WO2018066953A1 - Dispositif de culture cellulaire à goutte suspendue utilisant une membrane poreuse, procédé pour sa fabrication, procédé de culture cellulaire à goutte suspendue l'utilisant et dispositif d'automatisation de culture cellulaire à goutte suspendue - Google Patents

Dispositif de culture cellulaire à goutte suspendue utilisant une membrane poreuse, procédé pour sa fabrication, procédé de culture cellulaire à goutte suspendue l'utilisant et dispositif d'automatisation de culture cellulaire à goutte suspendue Download PDF

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WO2018066953A1
WO2018066953A1 PCT/KR2017/011043 KR2017011043W WO2018066953A1 WO 2018066953 A1 WO2018066953 A1 WO 2018066953A1 KR 2017011043 W KR2017011043 W KR 2017011043W WO 2018066953 A1 WO2018066953 A1 WO 2018066953A1
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cell culture
porous membrane
hydrophilic
droplets
hydrophilic portion
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PCT/KR2017/011043
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English (en)
Korean (ko)
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조윤경
김준영
아이작마이클
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울산과학기술원
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Publication of WO2018066953A1 publication Critical patent/WO2018066953A1/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
    • C12M23/04—Flat or tray type, drawers
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/22—Petri dishes
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12M23/34—Internal compartments or partitions
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

  • Cellular cell culture apparatus using porous membrane, preparation method thereof, cell culture method using same, and cell culture automated apparatus
  • the present invention relates to a wise culture device, a method for producing the same, a wise cell culture method using the same, and a wise cell culture automation device using the same.
  • Embodiments of the present invention to provide a cultivated cell culture device capable of replacing the culture medium, a method for preparing the same, a cultivated cell culture method using the same, and an automatic cell culture automated device using the same.
  • the porous membrane A hydrophobic part located in the porous membrane; And a hydrophilic portion surrounded by the hydrophobic portion, wherein the droplet of the cell culture solution is suspended in the hydrophilic portion to perform cell culture, and the culture medium in the droplet formed in the hydrophilic portion through the porous membrane can be exchanged and injected with an external substance.
  • the porous membrane may have hydrophilicity.
  • the porous membrane may include paper, nitrocellulose, silk, cotton fiber, polymer porous membrane, or a combination thereof.
  • the porous membrane may have a pore diameter of 0.2 ⁇ or more and 10 ⁇ or less.
  • the porous membrane may have a thickness of 0.04 mm or more and 0.5 kPa or less.
  • the porous membrane may be one in which fibrous particles are randomly arranged.
  • the hydrophilic portion may be 0.5mm or more and 8mm or less in diameter.
  • the length of the droplets suspended in the hydrophilic portion may be more than 0.5 mm 3 and less than 7 mm.
  • Droplets deposited on the hydrophilic portion may have a contact angle of 20 ° or more and 120 ° or less.
  • the droplets suspended in the hydrophilic portion may have a volume of 10 ⁇ l or more and 100 ⁇ 1 or less.
  • Cells cultured in the droplets suspended in the hydrophilic portion is a three-dimensional spheroid, may be 50 ⁇ or more in diameter and 2000 ⁇ or less.
  • the hydrophobic portion may be coated with a hydrophobic polymer.
  • the hydrophobic polymer may be a wax, a teflon, a photoresist, a silane compound, a polytetrafluoroethylene, a wax, a polydimethylsiloxane, PDMS, silicone (si 1 i cone), It may be a metal nanoparticle, a metal oxide nanoparticle, a silica nanoparticle, a polymer nanoparticle or a combination thereof.
  • the distance between each of the hydrophobic portion may be 1 ⁇ or more and 9 mm or less.
  • the channel is to connect two or more hydrophilic portion, the channel may be to enable interaction between the hydrophilic portion.
  • the width of the channel may be 0.1 mm or more and 2.5 mm 3 or less.
  • the hydrophilic portion may further include a bio-pass ivat ion coating layer.
  • the bio-passivat ion coating layer may have a thickness of 1 nm or more and 10 nm or less.
  • the surface in which the droplet is suspended in the hydrophilic portion may be a surface having a reduced hydrophilicity compared to the other surface.
  • the cells contained in the cell culture medium may be any one selected from a single cell line (pr imary cel ll ine) of mammalian cells, a cont inuous eel line, stem cells, tumor cells, immune cells, bacteria and microorganisms. It may be.
  • Two or more cultivated cell culture units are stacked, and the cultivated cell culture units comprise a porous membrane; A hydrophobic part located in the porous membrane; And a hydrophilic part surrounded by the hydrophobic part, wherein the porous medium can be exchanged with the culture medium in the droplet formed in the hydrophilic part and foreign material can be injected into the hydrophilic part of the cell culture unit located at the bottom of the cell culture medium.
  • Droplets of the cultivation may be a cell culture.
  • the at least one culturing cell culture unit further comprises a channel located in the porous membrane, the channel is to connect two or more hydrophilic portion, the channel may be to enable interaction between the hydrophilic portion.
  • the preparing of the extensible cell culture unit may include forming a channel connecting the hydrophilic part.
  • the preparing of the conventional cell culture unit may be performed by printing a hydrophobic material on a hydrophilic porous membrane.
  • the step of stacking two or more wise cell culture unit may be to stack the wise cell culture unit by folding so that the hydrophilic portion of the upper layer and the lower layer of the fold cell culture unit overlap.
  • Preparing a cell culture unit by forming a hydrophilic part and a hydrophobic part on the porous membrane; and then surface treating the hydrophilic part; It may be to include more.
  • Culturing the cells; injecting an external material through the porous membrane; may be further included.
  • Said cultivating cell culture device comprises a channel, said channel connecting at least two hydrophilic parts, said channel enabling interaction between said hydrophilic parts, and forming droplets of said cell culture fluid; More than one species of cell culture solution is added to each of the hydrophilic parts connected to the channel to form droplets of the cell culture solution, and culturing the cells; the droplets of the cell culture solution through the channels between the respective cell spheroids Interactions may be made and the cells may be cultured.
  • a culture sheet comprising a plurality of modern cell culture units; A driving motor for moving the culture sheet; A cell culture solution injector disposed above the culture sheet, and dropping the cell culture solution into a hydrophilic part of the hanging cell culture unit;
  • each of the cultivated cell culture units comprises a porous membrane; A hydrophobic part located in the porous membrane; And a hydrophilic portion surrounded by the hydrophobic portion, wherein the droplet of the cell culture solution is suspended in the hydrophilic portion to perform cell culture, and the exchange of the culture medium in the droplet formed in the hydrophilic portion through the porous membrane and the injection of external material are possible. It provides phosphorus, automatic cell culture automation device.
  • the culture sheet has a conveyor belt structure, it may be to move in the form of a chain up and down.
  • the cell culture solution may be dropped into the hydrophilic portion of the culture unit to form droplets from the cell culture solution input device.
  • droplets formed on the hydrophilic portion of the culture unit may be suspended by gravity.
  • the reagent may be added to the droplets.
  • the culture medium can be exchanged, it is possible to culture the cells for a long time.
  • it is possible to inject foreign materials it is applicable to the study of cell changes by the influence of foreign materials.
  • Figure 1 shows the structure of a cell culture device, an embodiment of the present invention
  • Figure 2 is an embodiment of the present invention, an experimental schematic diagram of cell cultivation technology using a hydrophobic wax-printed vesicle cell culture device hydrophobic membrane.
  • 3 is a result showing the relationship between various diameters of the hydrophilic portion and thus the acceptable droplet volume of the droplets suspended in a paper-based suspension culture device (PHDC, Paper Hanging Drop Chip) according to an embodiment of the present invention.
  • 4 is a relationship between droplet volume and droplet length according to a hydrophilic portion having a specific diameter.
  • 5 is an experimental result of the droplet maximum receiving volume and droplet stability by measuring the droplet contact angle of the hydrophilic portion.
  • Figure 6 is the result of the concentration gradient formation over time in the channel of various widths in the channel formed PHDC (Net worked PHDC, N-PHDC).
  • FIG. 7 shows an image of a PHDC which is an embodiment of the present invention assembled to various kinds of dishes.
  • FIG. 8 is a schematic diagram of a cell injection, culture and analysis automation device using PHDC and N-PHDC which is an embodiment of the present invention.
  • 9 is a schematic diagram of a method for manufacturing a conventional cell culture device according to an embodiment of the present invention.
  • Figure 11 is a schematic diagram of a manufacturing process of a conventional cell culture device according to an embodiment of the present invention.
  • FIG. 12 is a schematic view of the hydrophilic surface treatment step of a three-dimensional cell suspension culture apparatus using a hydrophobic polymer printed hydrophilic porous membrane. '
  • FIG. 13 shows experimental results of droplet retention time and degree of protein nonspecific binding after surface treatment on a hydrophilic part.
  • FIG. 14 is a schematic diagram of a method for culturing cells in accordance with an embodiment of the present invention.
  • Figure 15 is a chemotactic reaction in the concentration gradient of the three-dimensional spheroid in N-PHDC of one embodiment of the present invention.
  • FIG. 16 shows the results of experiments on general cells (CCD 1058sk) and tumor cells (MDA-MB 231) drug test and immuno reaction against anticancer drugs (5FU) using PHDC, which is an embodiment of the present invention.
  • 17 is an image of tumor spheroid formation in PHDC according to an embodiment of the present invention.
  • FIG. 18 is a comparison result between spheroids cultured in PHDC and spheroids cultured in Petr i-di sh, a conventional cultivation method.
  • FIG. 20 shows a case in which only breast cancer cells (MDA—MB 231) are monocultured and breast cancer cells (M-MB 231) and fibroblasts (MEF), which are manufactured by a lamination method and using a cell culture apparatus including a channel. Electron scanning microscopy images for comparison of cultured cell binding states when cultured in a mutually compatible state in the channel-formed hydrophilic region. [Specific contents to carry out invention]
  • the length of the drop (Height of drop) is used to mean the vertical length of the droplet relative to the surface of the porous membrane, it is used to mean the length of the vertical direction of the droplet suspended under the hydrophilic portion of the porous membrane Can be.
  • the width of the channel means a width of the channel in a length perpendicular to the direction in which the fluid moves.
  • the biggest difficulty with existing conventional culture techniques is the exchange of media from the droplets and the control of droplet size.
  • by printing a hydrophobic pattern, forming a limited hydrophilic space on the porous material, and microliterized cells including cells to be cultured The culture solution is suspended in a limited hydrophilic space by the strong capillary force of the porous membrane to form a three-dimensional spheroid.
  • periodic culture exchange is possible, and the culture period can be extended by removing cell waste and injecting a new culture solution.
  • the size of the droplets may be controlled by controlling the diameter of the hydrophilic portion of the porous membrane or by adding or removing a part of the culture solution through the porous membrane.
  • the present invention can be easily produced using a wax printing method for the channel that can interact with each droplet, there is an advantage that can be easily changed to the design according to the user's needs.
  • the injected foreign substance may be an experimental reagent, a fluorescent staining agent, a disease treatment agent such as an anticancer agent, an extracellular matrix protein such as a hydrogel, a cell, but is not limited thereto.
  • FIG. 1 shows a structure of a cell culture device, an embodiment of the present invention.
  • the present cell culture device may include a porous membrane (1), at least one hydrophobic portion (3) located in the porous membrane, and at least one hydrophilic portion (2) surrounded by the hydrophobic portion. More than 2 It may further comprise a channel (4) for connecting the hydrophilic portion to enable interaction.
  • Figure 2 is an experimental schematic diagram of cell cultivation technology using a hydrophobic wax-printed cultivation cell culture device hydrophobic membrane.
  • the hydrophobic portion pattern is printed on the hydrophilic porous membrane using wax printing. After dropping the cell culture onto the hydrophilic portion, turn the instrument upside down to suspend the cell culture droplets. The droplets are suspended in the hydrophilic region through the interaction of the capillary force of the porous membrane and the surface tension of the culture medium.
  • Periodic replacement of the culture medium through the upper portion of the porous material allows cell culture while supporting droplets for a longer period of time.
  • the porous membrane may be hydrophilic, specifically, paper, nitrocellulose, silk, cotton fiber, polystyrene, polytetrafluoroethylene
  • a polymer porous membrane may be made of any one selected from a porous membrane having a micro or nano-size pattern.
  • the porous membrane has a pore diameter of 0.2 ⁇ or more and 10 ⁇ or less, specifically, 0.5 ⁇ to 9 ⁇ , 0.7 ⁇ to 8 ⁇ , 1 ⁇ to 7 ⁇ , 3 ⁇ to 5 ⁇ , 0.7 ⁇ to 3 ⁇ , 4 ⁇ to 10 ⁇ , or 0.2 ⁇ to 6 ⁇ .
  • the pore diameter is too large, when the cell culture fluid is added to the hydrophilic part, the cells may be caught in the porous membrane, thereby reducing the number of cells used for spheroid formation. If the pore diameter is too small, it is difficult to exchange the nutrient medium through the porous membrane. Problems may occur, and if the pore range is satisfied, the cells may be prevented from being caught in the porous membrane, as well as the nutrient medium for cell culture, and the effect of facilitating reagent exchange for later assays. There is.
  • the porous membrane may have a thickness of 0.04 mm 3 or more and 0.5 mm 3 or less, and specifically, 0.05 mm or more and 0.4 mm or less, 0.07 mm or more and 0.3 mm or less, 0.1 mm or more and 0.2 mm.
  • the thickness of the porous membrane is thin, it is difficult to support more than a certain volume of droplets, and when the thickness of the porous membrane is too large, the volume of the solution that the porous membrane can absorb is too large. It can stably support the droplet volume required for cultivation, and it is possible to perform further analysis experiments even with a small reagent volume.
  • the porous membrane may be composed of f iber particles, for example, may be cellulose fibers. Specifically, silk fibers, cotton fibers and the like can be used.
  • the fibrous particles may be arranged in a line, but is not limited thereto, and may be in the form of randomly arranged fibrous particles.
  • fibrous particles having a random arrangement of fibrous particles there is an effect that signal communication between reagents and cells can be uniformly transmitted in all directions with uniform capillary force and diffusion without being affected by the direction of fiber arrangement.
  • Conventional cultivation technology has a problem that it is difficult to control the size of the droplets, according to one embodiment of the present invention by controlling the shape or diameter of the hydrophilic portion, it is possible to control the diameter, volume, contact angle, etc. of the droplets formed effectively effective cultivation This is possible.
  • the hydrophilic portion formed in the porous membrane may be 0.5mm or more and 8mm or less in diameter.
  • the droplets formed on the hydrophilic portion may have a volume of 10 ⁇ l or more and 100 ⁇ l or less, and specifically 10 ⁇ l or more and 90 ⁇ l or less, 10 ⁇ 1 or more and 80 ⁇ l or less, 20 ⁇ 1 or more. And 70 ⁇ l or less, 20 ⁇ l or more and 50 ⁇ l or less, 20 ⁇ l or more and 30 ⁇ l or less, 30 ⁇ l or more and 40 ⁇ 1 or less, 10 ⁇ l or more and 30 ⁇ l or less.
  • the volume of the droplets formed in the hydrophilic portion is related to the volume of the droplets, which is the space in which the cell is cultured.
  • the volume of the droplets formed in the hydrophilic portion is too large, due to the mass of the droplets, the droplets may not be stably formed in the hydrophilic portion, and if the volume of the droplets is too small, the cell culture may not be performed well. If the range is satisfied, the cell volume can be efficiently obtained by securing a sufficient volume of droplets, providing sufficient nutrients for cell culture, and preventing the culture liquid from drying out during the culture period, and through periodic culture medium exchange through a porous membrane. Stable droplet support for a long time
  • the diameter of the hydrophilic portion is related to the volume of droplets formed in the hydrophilic portion.
  • 3 is a paper-based suspension culture device (PHDC) which is an embodiment of the present invention Paper Hanging Drop Chip) was used to measure the volume of droplets that could be accommodated according to the diameter of the hydrophilic portion.
  • PHDC paper-based suspension culture device
  • the hydrophilic part is no longer supported by the hydrophilic part and separates and falls into water droplets.
  • it forms the most stable droplets for cultivation.
  • the diameter of the hydrophilic part is 3 ⁇ , the volume of the droplet is no longer supported by the hydrophilic part, but separates and falls into water droplets.
  • the diameter of the hydrophilic part is 4 mm, the volume of the droplet is no longer supported by the hydrophilic part and is separated. The droplet is dropped into the water droplet and forms the most stable droplet in the suspension culture at 30 ⁇ l. If the hydrophilic part is 5 mm in diameter, it forms the most stable droplet for suspension culture at 40 ⁇ l.
  • the volume of the droplets that can be seen increases, and the volume of droplets that is most stable for the culture of cultivation also increases.
  • the volume of the most stable droplets for culturing in the hydrophilic portion of a specific diameter is increased. It was confirmed that there is.
  • the longitudinal length of the droplet formed in the water-repellent part may be 0.5 mm or more and 7 mm or less, specifically, 0.5 mm or more and 4 mm or less, 0.5 mm or more and 3.7 mm or less, 0.5 mm or more and 3 mm or less, or 1.3 and at most 3 mm and at most 3 mm, at least 1.7 mm and at most 3 mm, at least 1.7 mm and at most 2.5 mm, or at least 0.5 mm and at most 4 mm.
  • Figure 4 shows the relationship between the volume of the droplets suspended in the hydrophilic portion and the longitudinal length of the droplets according to the diameter of the hydrophilic portion.
  • a droplet volume of 30 ⁇ showed a droplet length of about 4 mm at a diameter of 2 mm hydrophilic portion, and a droplet length of about 2 mm at a diameter of 5 mm hydrophilic portion.
  • the diameter of the droplets suspended in the hydrophilic portion is too large, the retention of the droplets may be unstable, and the problem of dropping and not supporting the hydrophilic portion may occur. There is a problem in that a sufficient space cannot be secured. Therefore, when the droplet diameter range is satisfied, stable droplets can be formed, and the droplets are divided. By securing the internal volume, not only the cell culture can be efficiently carried out by providing a rich nutrient to the cell culture and preventing the culture liquid from drying out during the culture period, but also the stable cell culture can be stable for a long time in the hydrophilic part.
  • Droplets deposited on the hydrophilic portion may have a contact angle of 20 ° or more and 120 ° or less.
  • the contact angle of the droplets deposited on the hydrophilic portion may vary depending on the diameter of the hydrophilic portion or the size of the droplets deposited, specifically, 20 ° or more and 120 ° or less, 40 ° or more and 110 ° or less, 60 ° or more and 110 ° or less, 80 ° or more and 110 ° or less, or 70 ° or more and 110 ° or less, 80 ° or more and 120 ° .
  • the maximum receiving volume and the stability of the droplet are shown by measuring the contact angle of the droplets suspended in the hydrophilic portion by varying the diameter of the hydrophilic portion according to the exemplary embodiment of the present invention.
  • the stability of the droplets in the various hydrophilic regions was shown by the conditions of the largest contact angle of the droplets, and the range of the droplets in the various hydrophilic conditions was also demonstrated by the conditions of the contact angle tendency.
  • the contact angle means a value ⁇ measured from the inside of the liquid to form an angle between the solid surface and the liquid surface as shown in FIG. 5B.
  • the contact angle of the droplets in the hydrophilic part is too large, the droplets may not be stably formed in the hydrophilic part, and cell culture may be difficult for a long time. If the contact angle is too small, the volume inside the formed droplets may be too small to be sufficient for culturing cell culture. There is a problem that cell culture cannot be effectively performed due to lack of space, and if the contact angle range of the droplet is satisfied, a sufficient volume of droplets can be stably formed in the cell culture, and a sufficient internal volume of the droplet can be secured for cell culture. By providing abundant nutrients to the cell and preventing the culture liquid from drying out during the culture period, not only can the cell culture be efficiently conducted, but also the cell culture can be stably maintained for a long time in the hydrophilic part.
  • Cells cultured in the droplets suspended in the hydrophilic portion is a three-dimensional spheroid, may be more than 50 im diameter and less than 2000 ⁇ . Specifically, 50 ⁇ ⁇ or more and 1800 ⁇ ⁇ or less, 50 ⁇ ⁇ or more, 1500 ⁇ ⁇ or less, 80 ⁇ ⁇ or more, 1000 ⁇ ⁇ or less, 50 im or more and 500 ⁇ ⁇ or less, or 50 ⁇ ⁇ or more and 1000 um or less It may be.
  • the hydrophilic portion formed in the porous membrane is formed in a circular shape, but is not limited thereto, and may be formed in various shapes such as a circle, a rectangle, and an oval. In addition, the hydrophilic portion may be one containing pores.
  • the hydrophobic portion formed in the porous membrane may be a hydrophobic polymer coated on the porous membrane, specifically, the hydrophobic polymer may be wax, Teflon, photoresist, silane compound, or polytetraflooroethylene, wax. , Poly dimethyl si loxane, silicon (si 1 i cone), metal nanoparticles, metal oxide nanoparticles, silica nanoparticles, polymer nanoparticles, or a combination thereof. It may be silver, copper, platinum, or the like. However, this is one embodiment of the present invention, and the present invention is not limited thereto, and a hydrophobic polymer that may be commonly used in the art suitable for the method of forming hydrophobic portions may be used.
  • One embodiment of the present invention discloses a method of printing a hydrophobic pattern on a porous membrane through a wax printing method as a hydrophobic polymer, but is not limited thereto.
  • the distance between each hydrophobic portion may be 1 mm or more and 9 mm or less, specifically 1 mm or more and 8 mm or less, 2 mm or more and 7 mm or less, 3 or more and 6 or less, 4 or more and 6 or less Or less than 4 mW and 9 mW or less, or 1 mW or more and 9 mW or less.
  • the distance between the hydrophobic regions is too narrow, there is a problem that the droplets located in the hydrophilic pattern are likely to collapse, and if the droplets are too wide, the number of droplets that can be formed per modern culture device is limited, so that high throughput analysis is possible.
  • the distance between the hydrophobic parts is satisfied, by securing the distance between the inside of the droplets, preventing the risk of overlap between the droplets, not only stable cell culture is possible, but also a large number of liquids. By forming enemies simultaneously, high throughput analysis is also available.
  • the present invention may further include a channel located in the porous membrane, the channel may be to have a hydrophilic, by connecting two or more hydrophilic portion is possible interaction between the hydrophilic portion and the droplet formed on the hydrophilic portion It may be to.
  • a conventional cell culture device including these channels By culturing the cells using a conventional cell culture device including these channels, it is possible to study the interaction of cultured cells inside different droplets.
  • the width of the channel may be greater than 0.1 mm 3 and less than 2.5 mm, specifically 0.1 mm or more and 1.5 mm or less, 0.1 mm or more and 1 mm or less, 0.3 mm or more and 2.0 mm or less, 0. It may be 1 mm or more and 0.7 mm or less, or 0.1 mm or more and 2.0 mm or less. If the width of the channel is too wide, it may be difficult to maintain the shape of the hydrophilic droplets due to the large capillary force, too narrow if the capillary force is difficult to move between fluids, if the above range is met, the appropriate capillary Due to the force, it is possible to maintain the shape of the hydrophilic droplets and to facilitate fluid-to-fluid movement through the channel.
  • PHDCXNet worked channel formed in accordance with an embodiment of the present invention Results in concentration gradient formation over time in channels of varying widths (PHDC, N-PHDC).
  • PHDC varying widths
  • N-PHDC interactable cell culture device
  • the concentration of fluorescent sodium salt can be determined by injecting a solution containing 0.5 mol / ml fluorescent sodium salt (f luorescein sodium sal t) into the central hydrophilic part (source part) and measuring the relative fluorescence intensity. This shows different diffusion coefficients through channels of various widths.
  • the length of the channel may be more than 0.5 mm 3 and less than 15 mm, by adjusting the length of the channel, it is possible to control the interaction between the hydrophilic portion and the droplet formed on the hydrophilic portion.
  • FIG. 10 is a result of utilizing the concentration gradient and drug test using N—PHDC in which a plurality of hydrophilic units are connected by a channel, which is an embodiment of the present invention.
  • E using the various channel widths, the effect of the 5FT 5-FLU0R0URACIL) anticancer drug of the three-dimensional spheroid was confirmed using PHDC and N-PHDC at various concentrations. After incubation for 48 hours to form a three-dimensional spheroid to the surrounding hydrophilic part, 5FU of 1000 mMo l concentration was injected into the central hydrophilic part (source part).
  • the antitumor effect of the three-dimensional tumor spheroid was confirmed by confirming the viability of tumor cells (vi abi li ty) by treating 5FU of 100, 500, and 1000 Mol e for 24 hours.
  • Various levels of cell death were identified by various concentrations of anticancer agents from intermediate sources, the lowest cell viability in 2 mm wide channels for 5FU anticancer agents, and the highest viability at 0.5 ⁇ . Seemed.
  • N-PHD Networked Paper Hanging Drop
  • the hydrophilic portion may further include a bio-passivat ion coating layer.
  • Non-specific binding of the porous membrane of the hydrophilic part and the protein or extracellular matrix used in cell culture can be prevented.
  • the bio-passivat ion coating layer may be to form a coating layer on an area including the surface of the hydrophilic portion and the pore inner surface without closing the pores of the hydrophilic portion.
  • the bio-passivat ion coating layer is bovine serum albumin (Bovine Serum Albumin, BSA), polyethylene oxide-polypropylene oxide-polyethylene oxide (poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide), PE0-PP0—PEO), polyethylene glycol (PEG), or agarose.
  • BSA bovine serum albumin
  • polyethylene oxide-polypropylene oxide-polyethylene oxide poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide)
  • PE0-PP0—PEO polyethylene glycol
  • PEG polyethylene glycol
  • the bio-passivat ion coating layer may have a thickness of 1 nm or more and 10 nm or less. If the coating thickness is too thick, there is a problem of changing the material properties of the hydrophilic part, and if too thin, the effect of preventing nonspecific binding of the protein or extracellular matrix may be insignificant.
  • the surface of the droplet in the hydrophilic portion may be a surface having a reduced hydrophilicity compared to the other surface. This can be done by performing a step of irradiating the hydrophilic part with ultraviolet rays.
  • the cells cultured in a modern cell culture device is a single cell line (pr imary cel ll ine) of the mammalian cells, cont inuous cel ll ine, or stem cells, tumor cells, immune cells, It may be a bacterium or a microorganism, but is not limited thereto.
  • the design of the hydrophobic portion pattern it is possible to form 1 to 384 spheroids in one cultivation device, and after the cell spheroid formation through the cultivation, an optional single spheroid can be obtained. have.
  • the cell culture device according to an embodiment of the present invention can be used by assembling a variety of dishes such as 35, 60, 90, 100 mm Petri dishes, 6, 12, 24, 48, 96, 384 well plate have.
  • FIG. 7 shows an image of a PHDC assembled in various kinds of dishes. Images of PHDC assembled into PC holders, 384 and 96wels assembled into PC holders, 3x3 PHDCs assembled into 60 mm Petri dishes and droplet images formed thereon, which facilitate PHPHs of various designs. It shows that it can be manufactured and assembled.
  • a cultivated cell culture device in which two or more cultivated cell culture units are stacked.
  • two or more culturing cell culture units are stacked, and the culturing cell culture unit comprises a hydrophobic part located in the porous membrane and a hydrophilic part enclosed by the hydrophobic part, and is positioned at the bottom
  • a cell culture device capable of performing cell culture by droplets of cell culture fluid being deposited on a hydrophilic portion of a cell culture unit, and exchanging of the culture medium in the droplets formed on the hydrophilic portion and injecting foreign substances through the porous membrane.
  • Each modern cell culture unit may be one or more hydrophilic moieties, and one or more hydrophobic moieties.
  • the at least one cultivated cell culture unit further comprises a channel located in the porous membrane, wherein the channel connects two or more hydrophilic parts, and the channel is connected between the hydrophilic parts connected by the channel. It may be to enable the interaction.
  • It provides a method for producing a conventional cell culture device comprising the steps of preparing a porous membrane, forming a hydrophilic portion and a hydrophobic portion in the porous membrane to prepare a cultivated cell culture unit, and stacking two or more of the cultivated cell culture unit.
  • the spatial arrangement of the cells can be controlled, and it was difficult to spatially culture various types of cells through the spatial arrangement in the existing conventional culture apparatus, but one of the present invention By virtue of embodiments it is readily possible to simulate the biome.
  • FIG. 9 is a schematic diagram of a method for manufacturing a conventional cell culture device according to an embodiment of the present invention.
  • the preparing of the extensible cell culture unit may include forming a channel connecting the hydrophilic part.
  • the stacking of two or more wise cell culture units may be to stack the wise cell culture unit by folding so that the hydrophilic portion of the upper layer and the lower layer of the fold cell culture unit overlap.
  • Existing cultivation Although it was difficult to demonstrate spatial culturing of various kinds of cells in a device through spatial arrangement, when using a conventional cell culture device according to an embodiment of the present invention, the spatial arrangement of cells can be controlled, and the interaction between cells can be simulated. You can do it more accurately.
  • the channel allows interaction between the hydrophilic portion and the droplets formed on the hydrophilic portion, thereby enabling the study of the interaction of cultured cells inside different droplets, as well as the study of the interaction between heterologous cells.
  • various types of cultivated cell culture devices can be manufactured, and can be utilized for various and effective cell interaction studies. .
  • the manufacturing of the cultivated cell culture unit may be performed by printing a hydrophobic part pattern on the porous membrane, and in one embodiment of the present invention, the method is used by printing wax on a paper that is a porous membrane having hydrophilicity.
  • the cell culture device was produced, and by the printing method, it is possible to easily manufacture the modern cell culture device.
  • the preparing of the cultivated cell culture unit comprises printing a pattern of a hydrophobic portion on a hydrophilic porous membrane using a hydrophobic polymer, heat-treating the porous membrane on which the hydrophobic polymer is printed, and culturing the cultivated cell on which the hydrophobic portion and the hydrophilic portion are formed. It may be through the step of obtaining a unit.
  • the heat treatment step; and further comprising the step of cutting the porous membrane can be.
  • FIG. 11 is a schematic diagram of the manufacturing process of the conventional cell culture device as an embodiment of the present invention.
  • Circular hydrophilic patterns with specific diameters of 2 ⁇ to 8 ⁇ are designed using commercially available software (corel draw x5).
  • the hydrophilic pattern is determined by the well plate (96 wells, 384 wells, 3x3 or 5x5) used in the experiment.
  • This design uses a Xerox solid wax printer (colorQube 8570) to print a hydrophobic polymer, Sol id wax, printed on the porous membrane according to the hydrophobic pattern, and to heat the solid wax printed porous membrane to melt the wax. To form a hydrophobic part through wax deformation.
  • the flowchart of the method for fabricating the conventional cell culture device of FIG. 11 is merely for illustrating the present invention, and the present invention is not limited thereto.
  • the hydrophilic portion surface treatment step may be further included after the step of washing the conventional cell culture unit.
  • the hydrophilic portion surface treatment step may be to treat the hydrophilic portion of the hydrophilic portion of the wise cell culture unit with 333 ⁇ 1011). This process can prevent non-specific adsorption of the biomaterial to the hydrophilic part.
  • the step of bio-passivation treatment is bovine serum albumin (Bovine Serum Albumin, BSA), polyethylene oxide—polypropylene oxide-polyethylene oxide (Poly (ethylene oxide) -IX) ly (propylene oxide) -poly (ethylene oxide), PP0—PE0 as PE, polyethylene glycol (PEG), agarose, and the like.
  • BSA bovine serum albumin
  • the hydrophilic portion surface treatment step is a hydrophilic portion of the conventional cell culture unit It may be ultraviolet irradiation. This process can control the hydrophilicity of the hydrophilic portion.
  • the hydrophilicity of the porous membrane can be adjusted.
  • the step of bio passivation (b i-pass ivat i on) and the step of irradiating ultraviolet light may be performed only one, or both steps may be performed.
  • the hydrophilicity of the hydrophilic portion can be increased by the step of bio passivation (bi o-pass ivat i on), and the hydrophilicity of the hydrophilic portion can be properly adjusted by reducing the hydrophilicity through ultraviolet irradiation. Therefore, when both the bio passivation treatment and the ultraviolet irradiation step are performed, the droplet retention time of the hydrophilic portion of the porous membrane may be significantly increased.
  • the hydrophilicity may be reduced by the ultraviolet irradiation treatment, when both the bio passivation treatment and the ultraviolet irradiation step are performed, the non-specific adsorption reduction effect of the biomaterial may also be remarkably improved.
  • FIG. 12 is a schematic view of the surface treatment step of the hydrophilic part according to an embodiment of the present invention, the step of sterilizing the cell culture unit with ethanol, bio passivation using the BSA solution in the hydrophilic part (bi o-pass i vat i on) processing, washing with PBS, and ultraviolet irradiation.
  • Preparing a cell culture device Preparing a cell culture device according to an embodiment of the present invention, dropping the cell culture medium containing the cells to be cultured to the hydrophilic portion to form a droplet of the cell culture medium, the cell culture medium dropping the culture medium is dropped Suspending droplets of cell culture, culturing the cells with droplets of cell culture,
  • the step of culturing the cells provides a cultivation method further comprising the step of exchanging the culture solution through the porous membrane.
  • FIG. 14 shows a schematic diagram of a method for culturing cells according to an embodiment of the present invention.
  • Another embodiment of the present invention may further comprise the step of injecting an external material through the porous membrane after the step of culturing the cells.
  • FIG. 15 shows the results of a chemotactic reaction in the concentration gradient of the three-dimensional spheroid in N-PHDC as an embodiment of the present invention.
  • FIG. Chemistry about the FBS of the three-dimensional spheroid was confirmed for 24 hours using N-PHDC, which is an embodiment of the present invention, through which the present invention can be used for experiments on spheroid changes caused by the influence of external chemicals. Confirmed that it can.
  • Figure 15 A the concentration gradient simulation results of the chemical reagents after 24 hours in a 0.5 ⁇ wide channel are shown. The quality of the solution was gradually blue, and the left area of the reagent injection was the highest red color, the middle area was blue, and the right area was dark blue.
  • 15B compares the experimental results and the simulation results of chemical reagents in channels 0.5 mm, 1.0 mm and 2.0 mm wide.
  • 15 C, D, E confirmed the chemotaxis of the cells according to the concentration gradient between 0% FBS and 10% FBS, showed a strong chemotaxis of the cells with a high concentration of FBS.
  • FIG. 16 shows the results of experiments on general cells (CCD 1058sk) and tumor cells (MDA-MB 231) drug test and immuno reaction against anticancer drugs (5FU) using PHDC, which is an embodiment of the present invention.
  • the bobbin cell culture device includes a channel, and forming droplets of the cell culture fluid is one or more. Dropping in to form droplets of the cell culture medium, and culturing the cells is a conventional cell culture method for culturing cells while interacting with each cell spheroid through the channel while the droplets of the cell culture solution are suspended. To provide.
  • a culture sheet including a plurality of culturing cell culture units, a driving motor for moving the culture sheet, and positioned above the culture sheet, A cell culture solution injecting device for dropping the cell culture solution into the hydrophilic part of the cell culture unit,
  • Each of the cultivated cell culture units includes a porous membrane, a hydrophobic portion located in the porous membrane, and a hydrophilic portion surrounded by the hydrophobic portion, and droplets of the cell culture solution are suspended in the hydrophilic portion to perform cell culture.
  • the present invention provides an automatic cell culture automation device capable of exchanging a culture solution in a droplet formed in the hydrophilic part and injecting an external substance through the porous membrane.
  • the culture sheet has a conveyor belt structure, it may be to move in the form of a chain up and down.
  • the cell culture solution may be dropped into the hydrophilic portion of the culture unit to form droplets from the cell culture solution input device.
  • droplets formed on the hydrophilic portion of the culture unit may be suspended by gravity and cell culture may be performed.
  • a reagent may be introduced into the droplets.
  • PHDC and N-PHDC can use a rotating system to automate imaging and analysis of cell injection, spheroid culture, and spheroid phenomena.
  • a small conveyor belt rotating pul ly
  • This system automates the movement and formation of droplets, and has a configuration that rotates from the upper layer to the lower layer, and from the lower layer to the upper layer, and drops the cell culture liquid onto the hydrophilic portion to form droplets.
  • Example 1 The present cell culture apparatus of Example 1, wherein the hydrophilic portion is patterned by printing with a wax as a hydrophobic polymer on a chromatographic filter paper, heating the wax at 120 0 C for 2 minutes, and forming a hydrophobic portion. was prepared.
  • Example 2 In order to control the hydrophilicity of the hydrophilic portion of Example 1 was carried out UV irradiation treatment for exposing the ultraviolet light of 18.52 mW / cm 2 intensity for 3 minutes to prepare a cell culture device of Example 2.
  • the hydrophilic part was applied in 1% bovine serum albumin (BSA), for one hour After treatment,-washed three times with phosphate-buffered saline (PBS) buffer, dried, and then to control the hydrophilicity of the hydrophilic part.
  • BSA bovine serum albumin
  • Ultraviolet irradiation treatment was performed for 3 minutes of ultraviolet light of 18.52 mW / cm 2 century to prepare a cell culture device of Example 3.
  • Cell culture droplets were then formed in the conventional cell culture device of each example and fixed in a 3D printed holder.
  • FIG. 12 shows a flow chart of the porous membrane treatment method for the cultivation of the cultivated cells, which is merely to illustrate the present invention, and the present invention is not limited thereto.
  • Fig. 13 shows the droplet retention time before and after the BSA and UV irradiation treatment on the hydrophilic part; And experimental results for protein nonspecific binding.
  • Example 1 in which the surface treatment of the hydrophilic portion was not performed, the droplet retention time was less than 1 minute, but when subjected to ultraviolet irradiation treatment (Example 2), the droplet retention time was increased to less than 10 minutes than before the ultraviolet irradiation treatment, and the BSA In the case where both treatment and ultraviolet irradiation treatment were performed (Example 3), it was confirmed that the droplet retention time was significantly improved by exceeding 10 minutes.
  • Example 4 in order to prevent protein-specific binding in a material that can interfere with nutrient supply through the medium and intercellular signal communication, the degree of protein nonspecific binding was confirmed using fluorescent streptavidin (st reptavidin).
  • st reptavidin fluorescent streptavidin
  • PHDC was prepared using a 200 ⁇ GE whatman Chrome 1 f i lter paper, and 2, 000 tumor cells were cultured in 30 ⁇ 1 droplets over 4 mm diameter to form tumor spheroids at various culture dates. 17 shows an example of tumor cell spheroid formation according to various culture dates using PHDC.
  • Example 7 is a schematic diagram of imaging a spheroid using a microscope, and shows a spheroid-specific image and fluorescence image of breast cancer cells at 1.25 magnification.
  • cells in the PHDC droplets can form three-dimensional spheroids. You can check it.
  • Example 5
  • MCF 10A Breast epithelial cells
  • MDA MB 231 breast cancer cells
  • MCF 10A DMEM F12 culture medium
  • MDA—MB 231 cells DMEM culture medium containing 10% FBS and 1% col lagen was used to remove 5 ⁇ 1 of the droplets per day. Spheroid formation for 5 days while injecting.
  • FIG. 18 is a comparison result between spheroids cultured in PHDC and spheroids cultured in Petr i-dish.
  • FIG. 18A Comparing the spheroids grown in Petri dish with one of the existing cultivation techniques and the spheroids grown in PHDC, similar trends in morphology and viability as shown in Figs. 18A and B are shown. Showed. Figure 18C confirmed the metastatic capacity of the spheroid cultured in PHDC through F-act in staining, a marker related to the viability of the spheroid cultured in PHDC through Calcein-am staining and tumor cell metastasis. As shown in FIG.
  • TGF- ⁇ secretion known as a growth factor of tumor cells As a result of comparing TGF- ⁇ secretion known as a growth factor of tumor cells according to the number of cells and the date of culture in PHDC, spheroids formed with a large number of cells over the culture date were formed with a small number of cells. It can be seen that the secretion of TGF- ⁇ is secreted over the culture date than spheroids.
  • MCF-7 tumor cells
  • CCD 1058 fibroblasts
  • the spheroid diameter according to the culture time was compared for 10 days at 2 days intervals, depending on the cell type, and According to the cell culture method, the tendency of the spheroid diameter change was confirmed.
  • a cultivated cell culture device comprising a channel prepared by a stacking method of folding so that the hydrophilic portion of the upper layer and the lower layer of the two cultivated cell culture units overlap.
  • Breast cancer cells (MDA-MB 231) are cultured in a single culture without interaction with other cells, and breast cancer cells (MDA—MB 231) are connected to one hydrophilic part that can be channeled and interact with fibroblasts (MDA-MB 231). Electron scanning microscope image for comparing the cultured cell binding state after the culture of MEF) is shown in Figure 20.
  • the first layer of culturing cell culture unit (B) had a hydrophilic and hydrophobic pattern for cell culturing, and the second layer of culturing cell culture unit (A) had a channel (X, y, z) connecting the hydrophilic part. Is further formed.
  • the two cultivated cell culture units (A, B) were folded and stacked so that the hydrophilic portions overlapped, and the hydrophilic portions (2, 5, 8) had breast cancer cells (MDA-MB 231) in the hydrophilic portion (1, 4, 7) cultivates fibroblasts (MEFs), each of which has a network that can induce interactions between heterogeneous spheroids. Formed.

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Abstract

L'invention concerne un dispositif de culture cellulaire à goutte suspendue pouvant échanger un milieu de culture et injecter une substance étrangère, un procédé pour sa fabrication et un procédé de culture cellulaire l'utilisant.
PCT/KR2017/011043 2016-10-07 2017-09-29 Dispositif de culture cellulaire à goutte suspendue utilisant une membrane poreuse, procédé pour sa fabrication, procédé de culture cellulaire à goutte suspendue l'utilisant et dispositif d'automatisation de culture cellulaire à goutte suspendue WO2018066953A1 (fr)

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