WO2022258037A1 - 一种培养装置及基于培养装置的培养方法 - Google Patents

一种培养装置及基于培养装置的培养方法 Download PDF

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Publication number
WO2022258037A1
WO2022258037A1 PCT/CN2022/098031 CN2022098031W WO2022258037A1 WO 2022258037 A1 WO2022258037 A1 WO 2022258037A1 CN 2022098031 W CN2022098031 W CN 2022098031W WO 2022258037 A1 WO2022258037 A1 WO 2022258037A1
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Prior art keywords
culture
plate body
hole
culture device
collection
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PCT/CN2022/098031
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English (en)
French (fr)
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王玄
王春峰
陈睿
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上海睿钰生物科技有限公司
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Publication of WO2022258037A1 publication Critical patent/WO2022258037A1/zh

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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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • 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
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/01—Drops
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion

Definitions

  • This description relates to the technical field of cell culture, in particular to a culture device and a culture method based on the culture device.
  • Cell culture refers to simulating the in vivo (for example, human or animal) environment (for example, sterility, suitable temperature, pH and certain nutritional conditions, etc.) in a culture chamber, so that the culture can survive, grow, reproduce and maintain the main structure and a method of function.
  • the culture (that is, the sample of cell culture) can be placed in the culture chamber, and the culture medium supplying nutrients can be added into the culture chamber, and the culture can reproduce and grow by absorbing the nutrients in the culture medium.
  • This specification provides a culture device capable of stably generating liquid droplets or hanging drops for culturing.
  • the embodiment of the description provides a culture device, which includes: a first plate body and a second plate body that can be separated; the first plate body is provided with a collection hole and a barrier structure that are sequentially arranged along a preset direction, and the collection The holes are used to accommodate the cultures for generating droplets; the second plate body is provided with droplet generating holes for generating droplets, and the droplet generating holes include matching parts and generating parts arranged in sequence along the preset direction; when the first plate When the body is mated with the second plate body, the collection hole is inserted into the mating portion, and a part of the barrier structure is in contact with the inner surface of the mating portion.
  • the barrier structure is a network structure formed by intersecting fibrous or strip structures, and the network structure includes at least two meshes, wherein the largest mesh unit area is ⁇ 1/2 the cross-sectional area of the barrier structure .
  • the height of the pooling hole along the predetermined direction is less than or equal to ⁇ *l*sin ⁇ /S ⁇ g; where ⁇ represents the surface tension coefficient of the culture liquid at the air interface, l represents the perimeter of the barrier hole, and S represents the barrier
  • ⁇ represents the surface tension coefficient of the culture liquid at the air interface
  • l represents the perimeter of the barrier hole
  • S represents the barrier
  • the pore area, the contact angle ⁇ of the culture liquid on the surface of the material represents the wettability, ⁇ is the density of the culture liquid, and g is the acceleration of gravity.
  • the height of the generating part along the predetermined direction is greater than ⁇ *l 1 *sin ⁇ 1 /S 1 ⁇ g; where ⁇ is the density of the culture liquid, g is the acceleration of gravity, and ⁇ represents the velocity of the culture liquid at the air interface Surface tension coefficient, l 1 represents the pore diameter of the end of the generating part away from the mating part, S 1 represents the orifice area of the end of the generating part far away from the mating part, ⁇ 1 represents the contact angle of the culture liquid on the surface of the material.
  • the culture liquid here refers to the commonly used in the art. cell culture medium.
  • a second culture solution inlet and outlet are opened on the cover plate, and the second culture solution inlet and outlet are provided through the first plate body and the second plate body, and communicate with the accommodation space, so that the culture medium can enter the accommodation space.
  • the first plate is removed, and a culture solution is added to the hole of the second plate, so that the top of the hanging drop is covered by the culture solution, and no culture solution is added to the containing space.
  • the embodiment of this specification also provides a cultivation method based on a culture device, which is used for culturing based on the above-mentioned culture device, including: feeding culture into the confluence hole of the first plate body; connecting the first plate body and the second plate body The body cooperates so that the culture in the pooling hole passes through the barrier structure and enters the droplet generating hole to generate hanging drops.
  • the embodiment of this specification also provides an application of the above-mentioned culture device and/or the above-mentioned culture method in the field of 2D cell or 3D cell culture.
  • Figure 1 is a schematic structural view of a culture device according to some embodiments of the present specification.
  • Figure 2 is a cross-sectional view of a culture device according to some embodiments of the present specification.
  • Fig. 3 is a partial enlarged view of part A in Fig. 2;
  • Fig. 4 is a schematic structural view of a culture device according to other embodiments of the present specification.
  • Fig. 5 is a schematic diagram of a culture method based on a culture device according to some embodiments of the present specification.
  • system means for distinguishing different components, elements, parts, parts or assemblies of different levels.
  • the words may be replaced by other expressions if other words can achieve the same purpose.
  • the culture device involved in the embodiment of this specification can be used for 2D cell culture or 3D cell mass culture.
  • 2D cell culture refers to the growth and propagation of the culture attached to the substrate of the culture chamber in the form of adhesion, or growth and propagation in the culture medium in the form of suspension.
  • 3D cell culture may refer to scaffold-based culture, and scaffold may refer to natural materials (such as natural hydrogels (such as protein-based natural hydrogels) Matrigel)) or synthetic materials such as synthetic gels (such as polyethylene glycol hydrogel), Matrigel can provide support for aggregation, proliferation and migration of cultures, that is, provide growth support for cultures.
  • the three-dimensional environment maintains the culture in a state of growing in the three-dimensional environment, and finally obtains a three-dimensional cell culture product (eg, an organoid).
  • matrigel can be used as a carrier for 3D cell culture.
  • the cultured cells can be mixed with liquid matrigel, and the liquid matrigel can undergo a phase transition under certain conditions, from liquid to solid state, and the solid matrigel can provide a three-dimensional environment for cell growth, and cells can grow in such a three-dimensional environment.
  • the environment can grow and migrate in all directions.
  • Matrigel may be a temperature-sensitive gel, and the temperature-sensitive gel may undergo a phase transition with temperature changes, for example, gradually changing from a liquid state to a solid state or from a solid state to a liquid state.
  • the cells are mixed in the liquid temperature-sensitive gel.
  • the temperature-sensitive gel changes from liquid to solid, the temperature-sensitive gel can provide the cells with a three-dimensional growth environment for subsequent three-dimensional cell culture.
  • 3D cell culture can also refer to culture without support, which relies on specialized culture vessels or promotes the self-aggregation of cells by attaching cells to the surface of the vessel, such as based on the forced floating method and the hanging drop method. cultivation.
  • Organoid culture usually needs to rely on the support of matrigel-like scaffold materials. Organoids are suspended in matrigel liquid and dropped into the culture container for cultivation. Due to the effect of gravity, the organoids are easy to settle to the culture container before the matrigel solidifies. In the process of cell expansion and growth, the attachment of organoids to the surface wall of the culture container will form a 2D adherent growth layer, which is not conducive to the development and growth of 3D tissues. Therefore, adherent growth should be avoided as much as possible during organoid culture.
  • the hanging drop method is a method of cultivating cells by using suspended droplets. It is generally used to form and cultivate 3D cell clusters.
  • the suspended droplets are not in contact with the culture container, which can avoid the growth of cells in the droplets and solidify the hanging droplets.
  • the nutrients needed for cell culture are limited, and the culture medium is needed to maintain the culture.
  • the hanging drop method needs to drop the mixed liquid successively to form multiple hanging drops, which reduces the efficiency of cell culture and may also lead to the failure of cell culture.
  • the cultured mouse intestinal organoids and mouse lung organoids are prepared; the mouse intestinal organoids and mouse lung organoids are digested and diluted with medium, and then added with an equal volume of the medium Matrigel, mixed evenly; the cultures of the experimental group were inoculated in the culture device involved in the embodiments of this specification, and the cultures of the control group were inoculated in ordinary culture plates; during the culture process, the growth state of the cells in the experimental group and the control group was observed with a microscope; After culturing for 5 days, the cell viability of the experimental group and the control group was tested by chemiluminescence. Among them, the cells in the experimental group grew in the hanging droplets.
  • the culture device involved in the embodiments of this specification has the potential to improve cell culture viability and is beneficial to maintain a three-dimensional cell growth state.
  • the culture referred to in the embodiments of this specification can be understood as the object of cell culture.
  • the culture includes at least a cell sample that can be used for cell culture, also referred to as cultured cells.
  • the culture may consist of cells mixed with Matrigel.
  • the culture can also be formed by mixing cells and culture fluid.
  • the culture can be liquid.
  • the culture can be a single cell mixed with a culture solution or Matrigel, or a plurality of cells can be mixed with a culture solution or Matrigel.
  • the plurality of cells can be a plurality of dispersed cells, or a cell mass.
  • the cell mass may include a cell sphere formed by directly mixing multiple cells, or may include a cluster formed by aggregating multiple cells.
  • a culture device with one or more holes capable of forming droplets is usually used for culturing, but during the preparation of droplets, the culture (or Matrigel) is in a fluid
  • the culture or Matrigel
  • the surface tension of the droplet is often abnormal or the volume is too high due to the tangential force generated by the flow of the culture (or matrigel). Larger values lead to premature dripping of droplets or failure of hanging drop formation, which in turn leads to culture failure.
  • the culture device may include a first plate body and a second plate body that are separably arranged, and the first plate body may be provided with a collection hole and a barrier structure arranged in sequence along a predetermined direction, and the collection The holes are used to accommodate cultures for generating droplets.
  • Droplet generating holes for generating droplets may be opened on the second plate body.
  • the droplet generating holes include matching parts and generating parts arranged in sequence along a preset direction.
  • the first plate body can cooperate with the second plate body, and the matching part can be used to cooperate with the collection well, and the culture is added dropwise to the collection well, and the barrier structure inside the collection well can prevent the culture form from forming droplets in advance
  • the first plate body is matched with the second plate body
  • the pooling hole is inserted into the matching part
  • a part of the blocking structure can contact the inner surface of the matching part
  • the cultures blocked by the blocking structure in the pooling hole can be generated by droplets
  • the pores form droplets.
  • the culture device involved in the embodiment of this specification can be used to avoid the phenomenon that the culture (or matrigel) is added to the collection hole to form a droplet, and the culture can form a droplet or a hanging drop in the droplet generation hole uniformly, It is convenient for further collection and cultivation.
  • Fig. 1 is a schematic structural view of a culture device according to some embodiments of the present specification.
  • Figure 2 is a cross-sectional view of a culture device according to some embodiments of the present specification.
  • Fig. 3 is a partial enlarged view of part A in Fig. 2 .
  • the culture device 100 may include a detachable first plate body 110 and a second plate body 120 , which include a separated state as shown in FIG. 1 and a mated state as shown in FIG. 2 .
  • the first plate body 110 may include one or more collection holes 111 and barrier structures 112 arranged sequentially along a predetermined direction.
  • the collection holes 111 may be arranged along a predetermined direction.
  • the preset direction of the collection hole 111 may be the direction of gravity on the culture.
  • the pooling well 111 can be used to hold the culture that produces the droplets.
  • the collection holes 111 may have the same or different cross-sectional shapes at any position, for example, the collection holes 111 may be formed in a cylindrical structure, a rectangular parallelepiped structure or other irregular structures. Exemplarily, the cross-sectional shape at any position in the collection hole 111 may be circular. In some embodiments, the collection hole 111 may have the same or different cross-sectional area at any position. For example, the collection hole 111 may be formed to have an hourglass-shaped, spindle-shaped or other irregular-shaped outer contour structure, which is not described herein. limit.
  • the cross-sectional area of the collection hole 111 along a preset direction gradually decreases.
  • the collecting hole 111 has a frustum-shaped outer contour on the side close to the second plate body 120 in the predetermined direction, and the diameter of the collecting hole 111 gradually increases in the direction close to the second plate body 120 in the predetermined direction. decrease.
  • the culture can flow into the confluence hole 111 away from the side of the barrier structure 112 , and the culture flows in the confluence hole 111 toward the barrier structure 112 along a predetermined direction, and is blocked by the barrier structure 112 .
  • the barrier structure 112 can be arranged at the end of the confluence hole 111 away from the inflow of the culture. In some embodiments, the barrier structure 112 can be used to restrict the flow of the culture (for example, by forming surface tension to block the liquid), that is, the barrier structure 112 can be used to prevent the first plate body 110 from pooling before mating the second plate body 120 The culture contained in the well 111 for the formation of droplets flows out through the barrier structure 112 . In some embodiments, the barrier structure 112 can prevent the culture from falling, that is, the culture can be carried on the barrier structure 112 .
  • the bearing capacity of the barrier structure 112 to the culture can be the vertical ratio between the gravity of the culture and the maximum flow tangential force. force in the vertical direction. It should be noted that the tangential force is related to the type of culture and the flow rate, so the specific carrying capacity of the barrier structure is not limited in this specification.
  • the barrier structure 112 may include at least one fibrous or strip-like structure arranged radially along the collection hole 111. After the culture contacts with the barrier structure 112, the barrier structure 112 may divide the contact surface between the culture and the into at least two parts.
  • the barrier ability of the barrier structure 112 to the culture may be related to the size of the mesh formed by the fibrous or strip-like structures.
  • the barrier ability of the barrier structure 112 to the culture may be linearly related to the perimeter and area of the largest mesh formed by the fibrous or strip-like structure.
  • the wettability of the surface of the barrier structure 112 to the culture and the density of the fibrous or strip-like structures can determine the load-bearing capacity of the barrier structure.
  • the barrier structure can be a network structure formed by intersecting fibrous or strip structures, the network structure includes at least two meshes, wherein the largest mesh unit area is ⁇ 1/2 the cross-section of the barrier structure area.
  • the barrier structure 112 may be a structure formed by non-intersecting arrangement of fibrous or strip-like structures.
  • the longest diameter of the cross-section of the fibrous or strip-like structure may range from 10 ⁇ m to 1 mm.
  • the longest diameter of the cross-section of the fibrous or strip-like structure may be 10 ⁇ m. In some embodiments, the longest diameter of the cross-section of the fibrous or strip-like structure may be 1 mm. In some embodiments, the longest diameter of the cross-section of the fibrous or strip-like structure may range from 50 ⁇ m to 0.1 mm.
  • the barrier structure 112 may be a porous structure, and the barrier structure 112 has a plurality of barrier holes 1121 , such as a plate-shaped structure with a plurality of holes.
  • the barrier ability of the barrier structure 112 to the culture can be related to the density and size of the barrier pores.
  • the barrier ability of the barrier structure 112 to the culture can be linearly related to the perimeter and area of the largest barrier hole.
  • the barrier holes 1121 on the barrier structure 112 are evenly distributed.
  • the multiple blocking holes 1121 on the blocking structure 112 may have the same hole area.
  • the areas of the plurality of barrier holes on the barrier structure 112 may be different.
  • the area of the barrier hole can affect the barrier ability of the barrier structure 112 to the culture. The larger the area of the barrier hole, the smaller the barrier capacity of the barrier hole to the culture.
  • the material of the barrier structure 112 includes a structural material and a surface material coated on the surface of the structural material.
  • the structural material of the barrier structure 112 may include at least one of the following: including nylon, polypropylene, polyester, polyethylene (PE), polypropylene (PP), polystyrene, polyamide (PA), Polymer materials such as polymethyl methacrylate and polyethylene terephthalate (PET).
  • the structural material of the barrier structure 112 may include at least one of the following: stainless steel, copper, platinum, gold and other stable metals or alloys.
  • the structural material of the barrier structure 112 may be a memory alloy, which has a shape memory effect.
  • the structural material of the barrier structure 112 may be nickel-titanium alloy, which is a type of memory alloy.
  • the barrier ability of the barrier structure 112 to the culture may be related to the surface material of the barrier structure 112 .
  • the side of the barrier structure 112 close to the second plate body 120 is covered with a hydrophilic material.
  • the barrier ability of the barrier structure 112 to the culture can be linearly related to the surface tension coefficient of the culture at the air interface.
  • it in the process of the culture flowing into the collection hole 111, in order to prevent the culture in the collection hole 111 from falling through the barrier structure, it can be realized not only by determining the barrier capacity of the barrier structure 112 to the culture, but also by It is realized by determining the structural size of the collecting hole 111 .
  • the structural size of the collection hole 111 can be determined according to the following process:
  • the liquid pressure P1 in the collection hole 111 is:
  • ⁇ is the liquid density of the culture
  • g is the acceleration of gravity
  • h represents the highest liquid level that the culture can form in the pooling hole.
  • the situation of the mesh and the barrier hole is similar, and the pressure generated by the culture at the mesh or barrier hole of the barrier structure 112 (usually based on the relevant data of the mesh or barrier hole with the largest cross-sectional area) P2 is :
  • ⁇ represents the surface tension coefficient of the culture liquid at the air interface
  • l represents the perimeter of the mesh or barrier hole
  • S represents the area of the mesh or barrier hole
  • the liquid pressure P 1 is less than the pressure P 2 generated by the culture at the grid or barrier holes of the barrier structure 112 , and the culture will not flow out through the barrier structure 112 .
  • the culture will flow out through the mesh or barrier hole of the barrier structure.
  • the contact angle ⁇ between the hole and the surface of the material increases gradually, and when ⁇ reaches the maximum advancing contact angle, ⁇ no longer increases.
  • the height of the collection hole 111 along the direction of gravity of the culture can be less than or equal to ⁇ *l*sin ⁇ /S ⁇ g, where 90 ° ⁇ 180°; preferably, 90° ⁇ 160°; the value range of ⁇ is 0.02-0.08N/m; the value range of ⁇ is 0.8-2.0g/cm3.
  • the end of the collection hole 111 away from the barrier structure 112 may also be provided with a diversion area 113, and the flow diversion area 113 can be used to guide the culture to the collection hole 111.
  • the diversion area 113 may be connected to the wall of the collecting hole 111 .
  • the diversion area 113 can be gradually gathered in the direction of the converging hole, forming a small horizontal cross-sectional area in the direction close to the second plate body 120 and a large horizontal cross-sectional area in the direction away from the second plate body 120, A structure that resembles a funnel-shaped outer profile.
  • the diversion area 113 may have a frustum-shaped outer contour, and the diameter of the diversion area 113 gradually decreases toward the collection hole.
  • the deflector area may include a deflector arranged obliquely, and the bottom of the deflector in the direction toward the confluence hole is connected to the confluence hole 111 .
  • the diversion area 113 may be a rounded structure that is away from the barrier structure 112 and connected to the side of the confluence hole 111 , and the diversion effect is achieved by the area formed by the rounded structure inclined toward the confluence hole 111 .
  • the flow guide area can also be formed in other structures, such as a helical structure, etc., which are not limited here.
  • the diversion area 113 may be covered with a hydrophilic material, which is beneficial for the culture to flow to the collection hole 111 through the diversion area 113 , so as to optimize the diversion effect of the diversion area 113 .
  • the hydrophilic material may include but not limited to hydrophilic cotton, hydrophilic fiber or hydrophilic leather, etc. or any combination thereof.
  • the flow guide area 113 may not be covered with hydrophobic material or covered with other materials, such as.
  • a plurality of collection holes 111 are opened on the first plate body 110 , and an overflow groove 114 is opened between adjacent collection holes 111 .
  • the overflow tank 114 is used to limit the volume of culture in the sump well 111 to no greater than that required to generate droplets.
  • the overflow groove 114 may communicate with adjacent collection holes 111 .
  • the overflow groove 114 may be sequentially connected with the diversion area 113 and the collection hole 111 and arranged through it.
  • a desired total amount of culture can be added to any pooling well 111 of several pooling wells 111 by multiplying the number of droplets required by the number of droplets required to generate a single droplet.
  • the volume of the culture is obtained by calculation.
  • the pooling hole 111 where the culture enters is filled with the volume of culture required to generate a single droplet, and then the excess culture overflows, and the overflowing culture flows into the adjacent pooling hole through the overflow tank 114 111, until there is a culture no larger than the volume required to generate a single droplet in each pooling hole 111 on the first plate body 110, during this period, the barrier structure 112 maintains a barrier to the culture in the pooling hole 111, The culture in the pooling well 111 is prevented from forming droplets and falling.
  • the pooling holes 111 on the first plate body 110 can be connected in groups, for example, the nine pooling holes 111 on the first plate body are divided into three groups, and the same The collection holes 111 in a group are connected through the overflow groove 114, and the collection holes 111 in different groups are not connected with each other.
  • different cultures are added dropwise or injected into the cultures in batches into the collection wells 111 connected in groups, so as to form a control culture with different culture components.
  • the culture device 100 may also be provided with a culture inlet 115 , and the culture inlet 115 is used to feed the culture solution into the collection hole 111 on the first plate body.
  • the culture access port 115 can communicate with any of the collection wells 111 .
  • multiple culture inlets 115 can be set on the culture device 100, and the multiple culture inlets 115 can be used to feed the same culture into any collection hole 111 at the same time to improve the collection efficiency, or multiple The culture inlet 115 can be used to feed different cultures into the connected collection holes 111 in groups, which is convenient for control experiments.
  • the culture inlet 115 may be provided on the first plate. In some embodiments, the culture inlet 115 may be disposed on the side wall of the first plate body 110 and communicate with any collection hole 111 of the first plate body 110 . In some embodiments, two culture inlets 115 can be set on the first plate body 110, one culture inlet 115 is arranged at a distance from the second culture inlet 115 along the direction in which the culture flows through the overflow tank 114 the furthest position.
  • the second plate body 120 is provided with a droplet generating hole 121 for generating a droplet, and the droplet generating hole 121 is used to receive a culture to form a droplet.
  • the droplet generating hole 121 includes a matching portion 1211 and a generating portion 1212 sequentially arranged along a preset direction, and the preset direction is the same as the preset direction on the first plate.
  • the first plate body 110 is matched with the second plate body 120, the collection hole 111 is inserted into the matching portion 1211, a part of the blocking structure 112 is in contact with the inner surface of the matching portion 1211, because a part of the blocking structure 112 is in contact with the matching portion 1211 Contact between the inner surfaces of the part 1211 generates intermolecular attraction, so that the culture in the collection hole 111 on the barrier structure 112 flows into the droplet generation hole 121 and forms a droplet in the generation part 1212 .
  • the first board 110 and the second board 120 are arranged along a predetermined direction.
  • the inner wall of the droplet generating hole 121 may be covered with a hydrophilic material, which may be the same as or different from the hydrophilic material in the flow diversion area.
  • the thickness of the barrier structure 112 along the predetermined direction may be small, and/or, the barrier structure 112 is close to the second One side of the plate body 120 may be covered with a hydrophilic material.
  • the matching part 1211 can be used to cooperate with the collection hole 111 to accept the culture in the collection hole.
  • the matching portion 1211 can accommodate a part of the collection hole close to the barrier structure 112 .
  • the inner contour of the matching portion 1211 can be adapted to the outer contour of the collection hole 111 .
  • the shape of the inner contour of the mating portion may be the same as the shape of the outer contour of the collecting hole, so that when the first plate body 110 and the second plate body 120 are mated, the fitting portion 1211 fits against the inner wall of the collecting hole 111 ,
  • the inner contour shape of the matching portion 1211 and the outer contour shape of the collecting hole 111 are both truncated circular shapes.
  • the inner contour shape of the matching portion 1211 may be different from the outer contour shape of the collecting hole 111, for example, the inner contour shape of the fitting portion 1211 may be a cylindrical shape, and the outer contour shape of the collecting hole 111 may be a truncated cone shape.
  • the matching part 1211 includes a platform structure 1213, the platform structure 1213 is arranged between the matching part 1211 and the generating part 1212, the blocking structure 112 divides the collection hole 111 into at least two blocking holes, the first plate When the 110 is mated with the second plate body 120 , a part of at least one blocking hole is in contact with the platform structure 1213 .
  • the contact between the barrier structure 112 and the platform structure 1213 generates intermolecular attraction, so that the culture in the collection hole 111 on the barrier structure 112 flows into the generation part 1212 through the barrier structure 112 .
  • the platform structure 1213 may be in contact with the barrier holes of the partial barrier structure 112 .
  • the platform structure 1213 may be formed as a protrusion protruding from the mating portion, and when the first plate body is mated with the second plate body, the protrusion abuts against a part of the barrier structure 112 .
  • the platform structure may be formed as a plurality of protrusions protruding from the mating portion.
  • the platform structure 1213 can be formed as an annular boss protruding from the matching portion 1211. When the first plate body 110 is mated with the second plate body 120, the annular boss can abut against the peripheral portion of the barrier structure 112. catch.
  • the platform structure 1213 in order to improve the attractiveness of the platform structure 1213 to the culture, can be made of a hydrophilic material, or the platform structure 1213 can be covered with a hydrophilic material.
  • the generation section 1212 may be used to form droplets.
  • the generating part 1212 can be configured such that its end away from the mating part 1211 is used to form droplets.
  • the generating part 1212 can be configured such that its end away from the mating part 1211 is used to form a hanging drop, and the hanging drop containing cells is suspended from the end of the generating part 1212 away from the mating part 1211 for cell culture.
  • the end of the generating part 1212 away from the mating part 1211 has a circular outer contour.
  • the generating part 1212 may have a frustum-shaped inner contour, and the inner diameter of the generating part 1212 gradually increases in a direction away from the mating part 1211, so as to further promote the formation of hanging droplets or liquid droplets.
  • the inner wall of the generating part 1212 may be covered with or made of hydrophilic materials.
  • a plurality of droplet generating holes 121 may be provided on the second plate body 120 , and the droplet generating holes 121 may be arranged in one-to-one correspondence with the collection holes 111 on the first plate body 110 .
  • the distance between ends of adjacent generating portions 1212 away from the mating portion 1211 is not less than a first preset distance threshold.
  • the first preset distance threshold may be 2 mm.
  • the distance between ends of adjacent generating portions 1212 away from the mating portion may be 3mm.
  • the distance between the ends of adjacent generating parts 1212 away from the mating part 1211 may be different. In some embodiments, the distance between ends of adjacent generating portions 1212 away from the mating portion 1211 may be less than 2 mm and greater than 1 mm.
  • the structural size of the generating part 1212 in order to allow the culture in the droplet generating hole 121 to flow out from the end of the generating part 1212 away from the mating part 1211, the structural size of the generating part 1212 can be determined according to the above-mentioned method for determining the structural size of the collecting hole 111.
  • the culture can flow out through the orifice at the end of the generating part 1212 away from the mating part 1211 to form droplets.
  • the height of the generating part along the gravity direction of the culture can be greater than ⁇ *l 1 *sin ⁇ 1 /S 1 ⁇ g.
  • the culture in the droplet generating hole 121 in order to allow the culture in the droplet generating hole 121 to form a hanging drop at the end of the generating part away from the mating part 1211, it is necessary to prevent the drop formed by the culture flowing out of the generating part 1212 from breaking.
  • the substance moves down in the generating part until the orifice at one end of the generating part 1212, and then with the increase of the liquid volume, the culture liquid gradually assumes a hemispherical shape, and may constrict or break.
  • whether the droplets formed from the generating part 1212 are broken depends on the volume, viscosity and surface tension of the droplets.
  • the correction coefficient (has nothing to do with liquid surface tension, material properties, liquid density and viscosity, etc., and is related to the volume of the liquid and the radius (R) of the lower opening, ⁇ is proportional to R/V, ⁇ is 0.5 to 1, and R is The value is 1mm ⁇ 5mm, the value of l can be calculated from R, and the value of V is 10 ⁇ L ⁇ 200 ⁇ L).
  • the value range of V and l can be determined so that ⁇ Vg ⁇ l* ⁇ * ⁇ , that is, the volume of the pooling hole
  • V/l ⁇ * ⁇ / ⁇ g is satisfied with the circumference of the lower opening of the generated hole.
  • FIG. 1 , FIG. 2 and FIG. 3 are for illustrative purposes only, and are not intended to limit the scope of this description.
  • the droplet generating hole 121 may have an arbitrarily formed outer contour.
  • the culture can be directly fed into the collection hole 111 without setting a culture inlet.
  • cultures can be added to the collection well 111 through the above-mentioned culture device 100 as needed, and when the first plate body 110 and the second plate body 120 cooperate, liquid droplets or hanging drops can be uniformly generated as needed, which is convenient Further collection and cultivation avoids the situation that the droplets drop in advance.
  • Fig. 4 is a schematic structural view of a culture device according to other embodiments of the present specification.
  • the culture device 200 may include a cover plate 240 , a first plate body 210 , a second plate body 220 and a base 230 .
  • the first plate body 210 and the second plate body 220 are similar to the first plate body 110 and the second plate body 120 shown in Fig. Let me repeat.
  • the base 230 and the second plate body 220 may enclose a receiving space in a predetermined direction.
  • the base 230 may be disposed on a side of the second board 220 away from the first board 210 .
  • the accommodating space can form a closed culture environment, which can prevent external bacteria or microorganisms from contaminating the culture, and can also block foreign objects (such as dust, etc.) in the external environment, thereby avoiding contamination of the cell culture environment.
  • the holding space can be used to hold PBS (Phosphate Buffered Saline) or culture medium to provide a suitable growth environment (such as maintaining fitness or providing nutrients) for cell culture in hanging drops, e.g., based on scaffold-free culture
  • PBS Phosphate Buffered Saline
  • the PBS or culture medium contained in the holding space can ensure a certain humidity and avoid the volatilization of liquid hanging drops.
  • the culture solution in the holding space contacts the solidified hanging drop, which can be the solidified hanging drop.
  • the cell culture provides nutrients.
  • the base 230 and the second board body 220 may be connected by mechanical means, including but not limited to clamping, bonding, welding, integral molding, connection by holding components, etc. or any combination thereof.
  • a first culture solution inlet and outlet may be opened on the base 230 , and the first culture solution inlet and outlet are used to pass the culture solution into the accommodation space for culturing.
  • the inlet and outlet of the first culture solution can communicate with the containing space.
  • the inlet and outlet of the first culture solution can also be arranged on the second board body, and the inlet and outlet of the first culture solution can be arranged through the second board body and communicate with the accommodation space.
  • a plurality of inlets and outlets for the first culture solution may be provided on the second plate body and/or the base 230, and a partition (not shown in the figure) may be provided in the accommodation space, and the partition divides the accommodation space into Several sub-accommodating spaces, multiple inlets and outlets of the first culture solution can be arranged in one-to-one correspondence with the sub-accommodating spaces, through which the first culture solution inlets and outlets are passed into the corresponding sub-accommodating spaces with culture solutions of different components to conduct control experiments.
  • the culture solution is a drug solution that can interact with the cells, and different drug solutions are passed into the corresponding sub-accommodation spaces through the first culture solution inlet and outlet, and the cell activity in each sub-accommodation space is measured after the culture is completed.
  • the effective drug for treating the disease corresponding to the cell is screened from the various drug solutions used, or the concentration with the best curative effect for the disease is screened from the different concentration solutions of the same drug.
  • the cover plate 240 can be used to block the contact of the outside world with the first plate body 210 . In some embodiments, the cover plate 240 may be disposed on a side of the first plate body 210 away from the second plate body 220 . In some embodiments, a culture access port may be provided through cover plate 240 . In some embodiments, the culture inlet can be formed as a hollow structure protruding from the cover plate 240 (eg, a funnel structure, etc.). In some embodiments, the culture inlet can be formed as a tubular structure protruding from the cover plate 240, so as to facilitate the insertion of the pipes leading into the culture. In some embodiments, the culture access port may be a hole-like structure extending through the cover plate.
  • a second culture solution inlet and outlet (not shown in the figure) can be opened on the cover plate 240, and the second culture solution inlet and outlet are arranged through the first plate body and the second plate body, and communicate with the accommodation space. So that the culture medium can enter the holding space.
  • the first culture solution inlet and outlet and the second culture solution inlet and outlet can be used in conjunction.
  • the inlet and outlet of the second culture solution can protrude from the cover plate, so as to facilitate the insertion of pipes.
  • the diagram provided in FIG. 4 is for illustrative purposes only, and is not intended to limit the scope of this description.
  • the base 230 of the culture device 200 may be an orifice plate, and grooves on the orifice plate are provided with inlets and outlets for the first culture solution.
  • the cultivation method based on the aforementioned culture device may include: passing the culture into the collection hole of the first plate body, and matching the first plate body and the second plate body so that the collection hole The culture of the method passes through the barrier structure and enters the droplet generation hole to generate hanging droplets.
  • feeding the culture into the collection well can be achieved by manually dropping the culture liquid, or by automatic liquid addition equipment such as a syringe pump and a peristaltic pump.
  • a plurality of pooling holes can be opened on the first plate body, and an overflow tank is opened between adjacent pooling holes; the overflow tank is used to limit the volume of the culture in the pooling hole to no more than Generate the volume required for the droplet; feed the culture into the pooling hole, and the culture method may also include: feeding the culture into any pooling hole on the first plate body, and the culture is evenly distributed in each pooling hole through the overflow tank .
  • the formation of multiple droplets is achieved simultaneously for one drop of the culture, and the volume of the drop culture is the product of the capacity of each pooling well and the required number of pooling wells.
  • the culture device can include a first base, the first base is similar to the base shown in Figure 4 and Figure 5, and the culture method can also include: after the hanging drop is solidified, the second plate body and the first base Cooperate to form an accommodation space; add culture solution into the accommodation space to form an environment for maintaining cultures for cultivation.
  • the culture solution in the holding space can be in contact with at least part of the culture, and the culture solution is used to provide culture nutrients for the culture.
  • Fig. 5 is a schematic diagram of a culture method based on a culture device according to some embodiments of the present specification. As shown in FIG. 5 , the culture device 300 is similar to the culture device 200 in FIG. 4 , wherein the base can be a well plate.
  • the orifice plate may include a plurality of grooves 331 for containing culture fluid.
  • the grooves 331 on the orifice plate may be arranged in one-to-one correspondence with the droplet generating holes 121 on the second plate body 320 .
  • the culture solutions contained in the plurality of grooves may not be exactly the same, so as to facilitate control experiments and/or screening experiments.
  • the orifice plate can cooperate with multiple second plate bodies 320 , for example, the orifice plate 330 can cooperate with four second plate bodies 320 in FIG. 5 .
  • the first base may be a well plate
  • the culture method based on the above culture device may further include: matching multiple second plates with the first base.
  • a plurality of first plates and a plurality of second plates can be matched respectively, and then matched with the first base, the groove of the first base is filled with culture liquid, and the grooves of the second plates
  • the droplet generating holes are arranged corresponding to the grooves on the first base to form a culture environment for the droplets in the droplet generating holes.
  • the droplet generating holes on the second plate form hanging drops, and then a plurality of second plates are directly transferred to the first base, so that the concave The slots are placed in correspondence with the droplet generation wells for cell culture.
  • the well plate in order to conduct culture experiments (such as drug screening or high-throughput analysis), may include a first well plate 330-1 and a second well plate 330-2.
  • the first base may be the first orifice plate 330-1
  • the second base may be the second orifice plate 330-2
  • the cultivation method based on the cultivation device may also include: after the cultivation, the second The board cooperates with the second base to enclose a test space.
  • the testing space is used to perform experiments (eg, drug screening or high-throughput analysis) on cultured hanging drops.
  • the culture solution and the test drug solution can be respectively introduced into the accommodation space and the test space through different first culture solution inlets and outlets on the second plate body.
  • a third orifice plate (not shown in the figure) may also be included. By transferring the second plate body to the third orifice plate, the test result of the second orifice plate 330-2 is verified or further analysis.
  • the culture can include 3D cells.
  • the culture process of 3D cells may include: first, preparing the culture to be inoculated, and then adding a quantitative amount of culture to the collection well through the culture inlet (for example, assuming that the capacity of each collection well is The volume is 50 microliters, so according to the culture requirements, if you need to culture 5 hanging drops of cells, add 250 microliters), the excess culture flows from the overflow tank to the adjacent collection wells, and fills the collection wells in turn until Fill the target number of droplet pooling wells. Due to the barrier effect of the barrier structure, the culture in each collection hole will not drip.
  • the barrier structure is in contact with the droplet generation hole, and the droplet falls to form a hanging drop.
  • the culture solution is passed into the culture solution accommodation space through the culture solution inlet and outlet (for example, the first culture solution inlet and outlet or the second culture solution inlet and outlet), to provide the nutrients needed for cell culture, and the culture solution
  • the amount of addition should ensure that the culture medium is in contact with the hanging drop.
  • culture medium or PBS phosphate-buffered saline
  • PBS phosphate-buffered saline
  • the culture may comprise suspension cells or cell pellets.
  • the culture process of suspension cells or cell clusters may include: preparing the culture to be inoculated, then adding a quantitative amount of culture to the collection well through the culture inlet, and the excess culture is discharged from the overflow tank to the adjacent
  • the pooling holes flow and fill up the pooling holes one by one until the target number of droplet pooling holes are filled. Due to the barrier effect of the barrier structure, the culture in each collection hole will not drip. After the first plate body and the second plate body are matched, the barrier structure is in contact with the droplet generation hole, and the droplet falls to form a hanging drop. .
  • the culture solution or PBS can be passed into the culture solution accommodation space through the culture solution inlet and outlet.
  • the addition of the culture solution or PBS should ensure that the liquid surface will not contact the formed hanging drop. If the hanging drop contacts with the culture solution or PBS, it will fusion, resulting in failure of hanging drop culture.
  • the culture medium or PBS provides a certain humidity for the cell culture, which can avoid the volatilization of the formed hanging droplets. After the cell culture is completed, the culture medium or PBS can be continuously added through the culture medium inlet and outlet to flush the hanging drop into the holding space to merge with the culture medium, and then the culture can be collected through the culture medium inlet and outlet.
  • culturing includes: perfusion culture, non-perfusion culture or air-liquid interface culture.
  • 3D cells or organoid cells can be cultured by the above-mentioned perfusion culture, non-perfusion culture or air-liquid interface method.
  • the culture method does not require other tools outside the culture device, and it is possible to choose whether to expose a part of the cells to the gas for culture according to actual needs, to simulate various culture scenarios, and to meet the needs of various experiments (such as drug screening).
  • the non-perfusion culture includes: passing the total amount of culture suspension needed to any collection hole on the first plate body through the culture inlet, the culture suspension includes culture (for example, organoid, cell or cell mass, etc.), culture medium, gel.
  • the proportion of the gel should ensure that the hanging drop can solidify at the phase transition temperature, and the environment (for example, pH conditions or temperature conditions) of the suspension should ensure that the hanging drop does not undergo a liquid to solid phase transition, for example, Matrigel applied It needs to be operated at a temperature of 4°C.
  • the culture suspension is collected into the corresponding collection hole through any diversion area on the first plate, and the culture suspension that exceeds the volume required to generate a single droplet flows to the adjacent collection hole through the overflow groove until the first plate No volume of culture suspension greater than that required to generate a single droplet is present in as many pooling wells as are required in vivo.
  • the culture suspension in the pooling hole flows downward and stops after being blocked by the barrier structure.
  • the barrier structure is in contact with the droplet generation hole, and the droplet falls to the generation part.
  • a pendant drop is formed at the end of the generating part away from the mating part. After the culture suspension forms a hanging drop, it is necessary to change the environment of the hanging drop to solidify the hanging drop.
  • the applied Matrigel needs to be transferred to a temperature of 37°C for 10 minutes to solidify; after the hanging drop is solidified, pass The culture solution inlet and outlet lead into the culture solution into the containing space, and make the culture solution contact with the hanging drop.
  • the perfusion culture includes: passing a culture suspension into any collection hole on the first plate, and the culture suspension includes culture (for example, organoids, cells or cell clusters, etc.), culture solution, and gel.
  • the proportion of the gel should ensure that the hanging drop can solidify at the phase transition temperature, and the environment (for example, pH conditions or temperature conditions) of the suspension should ensure that the hanging drop does not undergo a liquid to solid phase transition, for example, Matrigel applied Need to operate at a temperature of 4°C; after the culture suspension forms a hanging drop, the environment of the hanging drop needs to be changed to solidify the hanging drop, for example, the applied Matrigel needs to be transferred to a temperature of 37°C for 10 minutes Then solidification occurs; after the hanging drop is solidified, the culture medium is passed into the accommodation space, and the culture medium is in contact with the hanging drop; the culture medium enters and exits at a speed of ⁇ 1 mm/s to form a perfusion culture.
  • the air-liquid interface method cultivation includes: passing the culture suspension into any collection hole on the first plate body, the culture suspension includes culture (for example, organoids, cells or cell clusters, etc.), culture solution, condensate, etc.
  • the ratio of gel to gel should ensure that the hanging drop can solidify at the phase transition temperature, and the environment (for example, pH conditions or temperature conditions) of the suspension should ensure that the hanging drop does not undergo a liquid to solid phase transition, for example, the application Matrigel needs to be operated at a temperature of 4°C; after the culture suspension forms a hanging drop, the environment of the hanging drop needs to be changed to allow the hanging drop to solidify, for example, the applied Matrigel needs to be transferred to a temperature of 37°C Solidification occurs after 10 minutes; after the hanging drop is solidified, remove the first plate body, and add culture medium to the hole of the second plate body, so that the top of the hanging drop is covered by the culture medium, and no culture medium is added to the holding space , make the hanging drop directly contact with the air to form an air
  • the possible beneficial effects may be any one or a combination of the above, or any other possible beneficial effects.
  • numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of the embodiments use the modifiers "about”, “approximately” or “substantially” in some examples. grooming. Unless otherwise stated, “about”, “approximately” or “substantially” indicates that the stated figure allows for a variation of ⁇ 20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximations that can vary depending upon the desired characteristics of individual embodiments. In some embodiments, numerical parameters should take into account the specified significant digits and adopt the general digit reservation method. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of the range are approximations, in specific embodiments, such numerical values are set as precisely as practicable.

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Abstract

一种培养装置及基于培养装置的培养方法，培养装置包括：可分离设置的第一板体和第二板体；第一板体上开设有沿预设方向依次设置的汇集孔和阻隔结构，汇集孔用于容纳生成液滴的培养物；第二板体上开设有用于生成液滴的液滴生成孔，液滴生成孔包括沿预设方向依次设置的配合部和生成部；当第一板体与第二板体配合时，汇集孔插入配合部中，阻隔结构的一部分与配合部内表面接触。

Description

一种培养装置及基于培养装置的培养方法
优先权信息
本说明书要求2021年06月10日提交的申请号为202110648527.5的中国申请以及2021年09月17日提交的申请号为PCT/CN2021/119155的PCT申请的优先权，其全部内容通过引用并入本文。
技术领域
本说明书涉及细胞培养技术领域，特别涉及一种培养装置及基于培养装置的培养方法。
背景技术
细胞培养是指在培养腔室中模拟体内(例如，人体内或动物体内)环境(例如，无菌、适宜温度、酸碱度和一定营养条件等)，使培养物生存、生长、繁殖并维持主要结构和功能的一种方法。可以将培养物(即细胞培养的样本)放在培养腔室中，并向培养腔室中加入供给养分的培养基，培养物可以通过吸收培养基中的养分进行繁殖、生长。
本说明书提供一种培养装置，能够稳定的生成用于进行培养的液滴或悬滴。
发明内容
说明书实施例提供了一种培养装置，该培养装置包括：可分离设置的第一板体和第二板体；第一板体上开设有沿预设方向依次设置的汇集孔和阻隔结构，汇集孔用于容纳生成液滴的培养物；第二板体上开设有用于生成液滴的液滴生成孔，液滴生成孔包括沿预设方向依次设置的配合部和生成部；当第一板体与第二板体配合时，汇集孔插入配合部中，阻隔结构的一部分与配合部内表面接触。
在一些实施例中，阻隔结构为纤维状或条状结构交叉排列形成的网状结构，网状结构包括至少两个网孔，其中，最大网孔单元面积≤1/2阻隔结构的横截面积。
在一些实施例中，汇集孔沿预设方向的高度小于或等于γ*l*sinθ/Sρg；其中，γ表示培养物液体在空气界面的表面张力系数，l表示阻隔孔周长，S表示阻隔孔面积，培养物液体在材料表面的接触角θ表示润湿性，ρ为培养物液体密度，g为重力加速度。在一些实施例中，生成部沿预设方向的高度大于γ*l 1*sinθ 1/S 1ρg；其中，ρ为培养物液体密度，g为重力加速度，γ表示培养物液体在空气界面的表面张力系数，l 1表示生成部远离配合部一端的孔径，S 1表示生成部远离配合部一端的孔口面积，θ 1表示培养物液体在材料表面的接触角。发明人根据本领域中一般培养物液体的参数，通过创造性劳动发现这样的高度设置有利于防止汇集孔内的培养物液体通过阻隔结构下落，此处的培养物液体是指本领域中一般使用的细胞培养液。
在一些实施例中，盖板上开设有第二培养液进出口，第二培养液进出口贯穿第一板体、第二板体设置，与容纳空间连通，以使培养液可以进入容纳空间。
在一些实施例中，悬滴固化后，移除第一板体，向第二板体的配合部孔内加入培养液，使悬滴上方被培养液覆盖，而在容纳空间内不加入培养液，使悬滴直接与空气接触，形成气液界面培养，或向容纳空间通入培养液，悬滴的至少部分与空气直接接触。
本说明书实施例还提供了一种基于培养装置的培养方法，用于基于上述培养装置进行培养，包括：向第一板体的汇集孔内通入培养物；将第一板体和第二板体配合，以使汇集孔内的培养物通过阻隔结构，进入液滴生成孔内，进而生成悬滴。
本说明书实施例还提供了一种上述的培养装置和/或上述的培养方法在2D细胞或3D细胞培养领域的应用。
附图说明
本说明书将以示例性实施例的方式进一步说明，这些示例性实施例将通过附图进行详细描述。这些实施例并非限制性的，在这些实施例中，相同的编号表示相同的结构，其中：
图1是根据本说明书一些实施例所示的培养装置的结构示意图；
图2是根据本说明书一些实施例所示的培养装置的剖视图；
图3是图2中A部的局部放大图；
图4是根据本说明书另一些实施例所示的培养装置的结构示意图；以及
图5是根据本说明书一些实施例所示的基于培养装置的培养方法的示意图。
具体实施方式
为了更清楚地说明本说明书实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地，下面描述中的附图仅仅是本说明书的一些示例或实施例，对于本领域的普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图将本说明书应用于其它类似情景。除非从语言环境中显而易见或另做说明，图中相同标号代表相同结构或操作。
应当理解，本文使用的“系统”、“装置”、“单元”和/或“模块”是用于区分不同级别的不同组件、元件、部件、部分或装配的一种方法。然而，如果其他词语可实现相同的目的，则可通过其他表达来替换所述词语。
本说明书以及权利要求书中使用的“第一”“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分不同的组成部分。同样，“一个”或者“一”等类似词语也不表示数量限制，而是表示存在至少一个。除非另行指出，“前部”、“后部”、“下 部”和/或“上部”等类似词语只是为了便于说明，而并非限于一个位置或者一种空间定向。一般说来，术语“包括”与“包含”仅提示包括已明确标识的步骤和元素，而这些步骤和元素不构成一个排它性的罗列，方法或者设备也可能包含其它的步骤或元素。
本说明书中使用了流程图用来说明根据本说明书的实施例的系统所执行的操作。应当理解的是，前面或后面操作不一定按照顺序来精确地执行。相反，可以按照倒序或同时处理各个步骤。同时，也可以将其他操作添加到这些过程中，或从这些过程移除某一步或数步操作。
本说明书实施例涉及的培养装置可以用于2D细胞培养或3D细胞团培养。在一些实施例中，2D细胞培养是指培养物以贴壁的形式附着在培养腔室的基底上进行生长、繁殖，也可以是以悬浮的形式在培养液中生长、繁殖。在一些实施例中，3D细胞培养可以是指基于支架的培养，支架可以是指天然材料(如天然水凝胶(如基于蛋白的天然水凝胶
Figure PCTCN2022098031-appb-000001
基质胶))或合成材料(如合成凝胶(如聚乙二醇水凝胶))等基质胶，基质胶可以为培养物提供聚集、增值和迁移的支撑，即，为培养物提供生长的三维环境，使培养物维持在三维环境中生长的状态，最终得到三维细胞培养产物(例如，类器官)。在一些实施例中，基质胶可以作为3D细胞培养的载体。在一些实施例中，可以将培养细胞与液态基质胶混合，液态基质胶可以在一定条件下发生相转变，由液态转变为固态，固态基质胶可以提供细胞生长的三维环境，细胞在这样一个三维环境里面可以向各个方向生长、迁移。在一些实施例中，基质胶可以为温敏性凝胶，温敏性凝胶可以随温度变化发生相转变，例如，由液态逐渐转变为固态或者由固态转变为液态。在制备培养物时，将细胞混合在液态温敏性凝胶中，当温敏性凝胶由液态转变为固态后，温敏性凝胶可以为细胞提供三维生长环境，以便后续进行三维细胞培养。在一些实施例中，3D细胞培养还可以是指基于无支架的培养，其依赖于专门的培养器皿或通过组织细胞附着在器皿表面来促进细胞的自聚集，例如基于强制浮动法、悬滴法的培养。
本说明书实施例涉及的培养装置还可以用于类器官培养，为了便于描述，后文以类器官培养为示例进行描述。类器官培养通常需要依赖基质胶类的支架材料的支撑，将类器官悬浮在基质胶液体中滴加至培养容器内进行培养，由于重力的作用，在基质胶固化之前类器官容易沉降至培养容器表壁，而在细胞扩增和生长过程中，类器官贴附到培养容器表壁将形成2D贴壁生长层，不利于3D组织的发育和生长。因此，在类器官培养过程中应尽量避免贴壁生长。而悬滴法是利用悬挂的液滴以培养细胞的方法，一般用于形成和培养3D细胞团，悬挂的液滴不与培养容器接触，能够避免液滴内的细胞贴壁生长，悬滴固化后其中的细胞培养所需的营养成分有限，需要配合培养液以维持培养。当涉及大规模的3D细胞培养时，悬滴法细 胞培养需要逐次滴加混合液体才能逐次形成多个悬滴，如此降低了细胞培养的效率，还可能导致细胞培养的失败。
在一些实施例中，准备培养好的小鼠肠类器官与小鼠肺类器官；将小鼠肠类器官与小鼠肺类器官消化好后用培养基稀释，之后加入与培养基等体积的基质胶，混合均匀；实验组培养物接种于本说明书实施例涉及的培养装置中，对照组培养物接种于普通培养板内；培养过程中用显微镜观察实验组与对照组内的细胞生长状态；培养5天后，用化学发光法测试实验组与对照组的细胞活力。其中，实验组细胞生长于悬挂液滴内，由于悬滴不直接与培养耗材的表面接触，因此不存在细胞贴附于材料表面生长的情况，保持着良好的三维生长状态，而对照组细胞生长在培养板表面，随着细胞的扩增，基质胶内的细包最终会与材料表面接触，从三维生长转为贴壁生长，最终失去三维组织的特性，导致培养失效。故本说明书实施例涉及的培养装置具有改进细胞培养活力的潜力有利于维持三维细胞生长状态。
本说明书实施例涉及的培养物可以理解为是细胞培养的对象。在一些实施例中，培养物至少包括可以用于细胞培养的细胞样本，也可称之为培养细胞。在一些实施例中，培养物可以由细胞与基质胶混合而成。在一些实施例中，培养物还可以由细胞与培养液混合而成。在一些实施例中，培养物可以是液体。在一些实施例中，培养物可以是单个细胞与培养液或基质胶混合，也可以是多个细胞与培养液或基质胶混合。在一些实施例中，多个细胞可以是分散的多个细胞，也可以是细胞团。在一些实施例中，细胞团可以包括由多个细胞直接混合在一起形成的细胞球，也可以包括由多个细胞聚集在一起形成的团状物。
在进一些实施例的培养过程中，通常会使用带有一个或多个能够形成液滴的孔的培养装置进行培养，但在制备液滴的过程中，培养物(或基质胶)以流体的形态添加至培养装置中，由于在流体流动的过程中，会产生切向力，实际操作过程中，常由于培养物(或基质胶)流动产生的切向力导致液滴表面张力异常或体积过大导致液滴提前滴落或悬滴形成失败，进而导致培养失败。
有鉴于此，在一些实施例中，培养装置可以包括可分离设置的第一板体和第二板体，第一板体上可以开设有沿预设方向依次设置的汇集孔和阻隔结构，汇集孔用于容纳生成液滴的培养物，第二板体上可以开设有用于生成液滴的液滴生成孔，液滴生成孔包括沿预设方向依次设置的配合部和生成部。在一些实施例中，第一板体可以与第二板体配合，配合部可以用于配合汇集孔，向汇集孔滴加培养物，汇集孔内部的阻隔结构可以防止培养物形提前形成液滴滴落，当第一板体与第二板体配合时，汇集孔插入配合部中，阻隔结构的一部分可以与配合部内表面接触，被阻隔结构阻隔在汇集孔内的培养物可以通过液滴生成孔形成液滴。本 说明书实施例涉及的培养装置可以用于避免培养物(或基质胶)添加至汇集孔中即形成液滴滴落的现象，培养物可以统一在液滴生成孔内形成液滴或悬滴，便于进一步收集和培养。
图1是根据本说明书一些实施例所示的培养装置的结构示意图。图2是根据本说明书一些实施例所示的培养装置的剖视图。图3是图2中A部的局部放大图。
培养装置100可以包括可分离设置的第一板体110和第二板体120，其包括如图1所示的分离状态和如图2所示的配合状态。
在一些实施例中，第一板体110可以包括一个或多个开设沿预设方向依次设置的汇集孔111和阻隔结构112。
在一些实施例中，汇集孔111可以沿预设方向设置。在一些实施例中，为便于培养物在汇集孔111内汇集，汇集孔111的预设方向可以为培养物受到的重力方向。
在一些实施例中，汇集孔111可以用于容纳生成液滴的培养物。在一些实施例中，汇集孔111内可以具有任意位置上相同或不同的横截面形状，例如，汇集孔111可以形成为圆柱体结构、长方体结构或其它不规则结构。示例性的，汇集孔111内任意位置上的横截面形状可以为圆形。在一些实施例中，汇集孔111内可以具有任意位置上相同或不同的横截面面积，例如，汇集孔111可以形成为具有沙漏形、纺锤形或其它不规则形状外轮廓的结构，在此不作限制。在一些实施例中，为进一步便于培养物在汇集孔111内汇集，汇集孔111内沿预设方向的横截面面积逐渐减小。在一些实施例中，汇集孔111在预设方向上靠近第二板体120上的一侧具有圆台形外轮廓，且汇集孔111在预设方向上靠近第二板体120的方向上直径逐渐减小。在一些实施例中，可以通过汇集孔111远离阻隔结构112的一侧汇入培养物，培养物沿预设方向在汇集孔111内向阻隔结构112流动，并被阻隔结构112阻挡。
在一些实施例中，阻隔结构112可以设于汇集孔111远离培养物汇入的一端。在一些实施例中，阻隔结构112可以用于限制培养物的流动(例如通过形成的表面张力阻隔液体)，即阻隔结构112可以用于避免第一板体110与第二板体120配合之前汇集孔111内容纳的用于形成液滴的培养物通过阻隔结构112流出。在一些实施例中，阻隔结构112可以阻隔培养物的下落，即可以使得培养物承载于阻隔结构112之上。
在一些实施例中，为了避免培养物由于流动产生的切向力导致培养物穿过阻隔结构112，因此阻隔结构112对培养物的承载能力可以为培养物的重力与最大流动切向力在竖直方向上的分力。需要说明的是，切向力与培养物的类型和流速相关，因此在本说明书中不限制阻隔结构的具体承载能力。
在一些实施例中，阻隔结构112可以包括至少一根沿汇集孔111径向设置的纤维状或 条状结构，培养物与阻隔结构112接触后，阻隔结构112可以将培养物与其的接触面划分成至少两部分。在一些实施例中，阻隔结构112对培养物的阻隔能力可以与纤维状或条状结构形成的网孔大小有关。在一些实施例中，阻隔结构112对培养物的阻隔能力可以与纤维状或条状结构形成的最大网孔的周长及面积线性相关。在一些实施例中，阻隔结构112表面对培养物的润湿性及纤维状或条状结构的设置密度可以决定阻隔结构的承载能力，阻隔结构的润湿速度越慢、纤维状或条状结构的设置密度越高，阻隔结构的承载能力越大。在有其他外力(如吸引力)的影响下，培养物即可通过阻隔结构。在一些实施例中，阻隔结构可以为纤维状或条状结构交叉排列形成的网状结构，网状结构包括至少两个网孔，其中，最大网孔单元面积≤1/2阻隔结构的横截面积。在一些实施例中，阻隔结构112可以为纤维状或条状结构不交叉排列形成的结构。在一些实施例中，纤维状或条状结构的横截面最长径范围可以为10μm～1mm。在一些实施例中，纤维状或条状结构的横截面最长径可以为10μm。在一些实施例中，纤维状或条状结构的横截面最长径范围可以为1mm。在一些实施例中，纤维状或条状结构的横截面最长径范围可以为50μm～0.1mm。
在一些实施例中，阻隔结构112可以为多孔结构，阻隔结构112上多个阻隔孔1121，如开设有多个孔的板状结构。在一些实施例中，阻隔结构112对培养物的阻隔能力可以与阻隔孔的密度和大小有关。在一些实施例中，阻隔结构112对培养物的阻隔能力可以与最大阻隔孔的周长及面积线性相关。在一些实施例中，阻隔结构112上的阻隔孔1121均匀分布。在一些实施例中，阻隔结构112上的多个阻隔孔1121孔面积可以相同。在一些实施例中，阻隔结构112上的多个阻隔孔孔面积可以不同。在一些实施例中，阻隔孔孔面积可以影响阻隔结构112对培养物的阻隔能力，阻隔孔孔面积越大，该阻隔孔对培养物的阻隔能力越小。
在一些实施例中，阻隔结构112的材料包括结构材料及涂覆在结构材料表面的表面材料。在一些实施例中，阻隔结构112的结构材料可以包括以下中的至少一种：包括尼龙、丙纶、涤纶、聚乙烯(PE)、聚丙烯(PP)、聚苯乙烯、聚酰胺(PA)、聚甲基丙烯酸甲酯、聚对苯二甲酸乙二醇酯(PET)等高分子材料。在一些实施例中，阻隔结构112的结构材料可以包括以下中的至少一种：不锈钢、铜、铂、金等稳定的金属或合金。在一些实施例中，阻隔结构112的结构材料可以为记忆合金，记忆合金具有形状记忆效应。在一些实施例中，阻隔结构112的结构材料可以为镍钛合金，镍钛合金为记忆合金的一种。在一些实施例中，阻隔结构112对培养物的阻隔能力可以与阻隔结构112的表面材料有关。在一些实施例中，阻隔结构112靠近所述第二板体120的一侧覆盖亲水材料。
在一些实施例中，阻隔结构112对培养物的阻隔能力可以与培养物在空气界面的表面 张力系数线性相关。在一些实施例中，在培养物向汇集孔111流入的过程中，为了使汇集孔111内的培养物不通过阻隔结构下落，不仅可以通过确定阻隔结构112对培养物的阻隔能力实现，还可以通过确定汇集孔111的结构尺寸实现。在一些实施例中，汇集孔111的结构尺寸可以根据以下过程确定：
汇集孔111内的液体压强P 1为：
P 1＝ρgh    (1)，
式(1)中，ρ为培养物液体密度，g为重力加速度，h表示培养物在汇集孔内所能形成的最高液面高度。
在一些实施例中，网孔与阻隔孔的情况类似，培养物在阻隔结构112的网孔或阻隔孔(通常以最大横截面积的网孔或阻隔孔的相关数据为准)处产生的压强P 2为：
P 2＝γ*l*sinθ/S    (2)，
式(2)中，γ表示培养物液体在空气界面的表面张力系数，l表示网孔或阻隔孔周长，S表示网孔或阻隔孔面积，汇集孔内所能形成的最高液面高度h与材料(例如亲水或疏水材料)表面对液体的润湿性有关，其润湿性可以用培养物液体在材料表面的接触角θ表示。
液体压强P 1小于培养物在阻隔结构112的网格或阻隔孔处产生的压强P 2，培养物不会穿过阻隔结构112流出。当汇集孔111内液面上升至h>γ*l*sinθ/Sρg时，培养物穿过阻隔结构的网孔或阻隔孔流出，在这个过程中，液体压强P 1使得液体在网孔或阻隔孔与材料表面的接触角θ逐渐增大，当θ达到最大前进接触角后，θ不再增加。因此，在一些实施例中，为使汇集孔111内的培养物不通过阻隔结构112下落，可以使汇集孔111沿培养物的重力方向高度小于或等于γ*l*sinθ/Sρg，其中，90°＜θ＜180°；优选地，90°＜θ＜160°；γ的取值范围为0.02～0.08N/m；ρ的取值范围为0.8～2.0g/cm3。
在一些实施例中，为了便于将培养物汇集于汇集孔111内，汇集孔111远离阻隔结构112的一端还可以设有导流区113，导流区113可以用于将培养物导流至汇集孔111内。在一些实施例中，导流区113可以与汇集孔111孔壁衔接。在一些实施例中，导流区113可以沿朝汇集孔方向逐渐收拢，形成靠近第二板体120的方向上水平横截面面积小、远离第二板体120的方向上水平横截面面积大，类似漏斗形外轮廓的结构。在一些实施例中，导流区113可以具有圆台形外轮廓，导流区113在朝汇集孔方向上直径逐渐减小。在一些实施例中，导流区可以包括倾斜设置的导流板，导流板在朝汇集孔方向上的底部与汇集孔111衔接。在一些实施例中，导流区113可以是远离阻隔结构112与汇集孔111一侧衔接的倒圆结构，通过倒圆结构形成的向汇集孔111倾斜的区域实现导流效果。在一些实施例中，导流区还可以形成 为其它结构，例如，螺旋结构等，在此不做限制。
在一些实施例中，导流区113上可以覆盖亲水材料，有利于培养物通过导流区113流向汇集孔111，实现导流区113导流效果的优化。在一些实施例中，亲水材料可以包括但不限于亲水绵、亲水性纤维或亲水皮革等或其任意组合。在一些实施例中，导流区113上可以不覆盖疏水材料或覆盖其它特性的材料，如。
在一些实施例中，第一板体110上开设有多个汇集孔111，相邻汇集孔111之间开设有溢流槽114。在一些实施例中，溢流槽114用于限制汇集孔111内的培养物体积不大于生成液滴所需要的体积。在一些实施例中，溢流槽114可以连通相邻汇集孔111。在一些实施例中，溢流槽114可以依次与导流区113、汇集孔111衔接并贯通设置。在一些实施例中，可以向若干汇集孔111中的任意汇集孔111添加所需的培养物总量，所需的培养物总量可以通过所需液滴的数量乘以生成单个液滴所需培养物的体积计算获得，汇入培养物的汇集孔111内盛满生成单个液滴所需体积的培养物后溢出多余培养物，溢出的培养物再通过溢流槽114流入相邻的汇集孔111中，直至第一板体110上的每个汇集孔111内均存在不大于生成单个液滴所需体积的培养物，在此期间，阻隔结构112保持对汇集孔111内培养物的阻隔，避免汇集孔111内的培养物形成液滴下落。
在一些实施例中，为便于进行采用不同培养物的对照实验，第一板体110上的汇集孔111可以分组连通，例如，将第一板体上的9个汇集孔111分成三组，同一组内的汇集孔111通过溢流槽114连通，不同组的汇集孔111不相互连通。在一些实施例中，向分组连通的汇集孔111分别滴加不同的培养物或分批次注入培养物，以形成不同培养成分的对照培养。
参考图1，在一些实施例中，培养装置100上还可以设置培养物通入口115，培养物通入口115用于向第一板体上的汇集孔111通入培养液。在一些实施例中，培养物通入口115可以与任意汇集孔111连通。在一些实施例中，培养装置100上可以设置多个培养物通入口115，多个培养物通入口115可以用于同时向任意汇集孔111通入相同培养物，提高汇集效率，或者，多个培养物通入口115可以用于向分组连通的汇集孔111通入不同培养物，便于对照实验。
在一些实施例中，培养物通入口115可以设置于第一板体上。在一些实施例中，培养物通入口115可以设置于第一板体110的侧壁，并与第一板体110的任意汇集孔111连通。在一些实施例中，第一板体110上可以设置两个培养物通入口115，其一培养物通入口115设置于沿培养物通过溢流槽114流动方向的距离其二培养物通入口115的最远位置。
在一些实施例中，第二板体120上开设有用于生成液滴的液滴生成孔121，液滴生成 孔121用于承接培养物形成液滴。在一些实施例中，液滴生成孔121包括沿预设方向依次设置的配合部1211和生成部1212，预设方向与第一板体上的预设方向相同。在一些实施例中，第一板体110与第二板体120配合，汇集孔111插入配合部1211中，阻隔结构112的一部分地与配合部1211内表面接触，由于阻隔结构112的一部分与配合部1211内表面接触产生分子间引力，使得阻隔结构112上的汇集孔111内的培养物流动至液滴生成孔121内，并在生成部1212形成液滴。在一些实施例中，第一板体110与第二板体120配合时，第一板体110与第二板体120沿预设方向布置。在一些实施例中，为了提高液滴生成孔121对培养物的吸引力，液滴生成孔121内壁可以覆盖亲水材料，其亲水材料可以与导流区亲水材料相同也可以不同。在一些实施例中，为使培养物能够在液滴生成孔121的吸引力下穿过阻隔结构112，阻隔结构112沿预设方向的厚度可以较小，和/或，阻隔结构112靠近第二板体120的一侧可以覆盖亲水材料。
配合部1211可以用于配合汇集孔111以承接汇集孔内的培养物。在一些实施例中，配合部1211可以容纳汇集孔靠近阻隔结构112的部分。在一些实施例中，配合部1211的内轮廓可以与汇集孔111的外轮廓相适应。在一些实施例中，配合部的内轮廓形状可以与汇集孔的外轮廓形状相同，以便于在第一板体110和第二板体120配合时，配合部1211与汇集孔111内壁贴合，如配合部1211的内轮廓形状及汇集孔111的外轮廓形状均为圆台形状。在一些实施例中，配合部1211的内轮廓形状可以与汇集孔111的外轮廓形状不同，如配合部1211的内轮廓形状可以为圆柱形状，汇集孔111的外轮廓形状可以为圆台形状。
在一些实施例中，配合部1211包括平台结构1213，平台结构1213设置于配合部1211与生成部1212之间，阻隔结构112将所述汇集孔111分割为至少两个阻隔孔，第一板体110与第二板体120配合时，其中至少一个阻隔孔的一部分与平台结构1213接触。阻隔结构112与平台结构1213接触产生分子间引力，以使得阻隔结构112上的汇集孔111内的培养物穿过阻隔结构112流动至生成部1212内。在一些实施例中，平台结构1213可以与部分阻隔结构112的阻隔孔发生接触。
在一些实施例中，平台结构1213可以形成为凸出于配合部设置的凸块，第一板体与第二板体配合时，凸块与阻隔结构112的一部分抵接。在一些实施例中，平台结构可以形成为凸出于配合部设置的多个凸块。在一些实施例中，平台结构1213可以形成为凸出于配合部1211设置的环形凸台，第一板体110与第二板体120配合时，环形凸台可以与阻隔结构112的外围部分抵接。
在一些实施例中，为了提高平台结构1213对培养物的吸引力，平台结构1213可以由 亲水材料制成，或者平台结构1213上可以覆盖亲水材料。
生成部1212可以用于形成液滴。在一些实施例中，生成部1212可以被构造成其远离配合部1211的一端用于形成液滴。在一些实施例中，生成部1212可以被构造成其远离配合部1211的一端用于形成悬滴，使包含细胞的悬滴悬挂于生成部1212远离配合部1211的一端以进行细胞培养。在一些实施例中，为了促进悬滴或液滴的形成，生成部1212远离配合部1211的一端具备圆形外轮廓。在一些实施例中，生成部1212可以具有圆台形状内轮廓，且生成部1212在远离配合部1211的方向上其内径逐渐增大，以进一步促进悬滴或液滴的形成。在一些实施例中，为了提高生成部1212对培养物的吸引力，生成部1212内壁可以覆盖亲水材料，或由亲水材料制成。
在一些实施例中，第二板体120上可以设置多个液滴生成孔121，液滴生成孔121可以与第一板体110上的汇集孔111一一对应设置。在一些实施例中，为了防止相邻生成部1212形成的液滴发生聚集，相邻生成部1212远离配合部1211的一端之间的距离不小于第一预设距离阈值。在一些实施例中，第一预设距离阈值可以为2mm。例如，相邻生成部1212远离配合部的一端之间的距离可以为3mm。在一些实施例中，根据培养物在空气界面的表面张力系数不同，相邻生成部1212远离配合部1211的一端之间的距离可以不同。在一些实施例中，相邻生成部1212远离配合部1211的一端之间的距离可以小于2mm且大于1mm。
在一些实施例中，为了使液滴生成孔121内的培养物可以从生成部1212远离配合部1211的一端流出，生成部1212的结构尺寸可以根据上述确定汇集孔111结构尺寸的方法确定。在一些实施例中，生成部1212内的液面高度h 1大于γ*l 1*sinθ 1/S 1ρg时(其中，ρ为培养物液体密度，g为重力加速度，γ表示培养物液体在空气界面的表面张力系数，l 1表示生成部1212远离配合部1211一端的孔径，S 1表示生成部1212远离配合部1211一端的孔口面积，θ 1表示培养物液体在材料表面的接触角)，培养物可以通过生成部1212远离配合部1211一端的孔口流出形成液滴。因此，在一些实施例中，为使液滴生成孔121内的培养物可以从生成部远离配合部的一端流出，可以生成部沿培养物的重力方向高度大于γ*l 1*sinθ 1/S 1ρg。
在一些实施例中，为了使液滴生成孔121内的培养物可以在生成部远离配合部1211的一端形成悬滴悬挂，需要使培养物从生成部1212流出形成的液滴不发生断裂，培养物在生成部内向下运动直至生成部1212一端的孔口，再随着液量的增加，培养物液体逐渐呈现出半球状后，可能出现颈缩或断裂。在一些实施例中，从生成部1212流出形成的液滴是否发生断裂，可以取决于液滴的体积、粘度及表面张力，当培养物从生成部1212一端流出形成的液滴发生断裂时，存在mg＝l*γ*κ，其中，m为汇集孔汇集的培养物质量，(m＝ρV，V是汇 集孔所能容纳的最大体积)，l是液滴生成孔下开口周长，κ为矫正系数(与液体表面张力、材料性质、液体密度及粘度等因素无关，与液体的体积和下开口的半径(R)有关，κ正比于R/V，κ取值为0.5～1，R取值为1mm～5mm，由R可以算出l的取值，V的取值为10μL～200μL)。因此，在一些实施例中，为使培养物从生成部1212流出形成的液滴不发生断裂，可以通过确定V和l的取值范围，以ρVg<l*γ*κ，即汇集孔的体积与生成孔的下开口的周长满足关系V/l<γ*κ/ρg。
应当理解的是，图1、图2及图3所提供的示图仅是出于示例性说明目的，并无意限制本说明书的范围。对于领域内的技术人员而言，在本说明书的指导下可以进行各种变形和修改。例如，液滴生成孔121可以具有任意形成的外轮廓。又例如，可以不设置培养物通入口，直接向汇集孔111通入培养物。而这些变形和修改都将落入被申请的保护范围内。
在一些实施例中，通过上述培养装置100可以向汇集孔111内根据需要添加培养物，并在第一板体110和第二板体120配合时，根据需要统一生成液滴或悬滴，便于进一步收集和培养，避免了液滴提前滴落的情况。
图4是根据本说明书另一些实施例所示的培养装置的结构示意图。如图4所示，培养装置200可以包括盖板240、第一板体210、第二板体220及底座230。
第一板体210、第二板体220与图1-3所示的第一板体110、第二板体120类似，可以参见图1-3及本说明书其它部分的相关说明，在此不再赘述。
在一些实施例中，底座230与第二板体220在预设方向上可以围成容纳空间。在一些实施例中，底座230可以设置于第二板体220远离第一板体210的一侧。在一些实施例中，容纳空间可以形成封闭的培养环境，可以阻隔外部细菌或微生物污染培养物，也可以阻隔外部环境中的异物(如灰尘等)，从而避免污染细胞培养的环境。在一些实施例中，容纳空间可以用于容纳PBS(磷酸缓冲生理盐水)或培养液，为悬滴中的细胞培养提供适合的生长环境(如维持适度或提供养分)，例如，基于无支架培养时，容纳空间容纳的PBS或培养液可以确保一定的湿度，避免液态的悬滴挥发。再例如，当基于支架培养时(如混合有基质胶的培养物于生成部远离配合部的一端形成悬滴)，容纳空间中的培养液与固化的悬滴接触，可以为固化的悬滴中的细胞培养提供养分。在一些实施例中，底座230与第二板体220可以通过机械方式连接，机械方式包括但不限于卡接、粘接、焊接、一体成型、通过保持组件连接等或其任意组合。
在一些实施例中，底座230上可以开设有第一培养液进出口(图中未示出)，第一培养液进出口用于向容纳空间内通入培养液以进行培养。在一些实施例中，第一培养液进出口 可以与容纳空间连通。在一些实施例中，第一培养液进出口还可以设置于第二板体，第一培养液进出口可以贯穿第二板体设置，且与容纳空间连通。在一些实施例中，第二板体和/或底座230上可以开设多个第一培养液进出口，容纳空间内可以设有隔板(图中未示出)，隔板将容纳空间分割为若干子容纳空间，多个第一培养液进出口可以与若干子容纳空间一一对应设置，通过第一培养液进出口向对应的子容纳空间通入不同成分的培养液，以进行对照实验。示例性的，培养液为可以为与细胞相作用的药物溶液，通过第一培养液进出口向对应的子容纳空间分别通入不同的药物溶液，培养结束后测定各子容纳空间内的细胞活性下降程度或其他有效数据，根据有效数据从所用多种药物溶液中筛选出治疗该种细胞对应的疾病的有效药物，或从同种药物的不同浓度溶液中筛选出针对疾病疗效最优的浓度。
在一些实施例中，盖板240可以用于阻隔外界与第一板体210的接触。在一些实施例中，盖板240可以设于第一板体210远离第二板体220的一侧。在一些实施例中，培养物通入口可以贯穿盖板240设置。在一些实施例中，培养物通入口可以形成为凸出于盖板240设置的中空结构(例如，漏斗结构等)。在一些实施例中，培养物通入口可以形成为凸出于盖板240设置的管状结构，以便于通入培养物的管道的插接。在一些实施例中，培养物通入口可以为贯穿盖板的孔状结构。
在一些实施例中，盖板240上可以开设有第二培养液进出口(图中未示出)，第二培养液进出口贯穿第一板体、第二板体设置，与容纳空间连通，以使培养液可以进入容纳空间。在一些实施例中，第一培养液进出口和第二培养液进出口可以配合使用。在一些实施例中，第二培养液进出口可以凸出于盖板设置，以便于管道的插接。
应当理解的是，图4所提供的示图仅是出于示例性说明目的，并无意限制本说明书的范围。对于领域内的技术人员而言，在本说明书的指导下可以进行各种变形和修改。例如，培养装置200的底座230可以为孔板，孔板上的凹槽上均设置有第一培养液进出口。而这些变形和修改都将落入被申请的保护范围内。
在一些实施例中，基于前述培养装置的培养方法，其步骤可以包括：向第一板体的汇集孔内通入培养物，将第一板体和第二板体配合，以使汇集孔内的培养物通过阻隔结构，进入液滴生成孔内，进而生成悬滴。在一些实施例中，向汇集孔内通入培养物可以通过手动滴加培养物液体的方式实现，也可以通过注射泵、蠕动泵等自动加液设备实现。
在一些实施例中，培养装置中，第一板体上可以开设有多个汇集孔，相邻汇集孔之间开设有溢流槽；溢流槽用于限制汇集孔内的培养物体积不大于生成液滴所需要的体积；向汇集孔内通入培养物，培养方法还可以包括：向第一板体上任意汇集孔通入培养物，培养物通 过溢流槽均匀分布于各个汇集孔内。在一些实施例中，为一次滴加培养物同时实现多个液滴的形成，其滴加的培养物的体积为每个汇集孔的容量与所需汇集孔数量的乘积。
在一些实施例中，培养装置可以包括第一底座，第一底座与图4及图5所示的底座类似，培养方法还可以包括：待悬滴固化后，将第二板体与第一底座配合，围成容纳空间；向容纳空间内加入培养液，以形成维持培养物进行培养的环境。在一些实施例中，向容纳空间内的培养液可以与至少部分培养物接触，培养液用于为培养物提供培养养分。
图5是根据本说明书一些实施例所示的基于培养装置的培养方法示意图。如图5所示，培养装置300与图4中培养装置200类似，其中，底座可以为孔板。
在一些实施例中，孔板可以包括多个凹槽331，凹槽331用于盛装培养液。在一些实施例中，孔板上的凹槽331可以与第二板体320上的液滴生成孔121一一对应设置。在一些实施例中，多个凹槽内盛装的培养液可以不完全相同，以便于进行对照实验和/或筛选实验。在一些实施例中，孔板可以与多个第二板体320配合，如图5中孔板330可以与4个第二板体320配合。
在一些实施例中，第一底座可以为孔板，基于上述培养装置的培养方法还可以包括：将多个第二板体与第一底座配合。在一些实施例中，可以将多个第一板体与多个第二板体分别配合后，再与第一底座配合，第一底座的凹槽内盛装有培养液，第二板体上的液滴生成孔与第一底座上的凹槽对应设置，以形成液滴生成孔中液滴的培养环境。在一些实施例中，可以将第一板体与第二板体配合后，第二板体上的液滴生成孔形成悬滴，然后直接转移多个第二板体至第一底座，使凹槽与液滴生成孔对应放置，以进行细胞培养。
在一些实施例中，为了进行培养物的实验(如药物筛选或高通量分析)，孔板可以包括第一孔板330-1和第二孔板330-2。在一些实施例中，第一底座可以还是第一孔板330-1、第二底座可以还是第二孔板330-2，基于该培养装置的培养方法还可以包括：培养结束后，将第二板体与第二底座配合，围成测试空间。在一些实施例中，测试空间用于对培养完成的悬滴进行试验(如药物筛选或高通量分析)。在一些实施例中，可以通过第二板体上的不同第一培养液进出口分别向容纳空间及测试空间通入培养液、测试药液。
在一些实施例中，还可以包括第三孔板(图中未示出)，通过将第二板体转移至第三孔板，以对第二孔板330-2的试验结果进行校验或进一步分析。
在一些实施例中，培养物可以包括3D细胞。在一些实施例中，3D细胞的培养过程可以包括：首先，准备好待接种的培养物，然后将定量的培养物经培养物通入口加入汇集孔(示例性的，假设每个汇集孔的容纳体积为50微升，那么根据培养需求，如果需要培养5个悬滴 的细胞量就加入250微升)，多余培养物由溢流槽向相邻汇集孔流动，依次填满汇集孔中，直至充满目标数量的液滴汇集孔。由于阻隔结构的阻隔作用，各个汇集孔内培养物的不会发生滴落，待第一板体与第二板体配合后，即阻隔结构与液滴生成孔接触，液滴下落，形成悬滴。待悬滴固化后，经培养液进出口(例如，第一培养液进出口或第二培养液进出口)向培养液容纳空间内通入培养液，提供细胞培养所需要的营养物质，培养液的加入量要确保培养液与悬滴接触。待培养完成后，将培养装置转移至凝胶可液化的环境，待凝胶液化后，可以经培养物通入口加入培养液或PBS(磷酸缓冲生理盐水)将液化的悬滴冲入容纳空间与培养液汇合，之后经培养液进出口收集培养物。
在一些实施例中，培养物可以包括悬浮细胞或细胞团。在一些实施例中，悬浮细胞或细胞团的培养过程可以包括：准备好待接种的培养物，然后将定量的培养物经培养物通入口加入汇集孔，多余培养物由溢流槽向相邻汇集孔流动，依次填满汇集孔中，直至充满目标数量的液滴汇集孔。由于阻隔结构的阻隔作用，各个汇集孔内培养物的不会发生滴落，待第一板体与第二板体配合后，即阻隔结构与液滴生成孔接触，液滴下落，形成悬滴。可以经培养液进出口向培养液容纳空间内通入培养液或PBS，培养液或PBS的加入量要确保其液面不会与形成的悬滴接触，若悬滴与培养液或PBS接触会融合，造成悬滴培养失败。培养液或PBS为细胞培养提供一定的湿度，可以避免形成的悬滴挥发。待细胞培养完成后，可以经培养液进出口继续加入培养液或PBS将悬滴冲入容纳空间与培养液汇合，之后经培养液进出口收集培养物。
在一些实施例中，培养包括：灌流培养、非灌流培养或气液界面法培养。在一些实施例中，可以通过上述灌流培养、非灌流培养或气液界面法培养对3D细胞或类器官细胞的培养。通过该培养方法无需该培养装置外的其他工具，能够根据实际需要，选择是否将细胞的一部分暴露在气体中培养，模拟各种培养场景，满足各类实验(如药物筛选)的需求。
其中，非灌流培养包括：通过培养物通入口向向第一板体上的任意汇集孔通入所需的培养物悬液总量，培养物悬液包括培养物(例如，类器官、细胞或细胞团等)、培养液、凝胶。凝胶的比例需确保悬滴可以在相转变温度发生固化，悬液所处的环境(例如，pH条件或温度条件)需确保悬滴不发生液态向固态的相转变，例如，应用的基质胶需要在4℃的温度条件下操作。培养物悬液通过第一板体上的任一导流区汇集于对应汇集孔，超过生成单个液滴所需体积的培养物悬液通过溢流槽向相邻汇集孔流动，直至第一板体上所需的汇集孔内均存在不大于生成单个液滴所需体积的培养物悬液。汇集孔内的培养物悬液向下流动，被阻隔结构阻隔后停止流动，待第一板体与第二板体配合后，即阻隔结构与液滴生成孔接触，液滴下 落至生成部，于生成部远离配合部的一端形成悬滴。培养物悬液形成悬滴后，需要改变悬滴所处的环境以使悬滴固化，例如，应用的基质胶需转移至37℃的温度条件下10分钟后发生固化；悬滴固化后，通过培养液进出口向容纳空间内通入培养液，并使得培养液与悬滴接触。
其中，灌流培养包括：向第一板体上的任意汇集孔通入培养物悬液，培养物悬液包括培养物(例如，类器官、细胞或细胞团等)、培养液、凝胶。凝胶的比例需确保悬滴可以在相转变温度发生固化，悬液所处的环境(例如，pH条件或温度条件)需确保悬滴不发生液态向固态的相转变，例如，应用的基质胶需要在4℃的温度条件下操作；培养物悬液形成悬滴后，需要改变悬滴所处的环境以使悬滴固化，例如，应用的基质胶需转移至37℃的温度条件下10分钟后发生固化；悬滴固化后，向容纳空间内通入培养液，培养液与悬滴接触；培养液以≤1mm/s的速度进液和出液，以形成灌流培养。
其中，气液界面法培养包括：向第一板体上的任意汇集孔通入培养物悬液，培养物悬液包括培养物(例如，类器官、细胞或细胞团等)、培养液、凝胶，凝胶的比例需确保悬滴可以在相转变温度发生固化，悬液所处的环境(例如，pH条件或温度条件)需确保悬滴不发生液态向固态的相转变，例如，应用的基质胶需要在4℃的温度条件下操作；培养物悬液形成悬滴后，需要改变悬滴所处的环境以使悬滴固化，例如，应用的基质胶需转移至37℃的温度条件下10分钟后发生固化；悬滴固化后，移除第一板体，向第二板体的配合部孔内加入培养液，使悬滴上方被培养液覆盖，而在容纳空间内不加入培养液，使悬滴直接与空气接触，形成气液界面培养，或向容纳空间通入培养液，培养液不至于完全浸没悬滴，即，悬滴的至少部分与空气直接接触。
需要说明的是，不同实施例可能产生的有益效果不同，在不同的实施例里，可能产生的有益效果可以是以上任意一种或几种的组合，也可以是其他任何可能获得的有益效果。
上文已对基本概念做了描述，显然，对于本领域技术人员来说，上述详细披露仅仅作为示例，而并不构成对本说明书的限定。虽然此处并没有明确说明，本领域技术人员可能会对本说明书进行各种修改、改进和修正。该类修改、改进和修正在本说明书中被建议，所以该类修改、改进、修正仍属于本说明书示范实施例的精神和范围。
同时，本说明书使用了特定词语来描述本说明书的实施例。如“一个实施例”、“一实施例”、和/或“一些实施例”意指与本说明书至少一个实施例相关的某一特征、结构或特点。因此，应强调并注意的是，本说明书中在不同位置两次或多次提及的“一实施例”或“一个实施例”或“一个替代性实施例”并不一定是指同一实施例。此外，本说明书的一个或多个实施例中的某些特征、结构或特点可以进行适当的组合。
此外，除非权利要求中明确说明，本说明书所述处理元素和序列的顺序、数字字母的使用、或其他名称的使用，并非用于限定本说明书流程和方法的顺序。尽管上述披露中通过各种示例讨论了一些目前认为有用的发明实施例，但应当理解的是，该类细节仅起到说明的目的，附加的权利要求并不仅限于披露的实施例，相反，权利要求旨在覆盖所有符合本说明书实施例实质和范围的修正和等价组合。例如，虽然以上所描述的系统组件可以通过硬件设备实现，但是也可以只通过软件的解决方案得以实现，如在现有的服务器或移动设备上安装所描述的系统。
同理，应当注意的是，为了简化本说明书披露的表述，从而帮助对一个或多个发明实施例的理解，前文对本说明书实施例的描述中，有时会将多种特征归并至一个实施例、附图或对其的描述中。但是，这种披露方法并不意味着本说明书对象所需要的特征比权利要求中提及的特征多。实际上，实施例的特征要少于上述披露的单个实施例的全部特征。
一些实施例中使用了描述成分、属性数量的数字，应当理解的是，此类用于实施例描述的数字，在一些示例中使用了修饰词“大约”、“近似”或“大体上”来修饰。除非另外说明，“大约”、“近似”或“大体上”表明所述数字允许有±20％的变化。相应地，在一些实施例中，说明书和权利要求中使用的数值参数均为近似值，该近似值根据个别实施例所需特点可以发生改变。在一些实施例中，数值参数应考虑规定的有效数位并采用一般位数保留的方法。尽管本说明书一些实施例中用于确认其范围广度的数值域和参数为近似值，在具体实施例中，此类数值的设定在可行范围内尽可能精确。
针对本说明书引用的每个专利、专利申请、专利申请公开物和其他材料，如文章、书籍、说明书、出版物、文档等，特此将其全部内容并入本说明书作为参考。与本说明书内容不一致或产生冲突的申请历史文件除外，对本说明书权利要求最广范围有限制的文件(当前或之后附加于本说明书中的)也除外。需要说明的是，如果本说明书附属材料中的描述、定义、和/或术语的使用与本说明书所述内容有不一致或冲突的地方，以本说明书的描述、定义和/或术语的使用为准。
最后，应当理解的是，本说明书中所述实施例仅用以说明本说明书实施例的原则。其他的变形也可能属于本说明书的范围。因此，作为示例而非限制，本说明书实施例的替代配置可视为与本说明书的教导一致。相应地，本说明书的实施例不仅限于本说明书明确介绍和描述的实施例。

Claims (63)

  1. 一种培养装置，其特征在于，所述培养装置包括：
    可分离设置的第一板体和第二板体；
    所述第一板体上开设有沿预设方向依次设置的汇集孔和阻隔结构，所述汇集孔用于容纳生成液滴的培养物；
    所述第二板体上开设有用于生成液滴的液滴生成孔，所述液滴生成孔包括沿所述预设方向依次设置的配合部和生成部；
    当所述第一板体与所述第二板体配合时，所述汇集孔插入所述配合部中，所述阻隔结构的一部分与所述配合部内表面接触。
  2. 如权利要求1所述的培养装置，其特征在于：
    所述配合部包括平台结构，所述平台结构位于所述配合部与所述生成部之间；所述第一板体与所述第二板体配合时，所述阻隔结构上设有至少两个阻隔孔，其中至少一个所述阻隔孔的一部分与所述平台结构接触。
  3. 如权利要求2所述的培养装置，其特征在于：
    所述配合部的内轮廓与所述汇集孔的外轮廓相适应。
  4. 如权利要求2所述的培养装置，其特征在于：所述平台结构形成为凸出于所述配合部设置的凸块，所述第一板体与所述第二板体配合时，所述凸块与所述阻隔结构的一部分抵接。
  5. 如权利要求2所述的培养装置，其特征在于：
    所述平台结构由亲水材料制成，或者
    所述平台结构上覆盖亲水材料。
  6. 如权利要求1～5中任一种所述的培养装置，其特征在于：所述液滴生成孔内壁上覆盖亲水材料。
  7. 如权利要求1～5中任一种所述的培养装置，其特征在于：所述阻隔结构设于所述汇集孔远离培养物汇入的一端。
  8. 如权利要求1～5中任一种所述的培养装置，其特征在于：
    所述阻隔结构包括至少一根延所述汇集孔径向设置的纤维状或条状结构。
  9. 如权利要求8所述的培养装置，其特征在于：所述阻隔结构为纤维状或条状结构交叉排列形成的网状结构，其中，最大网孔单元面积≤1/2阻隔结构的横截面积。
  10. 如权利要求8所述的培养装置，其特征在于：
    所述纤维状或所述条状结构的横截面最长径范围为10μm～1mm。
  11. 如权利要求8所述的培养装置，其特征在于：
    所述纤维状或所述条状结构的横截面最长径范围为50μm～0.1mm。
  12. 如权利要求1～5中任一种所述的培养装置，其特征在于：
    所述阻隔结构为多孔结构。
  13. 如权利要求1～5中任一种所述的培养装置，其特征在于：
    所述阻隔结构为开设有多个孔的板状结构。
  14. 如权利要求1～13中任一种所述的培养装置，其特征在于：
    所述阻隔结构的结构材料包括以下中的至少一种：尼龙、丙纶、涤纶、聚乙烯、聚丙烯、聚苯乙烯、聚酰胺、聚甲基丙烯酸甲酯、聚对苯二甲酸乙二醇酯、不锈钢、铜、铂、金、或记忆合金。
  15. 如权利要求1～14中任一种所述的培养装置，其特征在于：所述阻隔结构靠近所述第二板体的一侧覆盖亲水材料。
  16. 如权利要求1～15中任一种所述的培养装置，其特征在于：
    所述汇集孔内的任意位置处垂直于所述预设方向的横截面形状均相同。
  17. 如权利要求1～15中任一种所述的培养装置，其特征在于：
    所述汇集孔内的任意位置处垂直于所述预设方向的横截面形状不完全相同。
  18. 如权利要求1～15中任一种所述的培养装置，其特征在于：
    所述汇集孔形成为具有沙漏形或纺锤形。
  19. 如权利要求1～15中任一种所述的培养装置，其特征在于：
    所述汇集孔形成为圆柱体结构或长方体结构。
  20. 如权利要求1～19中任一种所述的培养装置，其特征在于：
    所述汇集孔沿所述预设方向的高度小于或等于γ*l*sinθ/Sρg；
    其中，γ表示培养物液体在空气界面的表面张力系数，l表示阻隔孔周长，S表示阻隔孔面积，培养物液体在材料表面的接触角θ表示润湿性，ρ为培养物液体密度，g为重力加速度。
  21. 如权利要求20所述的培养装置，其特征在于：
    所述表面张力系数γ的取值范围为0.02～0.08N/m；所述接触角θ的取值范围为90°＜θ＜180°；所述培养物液体密度ρ的取值范围为0.8～2.0g/cm 3。
  22. 如权利要求1～21中任一种所述的培养装置，其特征在于：
    所述生成部沿所述预设方向的高度大于γ*l 1*sinθ 1/S 1ρg；其中，ρ为培养物液体密度，g为重力加速度，γ表示培养物液体在空气界面的表面张力系数，l 1表示所述生成部远离所述配合部一端的孔径，S 1表示所述生成部远离所述配合部一端的孔口面积，θ 1表示培养物液体在材料表面的接触角。
  23. 如权利要求的1～22中任一种所述的培养装置，其特征在于：
    所述汇集孔的体积与所述生成部远离所述配合部一端的孔口周长满足：V/l<γ*κ/ρg；
    其中，V为所述汇集孔的体积，l为所述孔口周长，κ为矫正系数；κ与R/V正相关，R为所述孔口的半径。
  24. 如权利要求23所述的培养装置，其特征在于：
    其中，所述矫正系数κ的取值范围为0.5～1；所述孔口的半径R的取值范围为1mm～5mm；所述汇集孔的体积V的取值范围为10μL～200μL。
  25. 如权利要求1～15中任一种所述的培养装置，其特征在于：
    所述汇集孔在预设方向上靠近所述第二板体上的一侧具有圆台形外轮廓；所述汇集孔在 靠近所述第二板体的方向上直径逐渐减小。
  26. 如权利要求1～23中任一种所述的培养装置，其特征在于：
    所述汇集孔远离所述阻隔结构的一端还设有导流区；
    所述导流区用于将所述培养物汇集至所述汇集孔内。
  27. 如权利要求26所述的培养装置，其特征在于：所述导流区沿朝所述汇集孔方向逐渐收拢，形成靠近所述第二板体的方向上水平横截面面积小、远离所述第二板体的方向上水平横截面面积大的结构。
  28. 如权利要求26所述的培养装置，其特征在于：所述导流区包括倾斜设置的导流板，所述导流板在朝所述汇集孔方向上的底部与所述汇集孔衔接。
  29. 如权利要求26所述的培养装置，其特征在于：所述导流区是远离所述阻隔结构与所述汇集孔一侧衔接的倒圆结构，通过所述倒圆结构形成向所述汇集孔倾斜的区域。
  30. 如权利要求26～29中任一种所述的培养装置，其特征在于：
    所述导流区上覆盖亲水材料。
  31. 如权利要求30中任一种所述的培养装置，其特征在于：
    所述第一板体上开设有多个汇集孔，相邻所述汇集孔之间开设有溢流槽；所述溢流槽用于限制所述汇集孔内的所述培养物体积不大于生成所述液滴所需要的体积。
  32. 如权利要求31所述的培养装置，其特征在于：
    所述溢流槽连通相邻所述汇集孔，且所述溢流槽与所述导流区、所述汇集孔依次衔接并贯通设置。
  33. 如权利要求31所述的培养装置，其特征在于：
    所述第一板体上开设有若干培养物通入口；所述培养物通入口与所述汇集孔连通。
  34. 如权利要求31所述的培养装置，其特征在于：
    所述第一板体的侧壁设置有所述培养物通入口，所述培养物通入口与所述第一板体的任意所述汇集孔连通。
  35. 如权利要求31所述的培养装置，其特征在于：所述第一板体上设置两个所述培养物通入口，其一所述培养物通入口设置于沿培养物通过所述溢流槽流动方向的距离其二所述培养物通入口的最远位置。
  36. 如权利要求1-35中任一种所述的培养装置，其特征在于：
    所述生成部远离所述配合部的一端具备圆形外轮廓。
  37. 如权利要求1-36中任一种所述的培养装置，其特征在于：
    所述培养装置还包括第一底座，所述第一底座与所述第二板体在预设方向上围成容纳空间。
  38. 如权利要求37所述的培养装置，其特征在于：
    所述第一底座上还开设有第一培养液进出口；所述第一培养液进出口用于向所述容纳空间内通入培养液以进行培养。
  39. 如权利要求38所述的培养装置，其特征在于：
    所述第一培养液进出口贯穿所述第二板体设置，且与所述容纳空间连通。
  40. 如权利要求37-39中任一种所述的培养装置，其特征在于：
    所述第一底座为孔板。
  41. 如权利要求37-40中任一种所述的培养装置，其特征在于：
    所述第一底座与所述第二板体通过机械方式连接，所述机械方式包括但不限于卡接、粘接、焊接、一体成型。
  42. 如权利要求1-41中任一种所述的培养装置，其特征在于：
    所述培养装置还包括盖板，所述盖板盖设于远离所述第一板体远离所述第二板体的一侧。
  43. 如权利要求42所述的培养装置，其特征在于：
    所述盖板上贯穿设置有所述培养物通入口。
  44. 如权利要求42或43所述的培养装置，其特征在于：
    所述盖板上开设有所述第二培养液进出口，所述第二培养液进出口贯穿所述第一板体、所述第二板体设置，与所述容纳空间连通。
  45. 如权利要求1-44中任一种所述的培养装置，其特征在于：
    所述培养装置还包括第二底座，所述第二板体与所述第二底座配合，围成测试空间，所述测试空间用于对培养完成的悬滴进行试验。
  46. 如权利要求1-45中任一种所述的培养装置，其特征在于：
    所述培养装置还包括第三孔板，所述第三孔板与所述第二板体配合，用于对所述第二底座内的试验结果进行校验或分析。
  47. 一种基于培养装置的培养方法，其特征在于，基于权利要求1-46中的任一项所述的培养装置进行培养；包括：
    向所述第一板体的汇集孔内通入培养物；
    将第一板体和第二板体配合，以使所述汇集孔内的培养物通过所述阻隔结构，进入液滴生成孔内，进而生成悬滴。
  48. 如权利要求47所述的培养方法，其特征在于，所述方法还包括：
    所述悬滴固化后，移除所述第一板体，向所述第二板体的所述配合部孔内加入培养液或向所述容纳空间通入培养液，悬滴的至少部分与空气直接接触。
  49. 如权利要求47或48任一种所述的培养方法，其特征在于，相邻所述汇集孔之间开设有溢流槽；所述方法还包括：
    向所述第一板体上任意所述汇集孔通入培养物，培养物通过所述溢流槽均匀分布于各个所述汇集孔内。
  50. 如权利要求47-49任一种所述的培养方法，其特征在于，所述培养装置还包括第一 底座；所述方法还包括：
    待所述悬滴固化后，将所述第二板体与所述第一底座配合，围成容纳空间；
    向所述容纳空间内加入培养液，以形成维持培养物进行培养的环境。
  51. 如权利要求50所述的培养方法，其特征在于：所述第二板体上贯穿设置所述第一培养液进出口，所述方法还包括：
    通过所述第一培养液进出口向所述容纳空间加入培养液。
  52. 如权利要求50所述的培养方法，其特征在于：所述容纳空间内设置用于将所述容纳空间分割为若干子容纳空间的隔板，所述第二板体上的多个所述第一培养液进出口可以与所述若干子容纳空间一一对应设置；所述方法还包括：
    分别通过多个所述第一培养液进出口向所述若干子容纳空间通入不同培养液。
  53. 如权利要求50-52中任一种所述的培养方法，其特征在于：所述培养装置还包括盖板，所述盖板上开设第二培养液进出口，所述第二培养液进出口贯穿第一板体、第二板体设置，所述方法还包括：
    通过所述第二培养液进出口向所述容纳空间加入培养液。
  54. 如权利要求50-52中任一种所述的培养方法，其特征在于，所述第一底座为第一孔板；包括：
    将多个所述第二板体与所述第一底座配合。
  55. 如权利要求54所述的培养方法，其特征在于，所述第一孔板上的凹槽上均设置有第一培养液进出口，包括：分别通过各所述第一培养液进出口向各所述凹槽内通入培养液。
  56. 如权利要求47-55中任一种所述的培养方法，其特征在于，所述培养装置还包括第二底座；所述方法还包括：
    培养结束后，将所述第二板体与所述第二底座配合，围成测试空间；所述测试空间用于对培养完成的悬滴进行试验。
  57. 如权利要求56所述的培养方法，其特征在于，所述方法还包括：
    所述测试空间内的所述悬滴液化后，使所述悬滴与培养液混合，以收集所述悬滴中含有的培养物。
  58. 如权利要求57所述的培养方法，其特征在于，所述第二底座为第二孔板，所述第二板体与所述第二底座配合；包括：
    向所述第二孔板上的各凹槽通入不同的测试药液。
  59. 如权利要求58所述的培养方法，其特征在于，培养装置还包括第三孔板；包括：
    通过将所述第二板体转移至所述第三孔板，以对所述第二孔板的试验结果进行校验或分析。
  60. 如权利要求47-59中任一种所述的培养方法，其特征在于，所述培养装置中，所述第一板体上开设有多个汇集孔，相邻所述汇集孔之间开设有溢流槽；所述溢流槽用于限制所述汇集孔内的所述培养物体积不大于生成所述液滴所需要的体积；所述向汇集孔内通入培养物，包括：
    向所述第一板体上汇集孔通入培养物，所述通入的培养物的体积为所述悬滴的体积与流通通道数量的乘积。
  61. 如权利要求47-60中任一种所述的培养方法，其特征在于：
    所述培养物包括3D细胞和/或2D细胞。
  62. 如权利要求47-61中任一种所述的培养方法，其特征在于，所述培养包括：
    灌流培养、非灌流培养或气液界面法培养。
  63. 一种如权利要求1-46中任意一项所述的培养装置和/或一种如权利要求47-62中任意一项所述的培养方法在2D细胞或3D细胞培养领域的应用。
PCT/CN2022/098031 2021-06-10 2022-06-10 一种培养装置及基于培养装置的培养方法 WO2022258037A1 (zh)

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