CN113355238A

CN113355238A - Culture device and culture method based on culture device

Info

Publication number: CN113355238A
Application number: CN202110648527.5A
Authority: CN
Inventors: 王玄
Original assignee: Shanghai Ruiyu Biotech Co Ltd
Current assignee: Shanghai Ruiyu Biotech Co Ltd
Priority date: 2021-06-10
Filing date: 2021-06-10
Publication date: 2021-09-07
Also published as: WO2022257296A1; WO2022258037A1

Abstract

The specification relates to the technical field of cell culture, in particular to a culture device and a culture method based on the culture device. The culture device comprises a liquid drop generating plate and a base, wherein a plurality of flow channels are formed in the liquid drop generating plate along the thickness direction; the base and the liquid drop generating plate enclose an accommodating space; each flow channel sequentially comprises a collecting hole and a liquid drop generating hole in the vertical direction, the collecting hole in each flow channel is communicated with the liquid drop generating hole, and the collecting hole is used for accommodating a culture for forming liquid drops; an overflow groove is arranged between the adjacent flow channels; the overflow channel serves to limit the volume of culture in the collection well to no more than that required to generate the droplets. The culture device can form a plurality of uniform droplets by a single drop of liquid.

Description

Culture device and culture method based on culture device

Technical Field

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

Background

The cell culture consumables in the current market need to be gradually dripped and inoculated one by one. Since some of the matrigel used in cell culture is temperature sensitive, it is easy to solidify the matrigel solution before the addition is complete during routine laboratory procedures. It is therefore desirable to provide a device that can form multiple uniform droplets in a single drop.

Disclosure of Invention

One embodiment of the present specification provides a culture apparatus. The culture device includes: the liquid drop generating plate is provided with a plurality of flow channels along the thickness direction; a base, wherein the base and the liquid drop generating plate enclose an accommodating space which is used for accommodating a culture solution; each flow channel sequentially comprises a collecting hole and a liquid drop generating hole in the vertical direction, the collecting hole in each flow channel is communicated with the liquid drop generating hole, and the collecting hole is used for accommodating a culture for forming liquid drops; an overflow groove is formed between every two adjacent flow channels; the overflow launder is configured to limit the volume of culture in the collection well to no more than the volume required to produce the droplets.

One of the embodiments of the present specification provides a culture method using a culture apparatus. The culture device includes: the liquid drop generating plate is provided with a plurality of flow channels along the thickness direction; a base, wherein the base and the liquid drop generating plate enclose an accommodating space which is used for accommodating a culture solution; each flow channel sequentially comprises a collecting hole and a liquid drop generating hole in the vertical direction, the collecting hole in each flow channel is communicated with the liquid drop generating hole, and the collecting hole is used for accommodating a culture for forming liquid drops; an overflow groove is formed between every two adjacent flow channels; said overflow launder being adapted to limit the volume of said culture within said collection well to no more than the volume required to produce said droplets; the method comprises the following steps: introducing a culture into a collection hole of one of the flow channels so as to form a hanging drop in a drop generation hole of the flow channel, wherein the volume of the culture is the product of the volume of the hanging drop and the number of the flow channels; and adding a culture solution into the accommodating space so that the culture is at least partially contacted with the culture solution, wherein the culture solution is used for culturing the culture.

Drawings

The present description 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 disassembled view of a culture device according to some embodiments of the present disclosure;

FIG. 2 is a schematic structural view of an assembled state of a culture device according to some embodiments of the present disclosure;

FIG. 3 is a schematic diagram of a droplet generator plate according to some embodiments of the present disclosure;

FIG. 4 is a cross-sectional view of a droplet generator plate according to some embodiments of the present description;

FIG. 5 is a schematic view of a flow-through channel according to some embodiments herein;

FIG. 6 is a cross-sectional view of a droplet generator plate according to some embodiments of the present description;

FIG. 7 is a schematic diagram of a culture device according to some embodiments of the present disclosure;

FIG. 8 is a cross-sectional view of a culture device according to some embodiments of the present disclosure.

Description of reference numerals: 100-a culture device; 110-a droplet generator plate; 111-a flow-through channel; 1111-collection holes; 1112-droplet generation of holes; 1113-barrier structure; 1114-a flow guiding zone; 112-an overflow launder; 113-a via; 120-a base; 130-a receiving space; 131-a sub-accommodation space; 133-a separator; 140-a cover plate; 150-first culture fluid inlet and outlet; 160-culture access port; 170-second culture solution inlet and outlet.

Detailed Description

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

It should be understood that "system", "apparatus", "unit" and/or "module" as used herein is a method for distinguishing different components, elements, parts, portions or assemblies at different levels. However, other words may be substituted by other expressions if they accomplish the same purpose.

As used in this specification 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.

Flow charts are used in this description to illustrate operations performed by a system according to embodiments of the present description. It should be understood that the preceding or following operations are not necessarily performed in the exact order in which they are performed. Rather, the various steps may be processed in reverse order or simultaneously. Meanwhile, other operations may be added to the processes, or a certain step or several steps of operations may be removed from the processes.

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.

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 be formed by mixing cells with matrigel. In some embodiments, the culture may also be formed by mixing cells with a culture medium. In some embodiments, the culture may be a single cell mixed with a culture solution or matrigel, or a plurality of cells mixed with a culture solution or 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.

The culture solution refers to a nutrient solution for the growth and propagation of the culture, is prepared by combining different nutrient substances, and is used for providing nutrition for the culture of the culture or simulating different environments for cell culture. In some embodiments, the culture device can be used for 2D cell culture or 3D cell mass culture. In the 2D cell culture, a culture (for example, cultured cells) is attached to a substrate of a culture chamber in an adherent manner to grow and propagate, or may be grown and propagated in a suspension manner in a culture solution. 3D cell culture can refer to scaffold-based culture using natural materials (e.g., natural hydrogels (e.g., protein-based natural hydrogels))

Matrigel)) or a synthetic material (e.g., a synthetic gel (e.g., a polyethylene glycol hydrogel)) provides support for aggregation, proliferation, and migration of the culture, i.e., provides a three-dimensional environment for growth of the culture, maintains the culture in a state of growth in the three-dimensional environment, and ultimately results in a three-dimensional cell culture product (e.g., organoids). In some embodiments, matrigel may serve as a vehicle for 3D 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. 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 3D 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 state of growing in the three-dimensional environment, so that the culture can be cultured in three dimensions. 3D cell culture may also refer to scaffold-free based cultures, relying on specialized culture vessels or through tissueThe cells are attached to the surface of the vessel to promote self-aggregation of the cells, e.g., culture based on forced floating, hanging drop methods.

In some embodiments, the matrigel may be a temperature sensitive gel that may undergo a phase transition from one material state to another as the temperature changes. For example, 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 culture device may be used for organoid culture, which is described hereinafter as an example for ease of description. As mentioned above, organoid culture usually requires supporting with a matrigel scaffold material, suspending the organoid in a matrigel solution, and dropping the solution into the culture material, and after the matrigel solution is solidified, the culture solution is required to be matched to maintain the culture. Due to the action of gravity, the organoid is easy to settle to the surface of the culture material before the matrigel is solidified, and in the processes of cell amplification and growth before and after the organoid is attached to the surface of the culture material, a 2D adherent growth layer is formed, which is not beneficial 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 culturing cells using a hanging drop, and is generally used for forming and culturing a 3D cell mass. Due to the action of gravity, the cells in the liquid drop tend to grow downwards without contacting with the culture material, and the adherent growth of the cells can be well inhibited. However, in the suspension drop formed by the culture without the scaffold, the nutrient content is limited, and the nutrient content is difficult to be added into the liquid suspension drop.

However, in some embodiments, when large-scale 3D cell culture is involved, serial dropping is required to form multiple hanging drops, which reduces the efficiency of cell culture and may also lead to failure of cell culture. In view of the above, in some embodiments, a culture device 100 is provided that can form a plurality of uniform droplets by a single drop of liquid.

FIG. 1 is a disassembled view of a culture device according to some embodiments of the present disclosure. FIG. 2 is a schematic structural view of an assembled state of a culture apparatus according to some embodiments of the present disclosure.

As shown in fig. 1 and 2, in some embodiments, culture device 100 can include a droplet-generating plate 110, a base 120, and a cover plate 140.

In some embodiments, the base 120 may enclose an accommodation space 130 with the droplet generator plate 110. In some embodiments, the accommodating space 130 can prevent both external bacteria or microorganisms from contaminating the culture and foreign substances (such as dust, etc.) in the external environment from entering the culture main body, thereby avoiding contamination of the environment in which the organisms are cultured. In some embodiments, the receiving space 130 can be used to receive PBS (phosphate buffered saline) or culture solution to provide a suitable growth environment (e.g., to maintain adequate or provide nutrients) for cell culture in the hanging drop. For example, in the case of the culture without a scaffold, the PBS or the culture solution contained in the containing space 130 can ensure a certain humidity to prevent the liquid hanging drop from volatilizing. For another example, when culturing on a scaffold basis (e.g., a culture mixed with matrigel is added to the droplet-generating plate 110 to form a hanging drop and solidified), the culture fluid in the holding space 130 is contacted with the solidified hanging drop to provide nutrients for cell culture in the solidified hanging drop. The cover plate 140 may be disposed on a side of the droplet-generating plate 110 away from the base 120, and the cover plate 140 may be provided with a culture access opening 160. In some embodiments, the base 120 and the droplet-generating plate 110 may be attached by snapping, bonding, molding, or by a retaining assembly, among other means. In some embodiments, the droplet-generating plate 110 has a plurality of flow channels 111 formed along the thickness direction, and the flow channels 111 are used for forming the culture into droplets or hanging droplets. FIG. 3 is a schematic diagram of a droplet generator plate according to some embodiments of the present disclosure. FIG. 4 is a cross-sectional view of a droplet generator plate according to some embodiments of the present disclosure. FIG. 5 is a schematic view of a flow-through channel according to some embodiments of the present disclosure.

In some embodiments, each flow channel 111 may have a structure with the same cross-sectional area at any position. For example, as shown in fig. 3 and 4, the flow channel 111 may be formed in a cylindrical or rectangular parallelepiped structure. In some embodiments, each flow channel 111 may have a different cross-sectional area at any location. For example, as shown in fig. 5, it may be formed with a flow channel 111a having an hourglass-shaped outer contour, or a flow channel 111b having a rectangular top end and a circular bottom end in the vertical direction. In some embodiments, the flow channel 111 may also form a truncated cone, a spindle, or other shaped structure, which is not limited in this specification.

In some embodiments, as shown in fig. 4, each flow channel 111 includes a collection hole 1111 and a droplet generation hole 1112 in sequence in a vertical direction (in a gravity direction). In some embodiments, the collection well 1111 and the droplet generation well 1112 in each flow channel 111 are in communication, and the culture flows through the collection well 1111 to the droplet generation well 1112 at the end away from the collection well 1111 to form droplets.

In some embodiments, the pooling wells 1111 are used to contain the culture that forms the droplets.

In some embodiments, as shown in fig. 3 and 4, the end of the pooling well 1111 distal to the droplet-generating well 1112 is further provided with a flow-directing region 1114, and the flow-directing region 1114 may be used to pool the culture into the pooling well 1111. In some embodiments, the flow guiding region 1114 may be formed with a large cross-section at one end and a small cross-section at the other end, the large cross-section end of the flow guiding region 1114 may engage the surface of the droplet generation plate 110, and the small cross-section end of the flow guiding region 1114 may engage the collection hole 1111. In some embodiments, the flow guiding region 1114 may include a tilted flow guiding plate, the bottom end of which engages the collection hole 1111. In some embodiments, as shown in fig. 3, the flow guiding region 1114 may be a rounded structure at one end of the collecting hole 1111, and the flow guiding effect is achieved by an inclined surface formed by the rounded structure. Furthermore, in some embodiments, the flow guiding region 1114 may also be formed in other configurations, such as, for example, a spiral configuration, and the like, without limitation.

In some embodiments, the flow-guiding region 1114 may be covered with a hydrophobic material. For example, the hydrophobic material includes polytetrafluoroethylene, fluorinated polyethylene, fluorocarbon wax, polyolefin, polycarbonate, polyamide, polyacrylonitrile, polyester, or molten paraffin, or the like, or any combination thereof. In some embodiments, the flow-guiding region 1114 may be covered with a hydrophilic material. For example, the hydrophilic material may include hydrophilic cotton, hydrophilic fibers, hydrophilic leather, or the like, or any combination thereof. In some embodiments, when the flow-directing region 1114 is covered with a hydrophobic material, the cross-sectional area of the side of the flow-directing region 1114 distal to the pooling hole 1111 may be greater than when covered with a hydrophilic material to facilitate the flow of the culture to the pooling hole 1111 under the force of gravity. In some embodiments, the flow-guiding region 1114 may not be covered with a hydrophilic material, a hydrophobic material, or other materials.

In some embodiments, as shown in fig. 3 and 4, an overflow groove 112 is formed between adjacent flow channels 111. In some embodiments, the overflow channel 112 is used to limit the volume of culture in the collection well 1111 to no more than the volume required to generate droplets. The desired total amount of culture can be added to the pooling well 1111 of any one of the several flow channels 111 and can be calculated by multiplying the number of droplets required by the volume of culture required to generate a single droplet. Culture in excess of the volume required to produce a single droplet can flow through the overflow channel 112 to the collection well 1111 of the adjacent flow channel 111 until no more than the volume required to produce a single droplet is present in the collection well 1111 of the desired flow channel 111 on the droplet generator plate 110.

In some embodiments, as shown in fig. 3 and 4, the overflow channel 112 can be in communication with the collection hole 1111. In some embodiments, the overflow channel 112 can be in communication with a flow guide 1114, the flow guide 1114 can direct the culture to a collection well 1111, the collection well 1111 can be filled with the culture and can overflow, and the overflowing culture can then flow through the overflow channel 112 into the adjacent collection well 1111. It will be appreciated that when a culture is received in excess of the volume required to generate a single droplet, the culture will flow to the adjacent flow-directing region 1114 through an overflow chute 112 in communication with the flow-directing region 1114.

FIG. 6 is a cross-sectional view of a droplet generator plate according to some other embodiments of the present disclosure.

In some embodiments, the flow channels 111 on the droplet generation plate 110 may be in communication in groups, as shown in fig. 6, in which 9 flow channels 111 are divided into three groups, each group includes three flow channels 111, the collection holes 1111 of the same group are in communication with the flow guiding region 1114 through the overflow chutes 112 to generate droplets at the droplet generation holes 1112, and the overflow chutes 112 are not disposed between the flow channels 111 of different groups. In some embodiments, by grouping the flow channels 111, batch injection of cultures or separate feeding of different cultures can be realized to realize control culture of the cultures, etc.

In some embodiments, the distance between adjacent drop generating apertures 1112 is not less than a first predetermined distance threshold to prevent the droplets generated by adjacent drop generating apertures 1112 from coalescing. For example, the distance between adjacent droplet generation holes 1112 is not less than 2 mm. As another example, adjacent drop generating apertures 1112 are 3mm apart. In some embodiments, the distance between adjacent droplet generation apertures 1112 can be different depending on the culture liquid. For example, the distance between adjacent drop generating apertures 1112 may also be less than 2 mm.

In some embodiments, as shown in fig. 4 or 6, a droplet generation orifice 1112 is located at an end remote from the collection orifice 1111 for forming hanging droplets. In some embodiments, the end of the generating aperture distal from the pooling aperture 1111 is provided with a circular cross-section for forming a hanging drop.

In some embodiments, the size of the droplet-generating apertures 1112 may be determined according to the following process:

when the culture medium in the hole is small in amount, the liquid pressure P is higher₁(P₁Where ρ is the liquid density and g is the acceleration of gravity) is less than the pressure P generated at the drop outlet by the surface tension of the liquid₂(P₂γ × l sin θ/S). Wherein gamma represents the surface tension coefficient of the liquid at the air interface, l represents the perimeter of a liquid drop outlet, S represents the area of the liquid drop outlet, h represents the highest liquid level height which can be formed by the culture in the hole, and h is related to the wettability of the surface of the hole wall material to the liquid, and the wettability can be represented by the contact angle theta of the liquid on the surface of the material.

When the liquid level in the hole rises to h > γ × sin θ/S ρ g, the liquid will flow out of the outlet. In the process, the liquid pressure leads the contact angle theta of the liquid and the surface of the hole wall material at the outlet to be gradually increased, and the theta is not increased after the theta reaches the maximum advancing contact angle.

Therefore, in order to flow out the liquid from the droplet-generating holes 1112, it is necessary to determine the perimeter and area of the opening of the generating hole (if the opening is a circular opening, the radius of the opening), the maximum advancing contact angle of the liquid on the material surface, and the surface tension coefficient of the liquid. When the culture liquid is determined, the larger the generating well opening, the lower the highest liquid level that can be carried by the droplet generating well 1112.

Whether the liquid drops from the droplet-generating holes 1112 depends on the volume, viscosity, and surface tension of the liquid droplet. The liquid moves downward in the droplet forming holes 1112 until the lower surface of the liquid reaches the opening position, and necking or breaking may occur after the liquid gradually assumes a hemispherical shape as the amount of the liquid increases.

When the droplet formed by the liquid flowing out of the droplet formation hole 1112 is broken, mg ═ l ×. gamma. ×, κ exists, where m is the mass of the culture liquid pooled in the pooling hole 1111 and κ is the correction factor (independent of factors such as liquid surface tension, material properties, liquid density and viscosity). However, when the droplet formed by the liquid flowing out of the droplet forming hole 1112 is suspended and does not break or neck, the value ranges of m and l can be determined by using mg < l x γ x κ. It should be noted that the above calculation is only an example, and the perimeter and the cross-sectional area of the droplet generation holes 1112 are not limited in this specification.

In some embodiments, as shown in fig. 4, a blocking structure 1113 is provided between the collection well 1111 and the droplet generation well 1112, and the blocking structure 1113 is used to limit the speed at which the culture forms droplets, i.e., the blocking structure 1113 may be used to prevent the culture added to the collection well 1111 from dripping out of the droplet generation well 1112 before being dispersed to other collection wells 1111 through the overflow chute 112.

In some embodiments, the barrier structures 1113 comprise at least one fibrous or strip-like structure disposed radially along the droplet-generating apertures 1112. After the culture is contacted with the blocking structure 1113, the blocking structure 1113 comprises at least one fiber-shaped or strip-shaped structure, which can divide the contact surface of the culture and the blocking structure into at least two small areas, and the carrying capacity of the flow channel 111 provided with the blocking structure 1113 for the culture is larger than that of the flow channel 111 not provided with the blocking structure 1113. The wettability of the surface of the barrier structure 1113 to the culture liquid and the arrangement density of the fiber-shaped or strip-shaped structures can determine the residence time of the culture liquid on the barrier structure 1113, and the slower the wetting speed of the barrier structure 1113 is, the higher the arrangement density of the fiber-shaped or strip-shaped structures is, the longer the residence time of the culture liquid is, and the barrier effect disappears after the barrier structure 1113 is completely wetted. When the barrier effect disappears, the culture liquid forms a drop or a hanging drop.

In some embodiments, the barrier structures 1113 may be a fiber-like or a stripe-like structure with a cross-hatched structure, wherein the smallest grid cell area is ≦ 1/2 for the cross-sectional area of the collection holes 1111 or the droplet generation holes 1112. In some embodiments, the barrier structures 1113 may be formed by non-intersecting fiber-like or stripe-like structures. In some embodiments, the longest cross-sectional diameter of the fibrous or ribbon-like structure can range from 10 μm to 1 mm. In some embodiments, the longest cross-sectional diameter of the fibrous or ribbon-like structure may be 10 μm. In some embodiments, the maximum diameter of the cross-section of the fibrous or strip-like structure may range from 1 mm. In some embodiments, the longest cross-sectional diameter of the fibrous or ribbon-like structure can range from 50 μm to 0.1 mm. In some embodiments, the material of the barrier structures 1113 may include at least one of: nylon, polypropylene, terylene, Polyethylene (PE), polypropylene (PP), Polyamide (PA), polyethylene terephthalate (PET) and other polymer materials. In some embodiments, the material of the barrier structures 1113 may include at least one of: stainless steel, copper, platinum, gold, and other stable metals or alloys. In some embodiments, the material of the barrier 1113 may be a memory alloy that has a shape memory effect, and that lengthens when heated and recovers when cooled. The barrier structure 1113 made of memory alloy can keep a small grid at low temperature and/or room temperature, and has a good barrier effect on liquid. At the temperature of cell culture (e.g., 37 ℃), the barrier structures 1113 deform, the mesh becomes larger, and the blocked liquid falls through the barrier structures 1113 and forms a drop or a hanging drop at a specified position. In some embodiments, the material of the barrier structure 1113 may be a nickel titanium alloy, which is one of the memory alloys.

The distance from the barrier 1113 to the overflow trough 112 may determine the volume of culture fluid that the collection wells 1111 can collect. The height of the liquid contained in the collection wells 1111 is required to be greater than γ × l sin θ/S ρ g, and the culture liquid can flow downward. The blocking structure 1113 and the end of the droplet generation hole 1112 far from the collecting hole 1111 also need to be ensured with a certain distance, which is such that when the droplet flowing from the collecting hole 1111 stops falling, its hanging position is not higher than the lower surface of the blocking structure 1113, and if the droplet hanging position is higher than the blocking structure 1113, the culture such as cells, organoids and the like in the droplet may adhere to the blocking structure 1113 to form 2D culture, which results in failure of 3D culture.

FIG. 7 is a schematic diagram of the structure of culture device 100 according to some embodiments of the present disclosure. FIG. 8 is a cross-sectional view of a culture device 100 according to some embodiments of the present disclosure.

In some embodiments, as shown in fig. 7 and 8, the base 120 may further have a first culture solution inlet and outlet 150, and the first culture solution inlet and outlet 150 may be used for introducing a culture solution into the accommodating space 130 for culturing. In some implementations, the first culture fluid access 150 can also be disposed through the droplet-generating plate 110. In some embodiments, a partition 133 may be further disposed in the accommodating space 130, the partition 133 divides the accommodating space 130 into a plurality of sub-accommodating spaces 131, the plurality of sub-accommodating spaces 131 may be respectively provided with a first culture solution inlet/outlet 150, and different components of culture solutions may be respectively introduced into the plurality of sub-accommodating spaces 131 through the first culture solution inlet/outlet 150 corresponding to the sub-accommodating spaces 131, so as to perform a control experiment. Illustratively, the culture solution is a drug solution which can interact with the cells, different drug solutions are respectively introduced into the plurality of sub-accommodation spaces 131 through the first culture solution inlets and outlets 150 corresponding to the sub-accommodation spaces 131, and the cell activity reduction degree or other effective data corresponding to the sub-accommodation spaces 131 is measured after the culture is finished, so as to screen out effective drugs for treating diseases corresponding to the cells from the multiple drug solutions used according to the effective data, or screen out the concentration optimal for the disease curative effect from different concentration solutions of the same drug.

In some embodiments, as shown in fig. 1 and 2, the culture access port 160 can be positioned to mate with any of the pooling wells 1111 of the droplet-generating plate 110 such that the culture accessed from the culture access port 160 can enter the corresponding pooling well 1111. In some embodiments, the culture introducing port 160 may not be provided corresponding to any one of the pooling holes 1111 of the droplet-generating plate 110, and a drainage tube or a drainage groove may be provided between the culture introducing port 160 and any one of the pooling holes 1111, so that the culture introduced from the culture introducing port 160 may be introduced into the corresponding pooling hole 1111. In some embodiments, power may be provided by a powered system, such as a pump, to pass the culture to the culture access port 160. In some embodiments, the culture itself can also be caused to flow toward the culture access port 160 by its own weight. In some embodiments, the culture introduction port 160 may be formed as a hollow structure (e.g., a funnel structure, etc.) provided to protrude from the cap plate 140. In some embodiments, the culture access port 160 may be formed as a tubular structure disposed to protrude from the cover plate 140 (e.g., as shown in fig. 1 or 2) to facilitate insertion of a conduit into which the culture is accessed. In some embodiments, the culture access port 160 may not protrude from the cover plate 140, and the shape of the culture access port 160 may not be limited.

In some embodiments, as shown in fig. 1 and 2, the cover plate 140 has a second culture solution inlet/outlet 170, and the droplet-generating plate 110 has a through hole 113, wherein the through hole 113 is communicated with the second culture solution inlet/outlet 170 so that the culture solution can enter the accommodating space 130. In some embodiments, the first and/or

second broth ports

150, 170 may also be disposed through the cover plate 140 (i.e., the droplet-generating plate 110), and the first and/or

second broth ports

150, 170 may be used to input and/or discharge broth. In some embodiments, the first broth inlet/outlet 150 and the second broth inlet/outlet 170 can be used in combination. In some embodiments, the first culture fluid inlet/outlet 150 and/or the second culture fluid inlet/outlet 170 may be disposed to protrude from the cover plate 140 to facilitate the insertion of pipes.

It should be noted that, in some embodiments, the first culture fluid inlet/outlet 150 and the second culture fluid inlet/outlet 170 may include one or more, and when only one is included, the inlet and the outlet are sequentially implemented through the inlet/outlet; when a plurality of inlets and outlets are provided, each inlet and outlet is configured to perform the function of introducing or discharging the culture solution according to the requirement.

In some embodiments, the culture method based on the culture device 100 may include the following steps: introducing a culture into a collecting hole 1111 of one flow channel 111 of the plurality of flow channels 111 to form hanging drops through a drop generating hole 1112 of the flow channel 111, wherein the volume of the culture is the product of the volume of the hanging drops and the number of the flow channels 111; the accommodating space 130 is configured to maintain an environment in which a culture is cultured.

In some embodiments, configuring the receiving space 130 as an environment for maintaining a culture for cultivation further comprises: after the hanging drop is solidified, a culture solution is added to the accommodating space 130 so that the culture is at least partially in contact with the culture solution to perform, for example, culture of cell clusters.

In some embodiments, the culture may comprise 3D cells. In some embodiments, the culturing process of the 3D cells may include: first, a culture to be inoculated is prepared, then a quantitative culture is added to the pooling wells 1111 through the culture access port 160 (for example, if the volume of each pooling well is 50 microliters, 250 microliters is added according to the culture requirement if the cell amount of 5 hanging drops needs to be cultured), and the excess culture flows from the overflow tank 112 to the pooling wells 1111 of the adjacent flow channels 111, and the pooling wells 1111 are filled up in turn until the desired number of the drop pooling wells 1111 are filled. Due to the structure of the droplet-generating holes 1112 or the blocking effect of the blocking structure 1113 (e.g., a screen), the culture does not fall until it fills the droplet-collecting holes, and after the blocking effect disappears, the droplets fall to form hanging droplets. After the hanging drop solidifies, a culture solution is introduced into the culture solution accommodating space 130 through a culture solution inlet and outlet (e.g., the first culture solution inlet and outlet 150 or the second culture solution inlet and outlet 170) to provide nutrients required for cell culture, wherein the culture solution is added in an amount that ensures contact between the culture solution and the hanging drop. After the culture is completed, the culture apparatus 100 is transferred to an environment where the gel can be liquefied, after the gel is liquefied, a culture solution or PBS (phosphate buffered saline) may be added through the culture inlet 160 to flush the liquefied hanging drop into the accommodating space 130 to join with the culture solution, and then the culture is collected through a culture solution inlet and outlet (e.g., the first culture solution inlet and outlet 150 or the second culture solution inlet and outlet 170).

In some embodiments, the culture may comprise suspended cells or cell clumps. In some embodiments, the culturing process of the suspension cells or cell mass may include: the culture to be inoculated is prepared, and then a certain amount of the culture is added to the pooling hole 1111 through the culture inlet 160, and the excess culture flows from the overflow tank 112 to the pooling hole 1111 of the adjacent flow channel 111, and fills the pooling hole 1111 in turn until the desired number of the droplet pooling holes 1111 is filled. Due to the blocking effect of the structure of the droplet generation hole 1112 or the blocking structure 1113, the culture does not fall down before filling the droplet collection hole, and the droplet falls down to form a hanging droplet after the blocking effect disappears. During the culture process, the culture solution or PBS may be introduced into the culture solution accommodating space 130 through the culture solution inlet/outlet (e.g., the first culture solution inlet/outlet 150 or the second culture solution inlet/outlet 170), and the culture solution or PBS is added in an amount that ensures that the liquid surface thereof does not contact with the formed hanging drop, and the hanging drop may be fused if contacting with the culture solution or PBS, thereby causing the failure of the hanging drop culture. The culture solution or PBS provides certain humidity for cell culture, and the formed hanging drops can be prevented from volatilizing. After the cell culture is completed, the culture solution or PBS can be continuously added through the culture solution inlet and outlet to flush the hanging drop into the accommodating space 130 to be converged with the culture solution, and then the culture is collected through the culture solution inlet and outlet.

In some embodiments, culturing comprises: perfusion culture, non-perfusion culture or gas-liquid interface culture. In some embodiments, the culturing of the 3D cells or organoid cells can be by perfusion culture, non-perfusion culture, or gas-liquid interface culture as described above. According to the culture method, other tools outside the culture device 100 are not needed, whether a part of cells are exposed in gas for culture can be selected according to actual needs, various culture scenes are simulated, and the requirements of various experiments (such as drug screening) are met.

Wherein the non-perfusion culture comprises: the desired total amount of culture suspension, including culture (e.g., organoids, cells or cell clusters, etc.), culture fluid, gel, is introduced into the culture access port 160 of the cover plate 140. The proportion of gel is such that the hanging drop solidifies at the phase transition temperature, the environment in which the suspension is placed (e.g., pH or temperature conditions) is such that the hanging drop does not undergo a phase transition from liquid to solid, and, for example, the matrigel used is such that it operates at a temperature of 4 ℃. The culture suspension is collected in the corresponding collection well 1111 through any of the flow directing areas 1114 on the droplet generator plate 110, and the culture suspension in excess of the volume required to generate a single droplet flows through the overflow channel 112 to the collection well 1111 of the adjacent flow channel 111 until no more than the volume required to generate a single droplet is present in the collection well 1111 of the desired flow channel 111 on the droplet generator plate 110. The culture suspension in the collection well 1111 flows downward past the barrier 1113, which slows the suspension flow, and after completely wetting the barrier 1113 (e.g., a memory metal mesh), continues to flow downward in the droplet-forming wells 1112 and forms a hanging drop at the end of the droplet-forming wells 1112 remote from the collection well 1111. After the culture suspension forms the hanging drop, the environment of the hanging drop needs to be changed to solidify the hanging drop, for example, the matrigel applied needs to be transferred to the temperature condition of 37 ℃ for 10 minutes and then solidified; after the hanging drop is solidified, the culture solution is introduced into the accommodating space 130 through the first culture solution inlet/outlet 150 or the second culture solution inlet/outlet 170, and the culture solution is brought into contact with the hanging drop.

Wherein, perfusion culture includes: a culture suspension comprising a culture (e.g., organoids, cells, or cell clusters, etc.), a culture fluid, a gel is introduced into the pooling well 1111 of one of the plurality of flow-through channels 111. The proportion of gel is such that the hanging drop can solidify at the phase transition temperature, the environment in which the suspension is placed (e.g., pH or temperature conditions) is such that the hanging drop does not undergo a phase transition from liquid to solid, e.g., matrigel applications requiring 4 ℃ operation; after the culture suspension forms the hanging drop, the environment of the hanging drop needs to be changed to solidify the hanging drop, for example, the matrigel applied needs to be transferred to the temperature condition of 37 ℃ for 10 minutes and then solidified; after the hanging drop is solidified, introducing a culture solution into the accommodating space 130, wherein the culture solution is in contact with the hanging drop; the culture solution is fed in and discharged out at the speed of less than or equal to 1mm/s to form perfusion culture.

Wherein, the gas-liquid interface method culture comprises: introducing a culture suspension into the pooling well 1111 of one of the plurality of flow-through channels 111, the culture suspension including a culture (e.g., organoids, cells, or cell clusters, etc.), a culture fluid, and a gel in a proportion such that the hanging drop can solidify at a phase transition temperature, and the suspension is in an environment (e.g., pH or temperature conditions) such that the hanging drop does not undergo a phase transition from a liquid state to a solid state, e.g., the matrigel used is required to operate at a temperature of 4 ℃; after the culture suspension forms the hanging drop, the environment of the hanging drop needs to be changed to solidify the hanging drop, for example, the matrigel applied needs to be transferred to the temperature condition of 37 ℃ for 10 minutes and then solidified; after the hanging drop is solidified, the culture solution is introduced into the accommodating space 130, the culture solution can cover at least one end of the generation hole far away from the collection hole 1111, and the culture solution cannot completely submerge the hanging drop, namely, at least part of the hanging drop is in direct contact with air.

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

Also, the description uses specific words to describe embodiments of the description. 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 specification is included. 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 specification are not necessarily all referring to the same embodiment. Furthermore, some features, structures, or characteristics of one or more embodiments of the specification may be combined as appropriate.

Additionally, the order in which the elements and sequences of the process are recited in the specification, the use of alphanumeric characters, or other designations, is not intended to limit the order in which the processes and methods of the specification occur, unless otherwise specified in the claims. 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 present specification, 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 imply that more features than are expressly recited in a claim. Indeed, the embodiments may be characterized as having less than all of the features of a single embodiment disclosed above.

Numerals describing the number of components, attributes, etc. are used in some embodiments, it being understood that such numerals used in the description of the embodiments are modified in some instances by the use of the modifier "about", "approximately" or "substantially". Unless otherwise indicated, "about", "approximately" or "substantially" indicates that the number allows a variation of ± 20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximations that may vary depending upon the desired properties of the individual embodiments. In some embodiments, the numerical parameter should take into account the specified significant digits and employ a general digit preserving approach. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the range are approximations, in the specific examples, such numerical values are set forth as precisely as possible within the scope of the application.

For each patent, patent application publication, and other material, such as articles, books, specifications, publications, documents, etc., cited in this specification, the entire contents of each are hereby incorporated by reference into this specification. Except where the application history document does not conform to or conflict with the contents of the present specification, it is to be understood that the application history document, as used herein in the present specification or appended claims, is intended to define the broadest scope of the present specification (whether presently or later in the specification) rather than the broadest scope of the present specification. It is to be understood that the descriptions, definitions and/or uses of terms in the accompanying materials of this specification shall control if they are inconsistent or contrary to the descriptions and/or uses of terms in this specification.

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

Claims

1. A culture device, comprising:

the liquid drop generating plate is provided with a plurality of flow channels along the thickness direction;

a base enclosing an accommodation space with the droplet generator plate;

each flow channel sequentially comprises a collecting hole and a liquid drop generating hole in the vertical direction, the collecting hole in each flow channel is communicated with the liquid drop generating hole, and the collecting hole is used for accommodating a culture for forming liquid drops;

an overflow groove is formed between every two adjacent flow channels; the overflow launder is configured to limit the volume of culture in the collection well to no more than the volume required to produce the droplets.

2. A culture device according to claim 1, wherein:

a blocking structure is arranged between the collecting hole and the liquid drop generating hole; the barrier structure is configured to limit the rate at which the culture forms the droplets.

3. A culture device according to claim 2, wherein:

the barrier structure comprises at least one fibrous or strip-like structure radially disposed along the droplet-generating apertures.

4. A culture device according to claim 2, wherein:

the material of the barrier structure bottom comprises at least one of the following materials: including nylon, polypropylene, polyester, Polyethylene (PE), polypropylene (PP), Polyamide (PA), polyethylene terephthalate (PET), stainless steel, copper, platinum, gold, or memory alloys.

5. A culture device according to claim 1, wherein:

a flow guide area is further arranged at one end of the collecting hole, which is far away from the liquid drop generating hole; the flow guide region is used for collecting the culture into the collecting hole.

6. A culture device according to claim 5, wherein:

and the diversion area is covered with a hydrophobic material.

7. A culture device according to claim 1, wherein:

the drop generating aperture is configured at an end distal from the collection aperture for forming a hanging drop.

8. A culture device according to claim 7, wherein:

the end of the generating hole remote from the collecting hole is provided with a circular outer contour.

9. A culture device according to claim 1, wherein:

the base is also provided with a first culture solution inlet and outlet; the first culture solution inlet and outlet are used for introducing culture solution into the accommodating space for culture.

10. A culture device according to claim 9, wherein:

a partition plate is further arranged in the accommodating space; the accommodating space is divided into a plurality of sub accommodating spaces by the partition boards; the plurality of sub-accommodation spaces are respectively provided with the first culture solution inlet and outlet.

11. A culture device according to claim 1, wherein:

the culture device further comprises a cover plate; the cover plate is arranged on one side of the liquid drop generating plate, which is far away from the base; the cover plate is provided with a culture inlet.

12. A culture device according to claim 11, wherein:

the cover plate is provided with a second culture solution inlet and outlet; the liquid drop generating plate is provided with a through hole; the through hole is communicated with the second culture solution inlet and outlet so that the culture solution enters the accommodating space.

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

a base enclosing an accommodation space with the droplet generator plate;

each flow channel sequentially comprises a collecting hole and a liquid drop generating hole in the vertical direction, the collecting hole in each flow channel is communicated with the liquid drop generating hole, and the collecting hole is used for accommodating a culture for forming liquid drops; an overflow groove is formed between every two adjacent flow channels; said overflow launder being adapted to limit the volume of said culture within said collection well to no more than the volume required to produce said droplets; the method comprises the following steps:

introducing a culture into a collection hole of one of the flow channels so as to form a hanging drop in a drop generation hole of the flow channel, wherein the volume of the culture is the product of the volume of the hanging drop and the number of the flow channels;

the accommodating space is configured to be an environment for maintaining a culture for cultivation.

14. The method of claim 13, wherein the adding a culture solution to the holding space such that the culture is at least partially in contact with the culture solution, wherein the culture solution is configured to maintain an environment in which the culture is cultured, comprises:

and after the hanging drop is solidified, adding a culture solution into the accommodating space, so that the culture is at least partially contacted with the culture solution.

15. The method of claim 13, wherein:

the culture includes 3D cells.

16. The method of claim 13, wherein the culturing comprises:

perfusion culture, non-perfusion culture or gas-liquid interface culture.