sleeve structure 110 in rows and columns and are fully paved on the bottom plane, an inverted rectangular pyramid groove structure 130 is used in the embodiment of the invention, and a slit structure 140 for communicating and exchanging inner liquid and outer liquid is formed on the side wall of the inner sleeve.
In the mold processing stage, the inner sleeve structure 110 is fixed inside the single hole 120 of the orifice plate by three clamping jaws 150 uniformly distributed along the circumferential direction, so that the assembly stability in the centrifugal stage can be ensured (as shown in fig. 1). Three slit structures 140 are formed on the sidewall between each adjacent two of the jaws 150. The lower end of the slit structure 140 has a certain distance from the bottom of the inner sleeve structure, and the distance is set according to requirements.
During the inoculation stage, the interior of the inner housing structure 110 can retain a volume of 250 μ L of liquid (see fig. 2). After the liquid is injected into the single hole 120 of the pore plate by the liquid transfer device to exceed 1.3mL, the liquid level exceeds the lower end of the slit structure 140 of the inner sleeve, and the cell culture liquid in the inner sleeve can be communicated with the liquid in the pore plate, so that the cells cultured in the inner sleeve can exchange substances with all the culture liquid. In accordance with the presently contemplated operation, when a total volume of 2mL of liquid is injected, a slit structure 140 having a length of 3mm is submerged below the liquid surface.
After the cells are inoculated, the cells are uniformly stacked in the inverted rectangular pyramid groove structure 130 on the bottom surface of each inner sleeve by the centrifugal force of the centrifuge. The sidewalls of the inverted rectangular pyramid can provide mechanical force to support the cells, forming cell spheres by cell aggregation without the need for exogenous support materials.
In the liquid replacement stage, a liquid transfer device is used for sucking liquid from the space between the inner sleeve structure 110 and the single hole 120 of the hole plate, the liquid in the inner sleeve structure 110 uniformly flows out of the slit structure 140, and new culture liquid is replaced by adding liquid between the inner sleeve and the hole plate, so that the disturbance on cells cultured at the bottom of the inner sleeve is small; the bottom cells are not affected when the liquid is added.
The bottom of the inner sleeve is perforated schematically as shown in FIG. 4, the diameter is 16.3mm, the side length of the square hole is 600, 800, 1000, 1200 μm, the depth is 300, 400, 500, 600 μm, the groove is in the shape of an inverted pyramid, and the bottom of the inner sleeve is subjected to low adhesion treatment by using polydimethylsiloxane PDMS.
The state of the cells uniformly distributed in the inner liner structure after plating is shown in fig. 5, and the culture properties of the organoids of uniform size and low adhesion and gel cultured by the device of the present invention are shown in fig. 6. for example, human breast cancer cells were cultured in organoids using the culture device of the present invention, gel and low adhesion, respectively, and the results are shown in fig. 6 after 12 days of culture. Compared with a glue method and a low-adhesion method, the organoid cultured by the device has more uniform size and more controllable growth. The uniform size of the tumor microspheres (brightfield) cultured by the device of the present invention is shown in fig. 7, and the formation of uniform size tumor spheres can be observed by testing the sphere forming ability in the device using the mouse prostate cancer cell line, and removing the microspheres after 7 days of culture. The change of the characteristics (bright field) of the organoids cultured by the device of the present invention before and after administration is shown in FIG. 8, and the drug sensitivity test was performed using the cell culture device. As shown in FIG. 8, after the organoids cultured to 100 μm were stimulated with cisplatin and adriamycin for 120 hours, the organoids were significantly changed in properties, and it was observed that the boundary structure of the organoids was destroyed and the outer edge cells were apoptotic. This indicates that organoids cultured using the device of the present invention do not affect the sensitivity of the drug to it.
In addition, the device can be adapted to the existing culture cell culture pore plate without injection molding production of the specially treated micropore culture pore plate, is more flexible and convenient, and enlarges the application scene and the application range of the tumor microsphere culture.
The cell culture device can realize three-dimensional culture of cells only by physical support of the inner sleeve structure of the pore plate without using exogenous support materials (such as matrigel), and the shape and the size of the cell culture device are uniform and controllable.
The invention can be matched with cell culture pore plates of various types, and the micropore design of the bottom surface of the inner sleeve structure can realize multi-pore measurement in a single device, thereby improving the experimental efficiency. The application range of the protective inner sleeve is not limited to a 6-hole plate, a 12-hole plate, a 24-hole plate, a 48-hole plate, a 96-hole plate and the like, the shape range of the protective micro-hole is not limited to a V shape, a U shape and the like, the protective micro-hole structure is not limited to an inverted triangular pyramid, a rectangular pyramid, a pentagonal pyramid and the like, the diameter size of the protective micro-hole is 200-800 micrometers, the depth is 200-800 micrometers, and the protective micro-hole coating is not limited to low adhesion treatment by PDMS.
The culture device provided by the invention can reduce the influence on cells caused by liquid flow to the maximum extent and realize the safe liquid change of the culture medium.
The above-described embodiments are intended to illustrate rather than limit the invention, and any modifications and variations of the present invention are within the spirit and scope of the appended claims.
Claims (10)
1. A cell culture device of tumor microspheres is characterized in that the device is an inner sleeve structure for a cell culture pore plate, the bottom of the inner sleeve structure is provided with a plurality of identical connected inverted polygonal pyramid grooves, and the inverted polygonal pyramid grooves are fully paved on the bottom plane; the outer side wall is provided with a slit structure for the circulation and exchange of the inner liquid and the outer liquid.
2. The device for culturing tumor microsphere cells of claim 1, wherein the inverted polygonal pyramid-shaped groove is an inverted quadrangular pyramid-shaped groove.
3. The device for culturing tumor microspheres according to claim 1, wherein the inverted polygonal pyramid-shaped grooves are arranged in rows and columns at the bottom of the inner casing structure.
4. The device for culturing tumor microballoons according to claim 1, wherein the outer sidewall of the inner sleeve structure is installed inside a single hole of the cell culture well plate through a claw.
5. The device for culturing tumor microsphere cells according to claim 4, wherein there are three claws, and the claws are uniformly distributed along the circumferential direction.
6. The device for culturing tumor microballs according to claim 5, wherein there are several slit structures on the side wall between every two adjacent claws.
7. The device of claim 1, wherein the bottom of the inner sheath is treated with PDMS to reduce adhesion.
8. The device for culturing tumor microballs according to claim 1, wherein the lower end of the slit structure is spaced from the bottom of the inner sleeve structure by a certain distance, and the distance is set according to the requirement.
9. A cell culture method of a cell culture device based on the tumor microsphere of claim 1, which comprises the following steps:
(1) in the inoculation stage, injecting cell culture solution into the inner sleeve structure in a single hole of the cell culture pore plate through a liquid transfer device, and after inoculating cells, enabling the cells to be uniformly accumulated in the inverted polygonal pyramid groove at the bottom surface of each inner sleeve structure under the centrifugal force action of a centrifugal machine; each side wall of the inverted polygonal pyramid groove can provide mechanical force for supporting cells, and cell balls are formed through cell aggregation under the condition that exogenous supporting materials are not needed;
(2) and in the liquid replacement stage, a liquid transfer device is used for sucking liquid from the space between the inner sleeve structure and the cell culture pore plate, the liquid in the inner sleeve structure uniformly flows out from the slit, and the liquid feeding replacement of new culture liquid is completed between the inner sleeve and the pore plate.
10. The method according to claim 9, wherein when the liquid level of the cell culture fluid exceeds the lower end of the slit of the sidewall of the inner sheath, the fluid inside the inner sheath structure is in fluid communication with the interior of the cell culture well plate, thereby allowing the cells cultured inside the inner sheath structure to exchange substances with the entire cell culture fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210650823.3A CN114736803A (en) | 2022-06-10 | 2022-06-10 | Cell culture device and method for tumor microspheres |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210650823.3A CN114736803A (en) | 2022-06-10 | 2022-06-10 | Cell culture device and method for tumor microspheres |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114736803A true CN114736803A (en) | 2022-07-12 |
Family
ID=82287603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210650823.3A Pending CN114736803A (en) | 2022-06-10 | 2022-06-10 | Cell culture device and method for tumor microspheres |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114736803A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120484959A (en) * | 2025-07-14 | 2025-08-15 | 中国农业科学院果树研究所 | Cell accommodating and culturing device |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245313A (en) * | 2007-02-13 | 2008-08-20 | 刘青 | Three dimensional cell culture construct and apparatus for its making |
| CN101643703A (en) * | 2009-08-28 | 2010-02-10 | 雷震 | Three-dimensional system and related method of cell long-term culture and preservation |
| CN106164244A (en) * | 2014-05-22 | 2016-11-23 | 住友电木株式会社 | Cell mass culture vessel |
| CN113308376A (en) * | 2021-07-14 | 2021-08-27 | 苏州大学 | Clamping type multi-concave cell culture sheet |
-
2022
- 2022-06-10 CN CN202210650823.3A patent/CN114736803A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245313A (en) * | 2007-02-13 | 2008-08-20 | 刘青 | Three dimensional cell culture construct and apparatus for its making |
| CN101643703A (en) * | 2009-08-28 | 2010-02-10 | 雷震 | Three-dimensional system and related method of cell long-term culture and preservation |
| CN106164244A (en) * | 2014-05-22 | 2016-11-23 | 住友电木株式会社 | Cell mass culture vessel |
| CN113308376A (en) * | 2021-07-14 | 2021-08-27 | 苏州大学 | Clamping type multi-concave cell culture sheet |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120484959A (en) * | 2025-07-14 | 2025-08-15 | 中国农业科学院果树研究所 | Cell accommodating and culturing device |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | A review of manufacturing capabilities of cell spheroid generation technologies and future development | |
| CN113290844B (en) | A Multilevel Suspension Printing Method for Constructing Complex Heterogeneous Tissue/Organs | |
| EP1976972B1 (en) | Cell aggregation and encapsulation device and method | |
| JP6998215B2 (en) | Growth medium for 3D cell culture | |
| CN110903976B (en) | Orifice plate device for organoid sphere culture | |
| CN113773959A (en) | Organoid culture chip and organoid culture method | |
| Levinger et al. | Life is three dimensional—as in vitro cancer cultures should be | |
| WO2022001631A1 (en) | Device for preparing cell clusters, construction method therefor and application thereof | |
| CN113846016B (en) | A high-throughput porous array chip, device, preparation method and application | |
| JP6021802B2 (en) | Culture method and drug screening method | |
| CN108728356B (en) | Apparatus and co-culture method for pairing of different three-dimensional cell clusters | |
| CN103351484A (en) | Micropatterned hydrogel coating, its preparation method and use | |
| CN113278525B (en) | Stem cell ball or tumor ball culture device and culture method | |
| CN113755425B (en) | Preparation method of porous microcarrier for carrying three-dimensional islet beta cell aggregate | |
| CN112442484A (en) | Method for large-scale cell culture based on porous nanoscale temperature-sensitive soft colloid | |
| CN114456936A (en) | Chip, organoid model, construction method and construction device of organoid model and application of organoid model | |
| CN114736803A (en) | Cell culture device and method for tumor microspheres | |
| Sośniak et al. | 3D cell culture technology–a new insight into in vitro research–a review | |
| CN211713118U (en) | A orifice plate device for organoid spheroid is cultivateed | |
| CN120005801A (en) | A hydrogel microporous array and its mold and application | |
| CN111647509A (en) | Sitting drop type cell ball culture chip and its use method | |
| CN115678778A (en) | Micro-fluidic chip device for culturing three-dimensional cell clusters | |
| CN114606129A (en) | A large-scale cell three-dimensional culture device and its application | |
| CN114958725B (en) | Three-dimensional cell spheroid hanging drop culture and co-culture method based on hydrophilic and hydrophobic array chips | |
| JP2010200679A (en) | Cell culture container, method for performing cell culture, and method for evaluating cell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220712 |