CN113046244A

CN113046244A - Culture device and culture method using same

Info

Publication number: CN113046244A
Application number: CN202110344389.1A
Authority: CN
Inventors: 王玄
Original assignee: Shanghai Ruiyu Biotech Co Ltd
Current assignee: Shanghai Ruiyu Biotech Co Ltd
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2021-06-29
Anticipated expiration: 2041-03-30
Also published as: CN113046244B

Abstract

The embodiment of the application discloses culture apparatus, culture apparatus includes: a culture chamber comprising a substrate; the plug-in is matched with the culture chamber and is provided with a plurality of through holes; when the plug-in is matched and connected with the culture chamber, the through holes can form a plurality of accommodating spaces with the substrate of the culture chamber, and the accommodating spaces are used for accommodating cultures.

Description

Culture device and culture method using same

Technical Field

The application relates to the technical field of cell culture, in particular to a culture device and a culture method based on the culture device.

Background

Cell culture is an important means for studying signal transduction, anabolism, and growth and proliferation of cells. In the course of cell culture, culture inoculation is an important step, and particularly when large-scale cell culture is involved, the rate of culture inoculation affects the efficiency of cell culture and also affects the product quality of cell culture. Therefore, there is a need to provide a culture device to increase the inoculation rate of a culture.

Disclosure of Invention

One of the embodiments of the present application provides a culture device, including: a culture chamber comprising a substrate; the plug-in is matched with the culture chamber and is provided with a plurality of through holes; when the plug-in is matched and connected with the culture chamber, the through holes can form a plurality of accommodating spaces with the substrate of the culture chamber, and the accommodating spaces are used for accommodating cultures.

One of the embodiments of the present application provides a culture method based on a culture device, the culture device including: a culture chamber comprising a substrate; the plug-in is matched with the culture chamber and is provided with a plurality of through holes; when the plug-in is matched and connected with the culture chamber, the through holes and the substrate of the culture chamber can form a plurality of accommodating spaces, and the accommodating spaces are used for accommodating cultures; the method comprises the following steps: mating the insert with the culture chamber and adding the culture to the number of through holes on the insert; adding a culture medium to at least partially contact the culture with the culture medium, wherein the culture medium is used to provide a nutrient substrate to the culture.

Drawings

The present application will be further explained by way of exemplary embodiments, which will be described in detail by way of the accompanying drawings. These embodiments are not intended to be limiting, and in these embodiments like numerals are used to indicate like structures, wherein:

FIG. 1 is a schematic illustration of a culture device according to some embodiments of the present application with the insert not mated to the culture chamber;

FIG. 2 is a schematic diagram of a culture device with an insert mated to a culture chamber according to some embodiments of the present application;

FIG. 3 is a schematic diagram of a culture device inoculated with a culture according to further embodiments of the present application;

FIG. 4 is a schematic illustration of a culture device according to further embodiments of the present application wherein the insert is not mated to the culture chamber;

FIG. 5 is a schematic diagram of a culture device with an insert mated to a culture chamber according to further embodiments of the present application;

FIG. 6 is a schematic diagram of a culture device inoculated with a culture according to further embodiments of the present application;

FIG. 7 is a schematic structural view of an insert according to some embodiments of the present application;

FIG. 8 is a schematic drawing in section of a portion of a via according to other embodiments of the present application;

FIG. 9 is a schematic view of an insert shown at another angle according to other embodiments of the present application.

Reference numerals: a culture device 10; a culture chamber 20; a substrate 21; a bearing projection 23; an insert 30; a through hole 31; an accommodating space 33; a culture 40; a projection 50; an inverted conical ramp 60; an inverted conical structure 70.

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings used in the description of the embodiments will be briefly introduced below. It is obvious that the drawings in the following description are only examples or embodiments of the application, from which the application can also be applied to other similar scenarios without inventive effort for a person skilled in the art. Unless otherwise apparent from the context, or otherwise indicated, like reference numbers in the figures refer to the same structure or operation.

As used in this application and the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that steps and elements are included which are explicitly identified, that the steps and elements do not form an exclusive list, and that a method or apparatus may include other steps or elements. The term "based on" is "based, at least in part, on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment". Relevant definitions for other terms will be given in the following description.

In some embodiments, cell culture refers to a method of simulating an in vivo (e.g., human or animal) environment (e.g., sterility, suitable temperature, ph, and certain nutrient conditions, etc.) in a culture chamber, such that the culture (e.g., cultured cells) survive, grow, propagate, and maintain primary structure and function. In some embodiments, a culture (i.e., a sample of a cell culture) can be placed in the culture chamber and nutrient-supplying medium can be added to the culture chamber, and the culture can multiply and grow by absorbing the nutrients in the medium.

In some embodiments, the means of cell culture may include two-dimensional cell culture and three-dimensional cell culture. In the two-dimensional cell culture, a culture (for example, cultured cells) is adherently attached to a substrate of a culture chamber to grow and propagate. Three-dimensional cell culture may refer to culture of a culture in a carrier that provides a three-dimensional environment in which the culture is grown, such that the culture is maintained in a state of growth in the three-dimensional environment, resulting in a three-dimensional cell culture product (e.g., organoid). In some embodiments, matrigel can serve as a carrier for three-dimensional cell culture. In some embodiments, the culture may be mixed with a liquid matrigel prior to inoculation of the culture, the liquid matrigel may undergo a phase transition under conditions to change from a liquid state to a solid state, and the solid matrigel may provide a three-dimensional environment for growth of the culture.

A culture is understood to be the object of cell culture. In some embodiments, the culture includes at least a sample of cells, which may be used for cell culture, also referred to as cultured cells. In some embodiments, the culture may also be mixed with matrigel. In some embodiments, the culture may be formed by mixing cells with matrigel. In some embodiments, the culture may be a single cell mixed with matrigel or a plurality of cells mixed with matrigel. In some embodiments, the plurality of cells may be a dispersed plurality of cells or may be a cell mass. In some embodiments, the cell pellet may comprise a cell pellet formed by a plurality of cells mixed together directly, i.e., a dough formed by a plurality of cells aggregated together. In some embodiments, the culture may be formed by mixing three-dimensional cells with matrigel. Three-dimensional cells refer to the product of a three-dimensional cell culture, e.g., organoids. In some embodiments, the organoids may include brain organoids, colon organoids, liver organoids, tumor organoids, stomach organoids, and the like.

Matrigel refers to an extracellular matrix that provides a three-dimensional growth environment for a culture during three-dimensional cell culture. Specifically, the matrigel can provide a growth carrier for three-dimensional cell culture, the culture can grow and migrate in all directions in such a three-dimensional environment, and the culture is mixed in the matrigel so as to maintain the culture in a middle-growth state, so that the culture can be cultured in three dimensions.

In some embodiments, the matrigel may be a temperature-sensitive gel that may undergo a phase transition with a change in temperature, i.e., a transition from one material state to another, e.g., a gradual transition from a liquid state to a solid state or a transition from a solid state to a liquid state. When the culture is prepared, the culture is mixed in the liquid temperature-sensitive gel, and after the temperature-sensitive gel is changed from a liquid state to a solid state, the temperature-sensitive gel can provide a three-dimensional growth environment for the culture so as to carry out three-dimensional cell culture in the subsequent process.

In some embodiments, the temperature sensitive gel may transition from a liquid state to a solid state at temperatures in excess of 20 ℃. In some embodiments, the temperature sensitive gel may transition from a liquid state to a solid state at temperatures in excess of 15 ℃. In some embodiments, the temperature sensitive gel may transition from a liquid state to a solid state at temperatures in excess of 10 ℃. In some embodiments, the temperature at which the temperature-sensitive gel undergoes a phase transition is related to the type of temperature-sensitive gel, and different temperature-sensitive gels have different phase transition temperatures. In some embodiments, the temperature sensitive gel may include, but is not limited to, poly-N-isopropylacrylamide (PNIPAM), block copolymers of poly-N-isopropylacrylamide and polyethylene glycol (PNIPAM-PEG), polyethylene glycol (PEG), block copolymers of polylactic-co-glycolic acid (PLGA) (PEG-PLGA), PLGA-PEG-PLGA triblock polymers, PCLA (epsilon-caprolactone and L-lactide copolymer) -PEG-PCLA triblock polymers.

The culture medium refers to nutrient solution for the growth and reproduction of the culture and is prepared by combining different nutrient substances. An important step in cell culture is the inoculation of the culture, i.e. the addition of the culture to the culture chamber. In some embodiments, the common mode of inoculation for cell culture may include manual inoculation, e.g., where the operator inoculates the culture to the substrate row by row or row by row using an inoculation tool (e.g., a pin header). In some embodiments, the conventional inoculation methods have low inoculation efficiency and slow inoculation speed, and may affect the inoculated culture, and further affect the product quality of the cell culture.

In some embodiments, taking the temperature-sensitive gel as an example of the carrier, when large-scale three-dimensional cell culture is involved, the inoculation quantity is large, and the culture is inoculated one by one or row by row, which is not only slow, but also because the phase transition temperature range of Matrigel is narrow (for example, Corning Matrigel (Corning Matrigel) liquefies at 4 ℃ and gradually coagulates at more than 10 ℃), if the temperature is not properly controlled, the culture may already coagulate without being inoculated, the efficiency of cell culture is reduced, and the product of cell culture may not meet the culture requirements.

Some embodiments of the present application provide a culture device that may include an auxiliary inoculation device for increasing the inoculation rate. In some embodiments, the secondary inoculation device may be used to rapidly divide a mother culture sample (i.e., a larger volume of culture) into several sub-culture samples (i.e., a smaller volume of culture) and flow to a designated location to complete inoculation. In some embodiments, the inoculation assisting device at least comprises the plug-in unit in one or more embodiments of the present application, and when the plug-in unit is used for inoculation, the culture can be inoculated quickly and in batches, so that the inoculation speed of the culture is increased, the cell culture efficiency is improved, and the cell culture product quality is improved.

As shown in connection with fig. 1-4, in some embodiments, the culture device 10 can include a culture chamber 20 and an insert 30 that fits into the culture chamber 20; the culture chamber 20 may include a substrate 21, the plug-in unit 30 is provided with a plurality of through holes 31, when the plug-in unit 30 is coupled to the culture chamber 20, the plurality of through holes 31 may form a plurality of accommodating spaces 33 with the substrate 21 of the culture chamber 20, and the plurality of accommodating spaces 33 may be used for accommodating the culture 40.

The accommodating space 33 may be understood as a space for accommodating the culture 40. For example, a cavity which is open at one end and closed at one end is formed by the side wall of the through hole 31 and the base 21 of the culture chamber 20. Referring to fig. 1 to 6, in some embodiments, a plurality of through holes 31 may extend in the thickness direction of the insert 30, when the insert 30 is mated with the incubation chamber 20, one end surface of the insert 30 in the thickness direction thereof contacts the base 21 of the incubation chamber 20, the other end surface of the insert 30 faces away from the base 21, and the mother sample may be poured from the other end surface of the insert 30 and flow into the accommodating space 33. In some embodiments, the number of the through holes 31 on the insert 30 may be several, so that when the insert 30 is mated with the culture chamber 20, the insert 30 and the base 21 form several accommodating spaces 33, and when the mother sample flows through the insert 30, the mother sample flows into the several accommodating spaces 33 from the open end of each accommodating space 33, so as to divide the mother sample into several cultures 40, thereby achieving the purpose of rapid mass inoculation.

Culture chamber 20 refers to a device or structure that provides a living, growing, or breeding space for a culture.

In some embodiments, culture chamber 20 can be any structure or device capable of holding culture 40. For example, the culture chamber 20 may be any one of a petri dish (as shown in fig. 1-6), a culture flask, a culture tray, or a culture tubing string.

In some embodiments, the culture chamber 20 may comprise a base 21 and a chamber sidewall 25 connected with the base 21 to form an accommodating space, and the chamber sidewall 25 may comprise an outer surface facing away from the accommodating space and an inner surface facing toward the accommodating space. Further, the culture chamber 20 may further include a cap (not shown) for enclosing the culture chamber 20. In some embodiments, the side wall, base or top cover of the culture chamber 20 may be provided with a connection hole for connecting an external pipeline, and the culture medium or gas (such as oxygen) required for culture growth may be discharged into or out of the culture chamber 20 through the pipeline. In some embodiments, the top cover may be made of a flexible film material, which may include, but is not limited to, High Density Polyethylene (HDPE), Low Density Polyethylene (LDPE), polyvinyl chloride (PVC), Chlorinated Polyethylene (CPE), chlorosulfonated polyethylene (CSPE), plasticized polyolefin (ELPO), ethylene-propylene rubber (EPDM), neoprene (CBR), butylene rubber (PBR), thermoplastic synthetic rubber, chlorohydrin rubber. In some embodiments, the medium or gas may enter the conduit by the drive of a power unit, which may include a power pump for example.

The substrate 21 may be understood as the part of the culture chamber 20 that is in contact with the culture 40. For example, when the culture chamber 20 is a petri dish, the culture 40 may be placed on the bottom wall of the culture chamber 20 for easy observation, where the bottom wall of the culture chamber 20 is the base 21.

The insert 30 may be configured to mate with the culture chamber 20 to divide a mother culture sample (i.e., the sum of all cultures 40 that need to be inoculated) flowing through the insert 30 into several sub-culture samples for the purpose of large-scale, rapid inoculation of the cultures 40. Specifically, the mother culture sample flowing through the plug 30 is divided into several sub-culture samples and flows into the accommodating spaces 33, and compared with the way of inoculating one by one, the plug 30 can realize the fast and batch inoculation of the culture 40, thereby improving the inoculation rate.

In some embodiments, for convenience of description, the undispersed culture mother sample is referred to as a mother sample, and the culture sub-samples flowed from the culture mother sample into the respective accommodating spaces 33 through the inserts 30 are referred to as cultures 40.

Mating the insert 30 with the culture chamber 20 may mean that the insert 30 and the culture chamber 20 conform in shape, size, etc. to enable the two to be assembled or mated.

In some embodiments, insert 30 may mate with an interior surface of chamber sidewall 25 such that insert 30 can be received in culture chamber 20.

In some embodiments, the insert 30 may include a sidewall, and the shape (i.e., the shape enclosed by the sidewall) and the size (i.e., the size of the shape enclosed by the sidewall) of the sidewall of the insert 30 are less than or equal to the shape (i.e., the shape enclosed by the inner surface of the chamber sidewall 25) and the size (i.e., the size of the shape enclosed by the inner surface of the chamber sidewall 25) of the inner surface of the chamber sidewall 25, so that the sidewall of the insert 30 may be located inside (the side facing away from the outer surface) the inner surface of the chamber sidewall 25. Specifically, taking the embodiment shown in fig. 1 to 3 as an example, the culture chamber 20 is a barrel-shaped structure with one open end and one closed end, and the inner surface of the chamber side wall 25 is circular; the insert 30 is a discoid member, the side surface of which is the side wall of the insert 30, the shape enclosed by the side surface of the discoid member is circular, and the size of the side surface of the discoid member can be fitted with the size of the inner surface of the insert 30.

In some embodiments, the shape of the sidewall of the insert 30 may include, but is not limited to, circular, rectangular, polygonal, and the like. In some embodiments, the shape of the sidewall of the insert 30 may be determined according to the shape of the inner surface of the chamber sidewall 25. For example, if the inner surface of the chamber side wall 25 is polygonal in shape, the side wall of the insert 30 is also polygonal in shape.

In some embodiments, the size of the side wall of the insert 30 may be smaller than the size of the inner surface of the chamber side wall 25 so that the insert 30 may smoothly protrude into the culture chamber 20. In some embodiments, such as the embodiment shown in FIGS. 1-3, the size of the side walls of the insert 30 may be equal to the size of the inner surface of the chamber side walls 25, such that the side walls of the insert 30 conform to the inner surface of the chamber side walls 25 after the insert 30 is extended into the culture chamber 20. In some embodiments, the dimensions of the sidewall of the insert 30 may include the diameter or the length of the side wall of the insert 30. When the sidewall of the insert 30 is circular in shape, the sidewall of the insert 30 is sized to be the diameter of the circle; when the sidewall of the insert 30 has a polygonal shape, the sidewall of the insert 30 has a size of a side of the polygon.

In some embodiments, the insert 30 may mate with the outer surface of the chamber sidewall 25 such that the culture chamber 20 can be received in the insert 30.

In some embodiments, the insert 30 may be provided with a receiving groove (not shown) adapted to the culture chamber 20, the receiving groove including a bottom wall and a side wall connected to the bottom wall to form a space for receiving the culture chamber 20; the outer surface shape (i.e., the shape surrounded by the outer surface of the chamber sidewall 25) and the size (i.e., the size surrounded by the outer surface of the chamber sidewall 25) of the chamber sidewall 25 are matched with the shape (i.e., the shape surrounded by the sidewall of the receiving groove) and the size (i.e., the size surrounded by the sidewall of the receiving groove) of the sidewall of the receiving groove, so that the sidewall of the receiving groove is located outside (on the side facing away from the inner surface) the outer surface of the chamber sidewall 25. In this embodiment, the culture chamber 20 can extend into the accommodating groove from one end of the opening of the accommodating groove to mate with the insert 30, so that the base 21 of the culture chamber 20 and the through hole 31 form an accommodating space 33 (as shown in fig. 2).

In some embodiments, the shape of the side wall of the receiving groove may include, but is not limited to, a circle, a rectangle, a polygon, and the like. In some embodiments, the shape of the side walls of the receiving groove may be determined according to the shape of the outer surface of the chamber side wall 25. For example, if the outer surface of the chamber sidewall 25 is polygonal, the sidewall of the accommodating groove is also polygonal.

In some embodiments, the side walls of the receiving groove may have a size larger than the outer surface of the chamber side wall 25 so that the side walls of the culture chamber 20 can smoothly protrude into the receiving groove. In some embodiments, the size of the side walls of the receiving groove can be equal to the size of the outer surface of the chamber side walls 25, such that after the culture chamber 20 protrudes into the receiving groove, the side walls of the receiving groove contact the outer surface of the chamber side walls 25. In some embodiments, the dimensions of the side walls of the receiving groove may include the diameter or side length of the side walls of the insert 30. When the side wall of the accommodating groove is in a circular shape, the size of the side wall of the accommodating groove is the diameter of the circle; when the side wall of the accommodating groove is polygonal, the side wall of the accommodating groove has a side length of the polygon.

The culture device 10 can be formed when the plug-in 30 is coupled with the culture chamber 20 for cell culture, thereby effectively increasing the inoculation rate of the culture 40.

In some embodiments, the mating of the culture chamber 20 and the insert 30 may be performed manually. For example, the operator controls the insertion member 30 to extend into the culture chamber such that the insertion member 30 remains fixed relative to the culture chamber 20. In some embodiments, the docking of the incubation chamber 20 and the insert 30 may be accomplished by additional components, such as the connection of the insert 30 and the incubation chamber 20 to robotic arms, respectively, that are manipulated by a corresponding program to automatically control the docking of the insert 30 and the incubation chamber 20.

Since the insert 30 will be in direct contact with the culture 40, there are certain requirements on the material of the insert 30 in order to ensure that the culture 40 (e.g., cultured cells) can survive, grow, multiply and maintain the primary structure and function.

In some embodiments, the insert 30 may be fabricated from a biocompatible material. After the biocompatible material is contacted with cells, tissues and body fluid, the function of the cells and the tissues is not reduced, and inflammation, canceration and rejection reaction are not generated. In some embodiments, the biocompatible material may include, but is not limited to, natural chitosan, sodium alginate, polyethylene glycol, bioceramics.

In some embodiments, the sectional shape of the through-hole 31 refers to a shape in a section perpendicular to the axial direction of the through-hole 31. In some embodiments, the cross-sectional shape of the through-holes 31 may be regular, e.g., circular, square, triangular, oval, etc. In some embodiments, the cross-sectional shape of the through-hole 31 may also be irregular.

In some embodiments, the cross-sectional shape of the through-hole 31 may be circular, and when the cross-sectional shape of the through-hole 31 is circular, the contact area between the inoculated culture 40 and the culture medium may be increased, and the nutrient absorption rate may be increased.

In some embodiments, the through-hole 31 may be cylindrical, for example, cylindrical or prismatic. In some embodiments, the through hole 31 may be conical, for example, conical or pyramidal. In some embodiments, when the through-hole 31 is in a cone shape, an upper end face of the through-hole 31 (the end face of the through-hole 31 that is away from the base 21 when the insert 30 is attached to the culture chamber 20) may be set as an end having a smaller opening (i.e., a smaller opening diameter), and a lower end face of the through-hole 31 (the end face of the through-hole 31 that is close to the base 21 when the insert 30 is attached to the culture chamber 20) may be set as an end having a larger opening (i.e., a larger opening diameter).

In some embodiments, the insert 30 may disperse the mother specimen into several identical or nearly identical (e.g., identical or nearly identical in shape, size) cultures 40. In some embodiments, the through holes 31 opened on the insert 30 have the same shape and size, so that the through holes 31 and the substrate 21 form the accommodating space 33. In large scale cell culture, several cultures 40 of the same or approximately the same volume can be inoculated in rapid, batch quantities. In some practical scenarios, the total volume of the plurality of accommodating spaces 33 may be calculated, and then a mother sample having the same volume or approximately the same volume as the total volume of the plurality of accommodating spaces 33 may be prepared, so that the mother sample may flow into the plurality of accommodating spaces 33 and be evenly divided into a plurality of cultures 40 having the same volume or approximately the same volume when flowing through the insert 30.

In some embodiments, the plurality of through holes 31 may be arranged randomly. In some embodiments, the plurality of through holes 31 may be arranged in a regular pattern to facilitate batch manipulation of the culture 40 by an operator. In some embodiments, the distance between the axes of two adjacent through holes 31 of the plurality of through holes 31 is the same. When the distances between the axes of two adjacent through holes 31 in the plurality of through holes 31 are the same, it indicates that the intervals between the two adjacent through holes 31 are the same. When the insert 30 is removed, the solid culture 40 formed in the through hole 31 (i.e. the solidified culture 40 mixed with matrigel) can be uniformly distributed on the substrate 21, which is more convenient for the operator to perform batch operation on the solid culture 40. In addition, in some embodiments, the plurality of through holes 31 may be arranged linearly or uniformly around a certain point. The arrangement of the through holes 31 can be determined according to the requirement of cell culture, and the operator can select a proper arrangement to quickly inoculate the cultures 40 arranged according to a certain rule in batches.

In some embodiments, the size of the resulting culture 40 (e.g., cultured cells) for inoculation needs to be limited to a range to avoid an oversize that would affect nutrient uptake by the culture. Specifically, for example, when the culture 40 is mixed with matrigel, in the process of inoculating the culture 40, the matrigel flows into the through holes 31, and undergoes phase transition at a certain temperature, and changes from a liquid state to a solid state to finally form solid matrigel, the culture 40 in the solid matrigel can absorb nutrients of the culture medium to grow, and if the size of the solid matrigel is too large, the culture 40 in the central portion of the solid matrigel has a low rate of absorbing the culture medium, and the growth rate is affected. For example, in the case of a cylindrical solid matrigel, if the diameter of the cylindrical solid matrigel is too large or the height of the cylindrical solid matrigel is too high, the culture 40 located at the central portion of the cylindrical solid matrigel absorbs the culture medium at a low rate. In order to ensure that the culture 40 absorbs the culture medium more uniformly and more rapidly, there is a certain requirement for the size of the formed solid matrigel, which is related to the accommodating space 33 (i.e. the space formed by the through hole 31 and the substrate 21).

In some embodiments, the aperture of the plurality of through holes 31 may be set in a range of 2000 μm to 10 cm. Preferably, the aperture of the plurality of through holes 31 may be set in the range of 1500 μm to 7.5 cm. Preferably, the aperture of the plurality of through holes 31 may be set in the range of 100 μm to 6 cm. Further preferably, the aperture of the plurality of through holes 31 may be set in a range of 500 μm to 5 cm. In some embodiments, the aperture of the through-hole may be understood as the distance between the two farthest points on the cross-sectional shape of the through-hole. For example, if the cross-sectional shape of the through-hole is a circle, the diameter of the hole is the diameter of the circle. For another example, if the cross-sectional shape of the through-hole is a rectangle, the aperture is the length of the diagonal line of the rectangle.

In some embodiments, the height of the plurality of through holes 31 is in the range of 0.1mm to 50 mm. Preferably, the height of the plurality of through holes 31 is in the range of 0.5mm to 30 mm. Preferably, the height of the plurality of through holes 31 is in the range of 1mm to 10 mm. Preferably, the height of the plurality of through holes 31 is in the range of 2mm to 5 mm.

In inoculating the culture 40, the insert 30 is first mated with the culture chamber 20, and then a mother sample can be poured from the side of the substrate 21 facing away from the insert 30, and the mother sample can flow over the surface of the insert 30 into the plurality of receiving spaces 33. During this process, a small portion of the culture 40 may adhere to the surface of the insert 30. In some embodiments, the volume of the added mother specimen may be equal to the total volume of the plurality of receiving spaces 33, which may result in waste of matrigel and culture 40 (e.g., organoids) if culture 40 is attached to the surface of insert 30, increasing culture costs.

In some embodiments, the insert 30 may include a drain that can introduce the culture 40 attached to the surface of the insert 30 into the through-hole 31 sufficiently quickly to increase the inoculation rate.

Referring to FIGS. 1 to 8, in some embodiments, the connecting surface of the plurality of through holes 31 and one side of the insert 30 is an inverted conical slope 60, wherein the bottom of the inverted conical slope 60 (i.e., the end with the smaller diameter of the inverted conical slope 60 in FIG. 8) is connected to the through hole 31, and the top of the inverted conical slope 60 (i.e., the end with the larger diameter of the inverted conical slope 60 in FIG. 8) is connected to the upper end surface of the insert 30 (the end surface of the insert 30 away from the base 21 when the insert 30 is mated with the culture chamber 20). In this embodiment, when the insert 30 is mated with the culture chamber 20 (e.g., the culture chamber 20 in FIG. 1), the top of the inverted tapered ramp 60 is connected with the end face of the insert 30 that is away from the base 21 (e.g., the base 21 shown in FIG. 1). When the mother sample flows over the surface of the insert 30, the culture 40 will flow under gravity from the top of the inverted conical slope 60 to the bottom of the inverted conical slope 60 and then into the through-hole 31 due to the presence of the inverted conical slope 60.

It should be noted that the reverse tapered slope 60 is only one embodiment of the drainage portion, and after understanding the principle and method of the drainage portion, those skilled in the art can modify the reverse tapered slope to form a three-dimensional shape with gradually enlarged opening from top to bottom (i.e. from the through hole 31 to the end surface of the insert 30 far from the base 21) to achieve the purpose of sufficiently and rapidly guiding the culture 40 attached to the surface of the insert 30 into the through hole 31. Exemplary shapes may include, but are not limited to, hemispherical, semi-elliptical, and the like.

During the course of cell culture, it is necessary to add culture media to provide nutrients to the culture 40. In some embodiments, when the culture 40 is an adherently growing cultured cell (i.e., culture 40 in a two-dimensional cell culture mode), the culture medium can be added directly to the surface of the insert 30 and brought into contact with the cultured cell. In some embodiments, when the culture 40 is mixed with matrigel, the culture medium can be added after the matrigel in the accommodating space 33 is changed from a liquid state to a solid state, so that the solid matrigel is in contact with the culture medium, and the culture 40 in the solid matrigel can absorb nutrients in the culture medium for growth and reproduction. In some embodiments, since only the solid matrigel at the open end of the accommodating space 33 can contact with the culture medium when the solid matrigel is accommodated in the accommodating space 33, the contact area is small, so that the nutrient absorption rate of the culture 40 in the solid matrigel is slow, which results in slow growth and propagation speed, and affects the efficiency of cell culture. Thus, in some embodiments, the rate of nutrient uptake by the culture 40 in the solid matrigel can be increased by increasing the contact area of the solid matrigel with the culture medium.

As shown in connection with fig. 1-6, in some embodiments, the insert 30 is movable relative to the substrate 21 to at least partially separate the culture 40 from the number of through-holes 31.

Wherein at least partially separating may mean that the side walls of the through hole 31 are at least partially separated from the culture 40 in this through hole 31, such that the culture 40 is able to at least partially drain out of the through hole 31. For example, the solid-state matrigel formed in one accommodation space 33 is separated from the sidewall of the through-hole 31. When the culture 40 is separated from the sidewall of the through-hole 31, if a culture medium is added to the culture chamber 20, the portion of the solid matrigel exposed outside the through-hole 31 may contact the culture medium, increasing the contact area of the solid matrigel with the culture medium, thereby increasing the rate of nutrient absorption of the culture 40 in the solid matrigel.

In some embodiments, culture 40 may be partially separated from through-hole 31 or may be completely separated from through-hole 31. In some embodiments, the extent of separation of the culture 40 from the through-hole 31 depends on the distance the insert 30 moves relative to the base 21, e.g., the culture 40 may be completely separated from the through-hole 31 when the distance the insert 30 moves relative to the base 21 exceeds the dimension of the culture 40 in the direction of the axis of the through-hole 31.

In some embodiments, during the movement of the insert 30, the matrigel may adhere to the side walls of the through-hole 31, which may cause the shape and size of the formed solid matrigel to change, or the solid matrigel to be damaged, so that the culture 40 wrapped in the matrigel is exposed, and the absence of matrigel from the culture 40 as a carrier for three-dimensional growth may affect the quality of the cell culture product. Therefore, the matrigel is prevented from adhering to the side wall of the through-hole 31 as much as possible when the insert 30 is moved.

In order to prevent matrigel from adhering to the side walls of the through-hole 31, in some embodiments, the difference between the adhesion of the side walls of the through-hole 31 and the adhesion of the lower end surface of the insert 30 (the end surface of the insert 30 close to the base 21 when the insert 30 is mated with the culture chamber 20) may be increased. The adhesion force may refer to the ability of the culture 40 (e.g., the culture 40 mixed with matrigel) to adhere to the surface of the object, and the larger the difference in adhesion force, the more easily the culture 40 adheres to the lower end surface of the insert 30 rather than the sidewall of the through-hole 31.

In some embodiments, the difference in adhesion of the sidewalls of the through-holes 31 and the adhesion of the lower end surface of the insert 30 may be increased by reducing the adhesion of the sidewalls of the several through-holes 31.

Referring to FIGS. 1 to 6 and 9, in some embodiments, the through hole 31 may be formed as an inverted cone structure 70, wherein the bottom of the inverted cone structure 70 (i.e., the end of the inverted cone structure 70 having a larger diameter in FIG. 9) is connected to the lower end surface of the insert 30, and the top of the inverted cone structure 70 (i.e., the end of the inverted cone structure 70 having a larger diameter in FIG. 9 is connected to the upper end surface of the insert 30. when the insert 30 is mated with the culture chamber 20 (e.g., the culture chamber 20 shown in FIG. 1), the bottom of the inverted cone structure 70 is in contact with the base 21 (e.g., the base 21 shown in FIG. 1) to form a closed space.

In some embodiments, the sidewalls of the number of through holes 31 may be coated with a hydrophobic layer made of a hydrophobic material. The hydrophobic layer may reduce the adhesion of the sidewalls of the through-holes 31 to the matrigel, thereby reducing the matrigel adhering to the sidewalls of the through-holes 31. In some embodiments, the inverted cone structure 70 may be used in combination with a hydrophobic layer, for example, a hydrophobic layer coated on the sidewalls of the inverted cone structure 70 to further reduce the matrigel adhering to the sidewalls of the via 31. In some embodiments, the hydrophobic material may include PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene), and the like. In addition to applying a hydrophobic layer, in some embodiments, low surface energy substances may be applied to the sidewalls of the through-holes 31, which may also reduce adhesion. Exemplary low surface energy materials include silicone based nano-coatings and fluorocarbon based nano-coatings.

In some embodiments, the adhesion of the sidewalls of the through-hole 31 may also be reduced by reducing the surface roughness of the sidewalls of the through-hole 31, for example, by grinding the sidewalls of the through-hole 31 with an abrasive paste.

In some embodiments, the difference in adhesion of the sidewall of the through-hole 31 and the adhesion of the lower end surface of the insert 30 may be increased by increasing the adhesion of the lower end surface of the insert 30. In some embodiments, the means for improving the adhesion of the lower end surface of the insert 30 may include etching (e.g., soft etching, photo etching, one or a combination of electron beam etching, chemical etching, and laser etching) the lower end surface of the insert 30 or processing a regular micro-nano-pillar array or nano-pillar array on the lower end surface of the insert 30.

It should be noted that the methods of reducing the adhesion of the sidewall of the through hole 31 and increasing the adhesion of the lower end surface of the insert 30 described in one or more embodiments of the present application may be combined to further increase the difference in adhesion. For example, the side walls of the through-holes 31 may be coated with a hydrophobic layer and the lower end face of the insert 30 may be etched. For another example, the sidewall of the through-hole 31 may be polished using an abrasive paste, and a micro-nano pillar array arranged in a certain rule may be processed on the lower end surface of the insert 30. Such variations are within the scope of the protection sought herein.

In some embodiments, movement of the insert 30 may be achieved by manual control. In some embodiments, movement of the insert 30 may be controlled automatically by an external device to move the insert 30. The external device may be another device independent of the culture device 10, and may be used in conjunction with the culture device 10. In some embodiments, the external device may be a robotic arm.

In some embodiments, the insert 30 may be provided with an operating portion thereon, which may be configured for an external device or manual control for the purpose of moving the insert 30.

As shown in connection with fig. 1-6, in some embodiments, the handle portion may include a protrusion 50 disposed on one of the end surfaces of the insert 30. The movement of the insert 30 relative to the culture chamber 20 is controlled by the protrusion 50. Specifically, for example, in a manual operation, when the control insert 30 is required to move, an operator may grasp the protrusion 50 and apply a force thereto to control the movement of the insert 30 away from or toward the substrate 21.

In some embodiments, the protrusions 50 may be of various shapes, including but not limited to cylindrical, prismatic. Preferably, the protrusion 50 may be cylindrical. In some embodiments, the handle portion may be any structure that facilitates handling, in addition to the protrusion 50, including but not limited to a loop structure and a hook structure.

In some embodiments, the number of the protrusions 50 may be one or more, and the number of the protrusions 50 may be related to the position where the protrusions 50 are disposed. For example, when the axis of the protrusion 50 coincides with the central axis of the insert 30, that is, the protrusion 50 is disposed on the central axis of the insert 30, only one protrusion 50 needs to be disposed to ensure that the insert is balanced when moved without deviating from the axial direction of the through-hole. For another example, when the protrusion 50 is disposed at the edge of the insert 30, and the axis of the protrusion 50 is parallel to but not coincident with the central axis of the insert 30, it may be necessary to control a plurality of protrusions 50 simultaneously to ensure that the insert is balanced when moving, and the protrusions do not deviate from the axial direction of the through hole, so the number of protrusions may be multiple.

Furthermore, the number of the protrusions 50 can be two, the connecting line between the axes of the two protrusions 50 passes through the central axis of the plug-in unit 30, and the two protrusions 50 are controlled to ensure that the plug-in unit keeps balance in the movement process, so that the axis direction of the through hole is not deviated, and the cell culture efficiency is improved.

In some embodiments, the protrusion 50 may be integrally formed with the insert 30. In some embodiments, the protrusion 50 may be formed separately from the insert 30 and then the two assembled.

In some embodiments, the insert 30 is movable away from the base 21 in the axial direction of the plurality of through holes 31 (the direction of the arrow is parallel to the axial direction of the through holes 31 as indicated by the arrow in fig. 2).

In some embodiments, the distance that the insert 30 is able to move away from the base 21 in the direction of the axis of the number of through holes 31 may be the length of the through holes 31. In some embodiments, after the insert 30 is moved away from the base 21 in the axial direction of the plurality of through holes 31 by the length of the through holes 31, the insert 30 may be removed from the culture chamber 20, i.e., the insert 30 is completely detached from the culture chamber 20.

Referring to FIG. 5, in some embodiments, after the insert 30 is mated with the culture chamber 20, the insert 30 can move along the axial direction of the plurality of through holes 31 toward the base 21 (as indicated by the arrows in FIG. 5, the direction of the arrows is parallel to the axial direction of the through holes 31) in addition to moving away from the base 21 along the axial direction of the plurality of through holes 31.

In some embodiments, the substrate 21 is provided with a number of bearing protrusions 23 that fit into a number of through holes 31; the plurality of bearing protrusions 23 can extend into the plurality of through holes 31, the bearing protrusions 23 can position the insert 30 so that the insert 30 can be coupled with the culture chamber 20, and the insert 30 can move towards the base 21. Wherein, fitting with several through holes 31 may mean that the number of the bearing protrusions 23 is the same as the number of the through holes 31, and the cross-sectional shapes of the bearing protrusions 23 and the through holes 31 in the direction perpendicular to the axis of the through holes 31 are the same. In some embodiments, the bearing protrusions 23 may form accommodating spaces 33 with the through holes 31 when the culture chamber 20 is mated with the insert 30. Specifically, since the cross-sectional shapes of the bearing protrusion 23 and the through hole 31 are the same, the end surface of the bearing protrusion 23 facing away from the substrate 21 can be matched with the sidewall of the through hole 31 to close one end of the through hole 31 close to the substrate 21, so as to form an accommodating space 33 with an open end (i.e., an open end) and a closed end (i.e., a closed end), and the liquid matrigel can flow into the accommodating space 33 from the open end of the accommodating space 33 and be accommodated in the accommodating space 33.

In some embodiments, the height of the accommodating space 33 is related to the length of the bearing protrusion 23 extending into the through hole 31. It can be understood that, when the end surface of the bearing protrusion 23 facing away from the substrate 21 is parallel to the end surface of the through hole 31 near the end of the substrate 21, that is, the bearing protrusion 23 just contacts the through hole 31, the height of the accommodating space 33 formed by the bearing protrusion 23 and the through hole 31 is equal to the height of the through hole 31. As the length of the bearing protrusion 23 extending into the through hole 31 is longer, the height of the accommodating space 33 is smaller, the amount of the matrigel that can be accommodated is smaller, and the height of the finally formed solid matrigel is smaller. Therefore, by controlling the movement of the insert 30, the depth of the bearing protrusion 23 into the through hole 31 can be adjusted, and the height of the finally formed solid matrigel can be controlled, so as to meet different culture requirements.

In some embodiments, the insert 30 may be controlled to move closer to the base 21 in order to increase the contact area of the matrigel with the culture medium after the liquid matrigel has completely transformed into solid matrigel. During the movement of the insert 30, the bearing protrusions 23 push the solid matrigel in the accommodating space 33 to move relative to the insert 30, so that the solid matrigel is gradually separated from the insert 30, and as the solid matrigel is separated from the insert 30, the larger the surface area of the solid matrigel exposed outside the insert 30 is, the larger the surface area of the solid matrigel capable of contacting with the culture medium is.

In some embodiments, the height of the plurality of bearing protrusions 23 may be smaller than the length of the plurality of through holes 31, and at least a portion of the solid-state matrix glue on the end surface of the bearing protrusion 23 away from the base 21 can be separated from the insert 30 during the process of the bearing protrusion 23 approaching the base 21. In some embodiments, the height of the plurality of bearing protrusions 23 may exceed the length of the plurality of through holes 31. In this embodiment, if the height of the bearing protrusion 23 exceeds the height of the through hole 31, it indicates that the bearing protrusion 23 can penetrate through the through hole 31 to completely separate the solid matrigel on the end surface of the bearing protrusion 23 facing away from the base 21 from the insert 30, thereby increasing the contact area with the culture medium to the maximum.

In some embodiments, the height of the plurality of bearing protrusions 23 may be less than the depth of the culture chamber 20. The culture mixed with the matrigel needs to absorb nutrients in the culture medium, the culture medium is positioned in the culture chamber 20, and when the height of the bearing protrusions 23 is smaller than the depth of the culture chamber 20, solid matrigel formed by the matrigel on the end faces, facing away from the base 21, of the bearing protrusions 23 can contact with the culture medium to absorb the nutrients.

In some embodiments, the length of the protrusion 23 extending into the through hole 31 can be adjusted by controlling the movement of the insert 30, so as to control the volume of the culture 40 flowing into the accommodating space 33, and then the insert 30 needs to be kept fixed relative to the substrate 21 after the volume of the accommodating space 33 is determined.

In some embodiments, the culture device 10 may further comprise a holding assembly that may be used to hold the insert 30 relatively stationary with respect to the culture chamber 20.

In some embodiments, the holding assembly may include a snap (not shown) provided on the sidewall of the insert 30 and a snap groove (not shown) provided on the sidewall of the culture chamber 20 and adapted to the snap; the insert 30 is fixed relative to the base 21 when the latch is mated with the latch and the insert 30 is able to move relative to the base 21 when the latch is disengaged from the latch. In some embodiments, the retaining assembly may include a stopper (not shown) disposed on a sidewall of the culture chamber 20, and the stopper may limit the movement of the insert 30 relative to the base 21. In some embodiments, the stop may be a stop plate or a stop protrusion extending toward the center of the culture chamber 20, and when the insert 30 abuts against the stop plate or the stop protrusion, the insert 30 cannot move. In some embodiments, the stop protrusion or stop plate may have a certain elasticity, such that an operator may force the insert 30 to break through the limit of the stop by pushing or pulling the insert 30. In some embodiments, the retainer bump or retainer flap may be made of an elastic material. In some embodiments, the retaining assembly may also include a pinning assembly, a screw-nut assembly, or the like.

In some embodiments, the mating of the insert 30 to the culture chamber 20 may be detachable, i.e., the insert 30 may be removed after the mating of the insert 30 to the culture chamber 20. For example, the snap has a certain elasticity to allow disengagement from the snap groove for detaching the insert 30 from the culture chamber 20. In some embodiments, the mating of the insert 30 to the culture chamber 20 may not be removable, i.e., the insert 30 is not removable after the mating of the insert 30 to the culture chamber 20. For example, the insert 30 is adhesively mated with the culture chamber 20, and after mating, the insert 30 is fixed relative to the culture chamber.

Based on the culture device 10 of one or more of the foregoing embodiments, the method of cell culture may include the steps of:

first, the insert 30 is coupled to the culture chamber 20, the total amount of the inoculated culture 40 (i.e., the volume of the mother sample) is determined, and the mother sample is added to the insert 30, and the mother sample flows into the plurality of accommodating spaces 33 while passing through the insert 30, so as to be divided into a plurality of cultures 40; if the culture 40 is a liquid culture 40, adding a culture medium on the surface of the insert 30 to make the culture 40 contact with the culture medium to absorb nutrients required for growth and reproduction; if the culture 40 is mixed with matrigel, heating the matrigel to change the matrigel from a liquid state to a solid state, and when the matrigel is changed to the solid state, the matrigel can be used as a carrier for three-dimensional cell culture to provide a three-dimensional growth space for the culture 40 wrapped in the matrigel; after the matrigel in the plurality of accommodating spaces 33 is completely solidified, a culture medium is added on the surface of the insert 30 so that the culture 40 is in contact with the culture medium to absorb nutrients required for growth.

In some embodiments, if culture chamber 20 is a sealed structure (e.g., culture chamber 20 is enclosed by a cap), the culture medium and the gas (e.g., oxygen) required for growth of culture 40 may be driven by the power unit into culture chamber 20 via tubing.

In some embodiments, when the matrigel in the plurality of accommodating spaces 33 is completely solidified, the insert 30 may be controlled to move relative to the base 21 along the axial direction of the plurality of through holes 31 so that the matrigel is at least partially exposed out of the plurality of accommodating spaces 33, thereby increasing the contact area between the matrigel and the culture medium and increasing the culture medium absorption rate of the culture.

In some embodiments, when the matrigel in the plurality of accommodating spaces 33 is completely solidified, the insert 30 may be controlled to move away from the base 21 in the axial direction of the plurality of through holes 31 so that the matrigel is at least partially exposed out of the plurality of accommodating spaces 33. After the matrigel is at least partially exposed out of the plurality of accommodating spaces 33, a culture medium may be added to the culture chamber 20.

In some embodiments, the manner in which the insert 30 is controlled to move may be manually controlled, for example, by an operator directly holding the protrusion 50 with a hand to move the insert 30. In some embodiments, the manner in which the control insert 30 is moved may be automated. For example, the card 30 may be coupled to a robotic arm that is manipulated by a corresponding program to automatically control the movement of the card.

In some embodiments, when the insert 30 moves a distance that exceeds the height of the solid matrigel, the solid matrigel is completely separated from the insert 30 (except for the portion in contact with the base 21) (as shown in fig. 3).

In some embodiments, when the matrigel in the plurality of receiving spaces 33 is completely solidified, the control insert 30 moves close to the base 21 in the axial direction of the plurality of through holes 31 such that the solid matrigel is at least partially exposed out of the plurality of receiving spaces 33 (as shown in fig. 6). The bearing protrusion 23 in one or more of the foregoing embodiments may be combined with the present embodiment, specifically, before performing cell culture, the insert 30 needs to be first coupled with the culture chamber 20, so that the through hole 31 and the bearing protrusion 23 form an accommodating space 33; when the mother sample is added to the receiving space 33 and completely solidified, the control insert 30 is moved along the axial direction of the bearing protrusion 23 close to the substrate 21 so that the bearing protrusion 23 at least partially protrudes out of the through hole 31, leaving the culture at the end of the bearing protrusion 23 at least partially exposed out of the receiving space 33.

In some embodiments, the volume of the accommodating space can be adjusted by controlling the movement of the insert 30 before cell culture, and the closer the insert 30 is to the substrate 21, the smaller the volume of the accommodating space 33; the farther the insert 30 is from the base 21, the greater the volume of the receiving space 33.

In some embodiments, after the insert 30 is removed, the culture chamber 20 may be enclosed with a cover, and then the culture medium and the gas (e.g., oxygen) required for growth of the culture 40 driven by the power unit are introduced into the culture chamber 20 through the pipeline.

The beneficial effects that may be brought by the embodiments of the present application include, but are not limited to: (1) the auxiliary inoculation device (for example, a plug-in unit) is additionally arranged, so that the mother sample can be quickly divided into a plurality of cultures, and the auxiliary inoculation device can respectively accommodate the plurality of cultures in a plurality of accommodating spaces, so that the quick and batch inoculation of the cultures is realized, and the cell culture efficiency is improved; (2) the plug-in unit can be conveniently controlled by an operator by arranging the operation part (such as a bulge) on the plug-in unit; (3) the bearing bulge is arranged on the substrate, so that the bearing bulge and the through hole are matched to form the accommodating space, the volume of the accommodating space can be controlled by adjusting the height of the plug-in unit, and the volume of the culture accommodated in the accommodating space can be adjusted more conveniently; (4) by coating the hydrophobic layer on the side wall of the through hole, the culture attached to the side wall of the through hole when the plug-in moves relative to the culture can be reduced, the waste of the culture is reduced, and the cost is reduced; (5) by moving the plug-in unit, the culture is at least partially separated from the side wall of the through hole (or the accommodating space), the contact area between the culture and the culture medium is increased, the nutrient absorption rate of the culture is effectively improved, and the cell culture efficiency is further improved. It is to be noted that different embodiments may produce different advantages, and in different embodiments, any one or combination of the above advantages may be produced, or any other advantages may be obtained.

Having thus described the basic concept, it will be apparent to those skilled in the art that the foregoing detailed disclosure is to be considered merely illustrative and not restrictive of the broad application. Various modifications, improvements and adaptations to the present application may occur to those skilled in the art, although not explicitly described herein. Such modifications, improvements and adaptations are proposed in the present application and thus fall within the spirit and scope of the exemplary embodiments of the present application.

Also, this application uses specific language to describe embodiments of the application. Reference throughout this specification to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic described in connection with at least one embodiment of the present application is included in at least one embodiment of the present application. Therefore, it is emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various places throughout this application are not necessarily all referring to the same embodiment. Furthermore, some features, structures, or characteristics of one or more embodiments of the present application may be combined as appropriate.

Additionally, the order in which elements and sequences of the processes described herein are processed, the use of alphanumeric characters, or the use of other designations, is not intended to limit the order of the processes and methods described herein, unless explicitly claimed. While various presently contemplated embodiments of the invention have been discussed in the foregoing disclosure by way of example, it is to be understood that such detail is solely for that purpose and that the appended claims are not limited to the disclosed embodiments, but, on the contrary, are intended to cover all modifications and equivalent arrangements that are within the spirit and scope of the embodiments herein. For example, although the system components described above may be implemented by hardware devices, they may also be implemented by software-only solutions, such as installing the described system on an existing server or mobile device.

Similarly, it should be noted that in the preceding description of embodiments of the application, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure aiding in the understanding of one or more of the embodiments. This method of disclosure, however, is not intended to require more features than are expressly recited in the claims. Indeed, the embodiments may be characterized as having less than all of the features of a single embodiment disclosed above.

Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present application. Other variations are also possible within the scope of the present application. Thus, by way of example, and not limitation, alternative configurations of the embodiments of the present application can be viewed as being consistent with the teachings of the present application. Accordingly, the embodiments of the present application are not limited to only those embodiments explicitly described and depicted herein.

Claims

1. A culture device, comprising:

a culture chamber comprising a substrate;

the plug-in is matched with the culture chamber and is provided with a plurality of through holes;

when the plug-in is matched and connected with the culture chamber, the through holes can form a plurality of accommodating spaces with the substrate of the culture chamber, and the accommodating spaces are used for accommodating cultures.

2. The culture device of claim 1, wherein the insert is made of the biocompatible material.

3. The culture device of claim 1, wherein the insert is movable relative to the base to at least partially separate the culture from the number of through-holes.

4. The culture device of claim 3, wherein the insert is movable away from the base in a direction of an axis of the plurality of through-holes.

5. The culture device of claim 3, wherein the substrate is provided with a plurality of bearing protrusions fitting with the plurality of through holes; the bearing protrusions can extend into the through holes, so that the insert can move towards the substrate.

6. The culture device of claim 3, further comprising a retaining assembly for retaining the insert in fixed relation to the substrate.

7. The culture device of claim 6, wherein the holding assembly comprises a snap provided on the side wall of the insert and a catch provided on the side wall of the culture chamber to fit the snap;

when the buckle is matched and connected with the clamping groove, the plug-in piece and the base are relatively fixed, and when the buckle is separated from the clamping groove, the plug-in piece can move relative to the base.

8. The culture device of claim 1, wherein the plurality of through holes are circular in cross-sectional shape.

9. The culture device of claim 8, wherein the plurality of through holes have a diameter in the range of 100 μm to 5 cm.

10. The culture device of claim 1, wherein the connection surface of the plurality of through holes and the upper end surface of the insert is an inverted conical slope surface, wherein the bottom and the top of the inverted conical slope surface are respectively connected with one of the end surfaces of the plurality of through holes and the insert.

11. The culture device of claim 1, wherein the side walls of the plurality of through holes are coated with a hydrophobic layer made of a hydrophobic material.

12. The culture device of claim 1, wherein the culture is mixed with matrigel.

13. A culture method based on a culture device, characterized in that the culture device comprises:

a culture chamber comprising a substrate; the plug-in is matched with the culture chamber and is provided with a plurality of through holes; when the plug-in is matched and connected with the culture chamber, the through holes and the substrate of the culture chamber can form a plurality of accommodating spaces, and the accommodating spaces are used for accommodating cultures; the method comprises the following steps:

mating the insert with the culture chamber and adding the culture to the number of through holes on the insert;

adding a culture medium to at least partially contact the culture with the culture medium, wherein the culture medium is used to provide a nutrient substrate to the culture.

14. The culture device-based cultivation method according to claim 13, further comprising:

after the culture in the accommodating spaces is solidified, the insert is controlled to move along the axial direction of the through holes and away from the base so that the culture is at least partially exposed out of the accommodating spaces.

15. The culture device-based cultivation method according to claim 13, wherein the substrate is provided with a plurality of bearing protrusions fitted with the plurality of through holes; the culture method further comprises the following steps:

when the culture in the accommodating spaces is solidified, the insert is controlled to move close to the substrate, so that the bearing protrusions at least partially extend out of the through holes, and the culture is at least partially exposed out of the accommodating spaces.