KR20200111029A - Microwell Container for Cell Culture Camprising Functional Cap and the Cap therefor - Google Patents

Abstract

Disclosed are a microwell array container for cell culture comprising a functional cap; and the functional cap. The microwell array container of the present invention comprises: a main body in which a culture medium can be accommodated; and a cap which covers the main body to seal an inner space portion. The cap is provided with a guide portion for collecting cell colonies cultured in microwells during the process of collecting finally cultured cells, and a discharge hole for discharging the cell colonies collected through the guide portion is provided in the cap.

Description

Microwell Container for Cell Culture Camprising Functional Cap and the Cap therefor with a functional lid

The present invention relates to a container in which a microwell array is formed for cell cultivation, and a functional lid that allows a culture medium to be easily introduced into the microwell, and allows the cultured cell mass to be easily recovered from the microwell. It relates to a micro-well container provided with.

Microwell Arrays have been developed for culturing large amounts of uniform 3D cells and are widely used in research fields such as cell biology, anticancer drug development and regenerative therapy. Three-dimensional cell culture using a micro-well array, unlike a method of culturing cells in a medium, has an advantage of simulating a three-dimensional cell environment in a living body, and is therefore widely used to culture cells in an aggregated form.

The micro-well array is a hemispherical or U-shaped cross-sectional shape of concave wells arranged at regular intervals. Referring to FIG. 1, the micro-well array container 100 is a bottom plate on which a plurality of micro-wells 111 are formed. It is in the form of a plate composed of (110) and side walls (130) surrounding the bottom plate (110) to contain the cell culture solution. The surface of the micro-well 111 is surface-treated to prevent the cells from being deposited so that the cells are cultivated in a ball shape inside the micro-well 111.

To cultivate cells using micro-wells, (1) the researcher puts the culture medium containing the cells in a container and incubates them for a certain period of time under certain conditions. (2) During the culture process, the cells clump together in the micro-well. (3) At the end of the culture, the researcher collects the aggregated cell mass from the container.

There are some inconveniences in using a conventional microwell array vessel. Even if the culture solution is poured into the container, the air filled in the micro-well cannot be discharged and is trapped in the form of bubbles, and the culture solution cannot settle in the micro-well due to these bubbles. To solve this problem, the researchers repeat the so-called'Pipetting' process in which the culture solution is sucked up and discharged with a dropper or pipette, etc. You have no choice but to do the removal.

On the other hand, there is inconvenience even in the process of recovering the cultured cell mass from the microwell. Cell masses cultured three-dimensionally in micro-wells are not readily discharged from micro-wells. Therefore, in order for the cell mass to escape from the micro-well, researchers must shake the culture solution by repeatedly sucking up and discharging the culture solution with a dropper or the like. When the cell mass escapes from the micro-well by several times of pipetting and floats in the culture medium, the cell mass can be recovered.

An object of the present invention is to provide a micro-well container having a functional lid and the lid for allowing the culture medium to be easily introduced into the micro-well as well as easily recovering the cultured cell mass from the micro-well.

The microwell array container according to the present invention for achieving the above object includes a body and a lid. The body forms a space to accommodate the culture solution, and a microwell array is formed at the bottom of the space. The lid seals the space and has a discharge hole for providing a temporary negative pressure to the space. The negative pressure is used to remove air bubbles trapped in the micro-well when the culture solution is poured into the body.

Depending on the embodiment, the lid may further include a guide. The guide part is provided under the lid and inserted into the space of the main body, and the edge contacts the inner surface of the main body and is formed to be inclined toward the discharge hole in a funnel shape from the edge, thereby turning the main body over to recover the cell mass. In the cell mass is guided to the discharge hole.

In this case, it is preferable that the diameter of the discharge hole is larger than the size of the cell mass, and at least a size capable of delaying the discharge of the culture solution during the recovery process by the surface tension formed between the culture solution and the culture solution.

According to another embodiment, a vertical space part may be implemented in the guide part provided at a portion in contact with the discharge hole to provide a space for collecting the cell mass during the recovery process. The vertical space portion is formed to be concave and the shape of the concave groove in which the discharge hole is located in the middle, for example, a cross section cut into a plane including the central axis of the discharge hole may be implemented as a U-shaped concave groove.

According to another embodiment, it is preferable that the body is made of an elastic material that can be deformed by an external pressure applied to the floor, so that the culture solution can be discharged through the discharge hole by the external pressure during the recovery process. Do. In addition, an indicator may be formed in the main body to indicate the amount of the culture solution to be first poured into the main body in order to remove air bubbles trapped in the micro-well by the negative pressure.

According to another embodiment, the microwell array container according to the present invention may further include a multi-well plate for accommodating the main body in a plurality of grooves having an open top surface. In this case, the lid may cover the open groove of the multi-well plate to seal the space.

In the micro-well container according to the present invention, by closing the lid and providing a negative pressure therein, air bubbles trapped in the micro-well can be removed and the culture solution can be easily introduced into the micro-well.

In addition, the micro-well container according to the present invention can be discharged from the micro-well only by flipping the micro-well container after cultivation and gently contracting the container by hand.

Therefore, there is no inconvenience that researchers need to repeatedly use a pipette or the like for the purpose of removing air bubbles after pouring the culture solution into a micro-well container or for recovering a cell mass after culture.

1 is a view showing an example of a conventional microwell array container,
2 is a view showing a microwell array container according to an embodiment of the present invention,
3 is a cross-sectional view of the microwell array container of FIG. 2 in a recovery process;
4 is a view showing a microwell array container according to another embodiment of the present invention,
5 is a cross-sectional view of the microwell array container of FIG. 4 in a recovery process;
6 is a diagram provided to explain a method of using a microarray container, and
7 is a cross-sectional view of a container according to another embodiment of the present invention.

The present invention will be described in more detail below with reference to the drawings.

2, the microwell array container 200 of the present invention includes a main body 210 and a lid 230, and accommodates a culture medium to be used for cell culture.

The main body 210 forms a space portion 219 with an open upper surface to accommodate the culture solution. The main body 210 exemplarily illustrated in FIG. 2 is a cylinder implemented with a bottom 211 and a sidewall 213 surrounding the edge of the bottom 211, but is not limited thereto and may be implemented in various shapes. Even if it has a column shape, it is not limited to a cylindrical shape, and may be a square column, a pentagonal, or a hexagonal column. Meanwhile, the main body 210 may have a shape that is difficult to distinguish between the bottom and the sidewall, and the sidewall 213 may not be perpendicular to the bottom 211.

A micro well array 215 is formed on the bottom 211. In the micro-well array 215, a plurality of micro-wells 217 are arranged side by side, and cells are aggregated in each micro-well 217 to be cultured as a cell mass. The individual shape of the micro-well 217 may be any known conventionally. Since the micro-well 217 forms a three-dimensional space, cell culture is also carried out three-dimensionally, and is different from cell culture carried out flat in a medium.

The amount of the culture medium that the main body 210 can accommodate is determined by the size of the space portion 219, and may be implemented in various sizes. However, as described below, since the guide portion 235 of the lid 230 is inserted into the space portion 219, the maximum capacity of the microwell array container 200 is the space after the guide portion 235 is inserted. It can be determined based on the size of the part 219. When the culture solution is put in the main body 210, it is not essential because it is measured with a pipette, etc., but depending on the embodiment, a scale indicating the quantity or maximum amount may be displayed on the inner surface of the space part 219.

On the other hand, as described below, when the culture solution is placed in the main body 210 for cell culture according to the present invention, it is divided into two times, the first and the second stage, and at this time, an instruction indicating the amount of the culture solution to be poured first It is preferable that an additional is provided. In the bottom 211 of the main body 210 of FIG. 2, a jaw 221 is provided around the microwell array 215, and the height of the jaw 221 indicates the amount of the primary culture solution.

The body 210 may be implemented with a variety of materials, but it is preferable that the body 210 is implemented with an elastic material. For example, a silicone resin such as polydimethylsiloxane (PDMS) has a stable chemical structure, is not corrosive, does not oxidize, and has some elasticity, so it is preferable as a material for the body 210.

As described below, in the present invention, in the process of recovering the cell mass, the microwell array container 200 is turned over and the culture solution accommodated in the space 219 is discharged through the discharge hole 233 of the lid 230, at this time It is necessary to apply an external force in such a way that one side of the body 210 is pressed with a finger or the like to cause a shape deformation of the body 210. Through this morphological modification, the cell mass can be easily separated from the micro-well 217 and the culture solution can be easily discharged through the discharge hole 233.

Like the main body 210, the lid 230 is preferably made of a material having elasticity such as silicone resin, but if the main body 210 is made of a material having elasticity, the lid 230 must be made of a material having elasticity. It doesn't have to be.

The lid 230 covers the upper surface of the space part 219 to seal the space part 219. The shape of the lid 230 is determined according to the shape of the body 210. Therefore, the lid 230 may be any structure capable of sealing the space portion 219. However, when the lid 230 is implemented with a single material without other structures, in order to seal the space 219, a part of the lid 230 is in close contact with the inner or outer surface of the body forming the space 219 It should be in the form of

For example, in the case of the main body 210 having a cylindrical shape as shown in FIG. 2, the lid 230 includes an upper plate 231 and an insertion portion extending from the upper plate 231 and in close contact with the inner or outer surface of the side wall 213 It can be implemented as (234).

A discharge hole 233 for providing a temporary negative pressure to the space 219 is formed in the lid 230. In the case of FIG. 2, a discharge hole 233 is formed in the form of a through hole in the center of the top plate 231 of the lid 230. The negative pressure is used to remove air bubbles trapped in the micro-well 217 in the process of pouring the primary culture solution into the main body 210.

Example 1: Guide of the lid

As will be described again below, when the cell culture is completed in the culture medium accommodated in the main body 210, the'recovery process' of the cultured cell mass proceeds. In the recovery process of the present invention, the researcher turns over the microwell array container 200 with the lid 230 closed, and then recovers the cultured cell mass through the discharge hole 233.

The lid 230 may further include a guide part 235 for guiding the cell mass to the discharge hole 233 in the'recovery process'. The guide 235 is provided under the lid 230 and is inserted into the space 219 of the main body 210 to guide the space 219 to the discharge hole 233 of the lid 230. Accordingly, the guide portion 235 is formed so as to have an edge in contact with the inner surface of the space portion 219 and to be inclined toward the discharge hole 233 in a funnel shape from the edge.

In the embodiment of FIGS. 2 and 3, the guide part 235 is implemented integrally with the insertion part 234. The edge 237 of the guide portion 235 is formed in a cylindrical shape in close contact with the inner surface of the space portion 219 and is formed to be inclined from the edge 237 toward the discharge hole 233 located in the center of the upper plate 231 (219) is sealed in a conical shape. Therefore, when the microwell array container 200 is turned over during the recovery process, the aggregated-cultured cell mass in the microwell 217 escapes from the microwell 217 by gravity and the discharge hole 233 along the guide part 235 ), the cell mass is recovered.

At this time, the minimum diameter of the discharge hole 233 must be a size that allows the cell mass to pass. On the other hand, in order for the cell mass to escape from the micro-well 217 only by gravity without any other external force, a certain time is required, during which the culture medium is not discharged and must be accommodated in the space part 219. In other words, if the culture solution is discharged through the discharge hole 233 as soon as the microwell array container 200 is turned over in the recovery process, the cell mass cannot escape from the microwell 217. In order to solve this point, the microwell array container 200 of the present invention uses a surface tension that may be formed between the discharge hole 233 and the culture solution. Accordingly, the amount of the culture solution accommodated in the microwell array container 200 is determined such that the height of the culture solution is located in the discharge hole 233 when the lid 230 is closed as shown in FIG. 3A. If the size of the surface tension t1 formed between the discharge hole 233 and the culture solution can support the weight of the culture solution serving as the discharge hole 233, the discharge of the culture solution may be prevented or at least delayed. At this time, the smaller the size of the discharge hole 233 is, the more advantageous. In conclusion, the maximum diameter of the discharge hole 233 should be determined to an extent that can prevent or at least delay the discharge of the culture medium in the relationship between the weight of the culture medium acting on the discharge hole 233 and the surface tension, and the discharge hole 233 The maximum diameter of) can be obtained experimentally. In consideration of this point, the diameter of the discharge hole 233 is preferably about 2 mm.

On the other hand, the height of the culture solution is located in the discharge hole 233, the amount of the culture solution to overflow in the process of closing the lid 230, it is quite inconvenient in use. For this reason, it is possible to design the height of the culture solution to be located on the inclined surface of the guide portion 235. However, if the surface of the culture solution is located on the inclined surface of the guide portion 235 as shown in (b) of FIG. 3, it is not easy to obtain sufficient surface tension to prevent the discharge of the culture solution. As is known, the surface tension is affected by the angle formed between the culture medium (cf) and the inner surface of the body 210. Since the inclined surface of the guide part 235 is not perpendicular to the culture medium, the direction of the surface tension (t2) is It is not advantageous in stopping emissions.

Example 2: Guide portion 2 of the lid (Figs. 4 and 5)

A microwell array container 400 according to another embodiment of the present invention for solving a problem with the microwell array container 200 of FIG. 2 is disclosed in FIG. 4. 4 and 5, a vertical space portion 411 may be provided in a portion of the lid 410 in contact with the discharge hole 233. The vertical space part 411 is implemented in the shape of a concave groove in which the discharge hole 233 is located in the center, and preferably has a vertical inner wall. For example, as shown in FIG. 5, the vertical space part 411 may be implemented as a U-shaped concave groove having a cross section cut into a plane including the central axis of the discharge hole 233. Referring to FIG. 5, the surface tension t3 acting between the inner wall of the vertical space part 411 and the culture medium may be prevented from being discharged by acting in the upward direction. Accordingly, the amount of the culture medium accommodated in the microwell array container 400 is determined such that the height of the culture medium is located in the vertical space portion 411 when the lid 410 is closed.

Compared with the microwell array container 200 of FIG. 2, since the vertical space part 411 provides more empty space in a state filled with the culture medium, when closing the lid 410 again in FIG. 6(d) Reduce the risk of overflowing the culture medium. Meanwhile, the vertical space part 411 may also serve as a space in which cell masses are gathered.

Cell culture using a microwell array container (Fig. 6)

Hereinafter, a cell culture process will be described with reference to FIGS. 4 to 6 centering on the microwell array container 400.

<Sowing of culture solution: Fig. 6 (a)>

The researcher pours the primary culture solution into the body 210 not covered with the lid 410 using a dropper or pipette. At this time, when the culture solution is poured to fill the jaw 221 of the bottom 211 of the main body 210, it becomes the primary culture solution.

<Removal of air bubbles using negative pressure: (b) of FIG. 6>

As described in the prior art, in the process of pouring the primary culture solution, air in the microwell 217 is not discharged and is trapped in the form of bubbles (v). In the present invention, before filling the secondary culture solution, the lid 410 is closed to remove air bubbles (v) first. The researcher closes the lid 410 to seal the space 219. A negative pressure is provided to the space 219 through the discharge hole 233. The negative pressure may be provided by any method known in the art. For example, by repeating a simple operation of aligning the end of the pipette or the like 10 with the discharge hole 233 and removing air from the space part 219, the space part ( 219) may also provide a negative pressure. Bubbles (v) trapped in the micro-well 217 expand by negative pressure and escape from the micro-well 217 and are eventually discharged from the air.

<Cultivation of cell mass: Figure 6 (c)>

When the air bubbles (v) trapped in the micro-well 217 are removed, the lid 410 is removed and the secondary culture solution is poured into the main body 210 to put a quantitative amount of the culture solution (cf) in the main body 210, and then the cell culture Start.

<Recovery of cell mass: Figure 6 (d) and (e)>

When the cell culture is complete, the researcher proceeds with a recovery process to recover the cell mass. First, the lid 410 is closed again to seal the space portion 219, and then the microwell array container 400 is turned over and waited for a predetermined time. When the lid 410 is closed, a portion of the vertical space portion 411 is filled with the culture solution.

As described above, because of the surface tension acting between the culture solution cf and the vertical space portion 411, the culture solution cf is not immediately discharged through the discharge hole 233 even when the microwell array container 400 is turned over. Instead, the cell mass cultured in the micro-well 217 falls downward by gravity and is guided by the guide part 235 to collect into the vertical space part 411.

After a certain period of time has elapsed with the microwell array container 400 turned upside down, if the shape of the body 210 is changed by pressing the bottom 211 of the body 210, the influence of the surface tension is removed and the culture solution cf is discharged. It pours out through the hole 233.

Cell culture using the microwell array container 400 of the present invention is performed in the above manner.

Example 3: Another example of a microwell array container (Fig. 7)

Conventionally, a multi-well plate has been used to hold a plurality of micro-well containers. The multi-well plate 710 includes a plurality of grooves 711 whose upper surfaces are open to accommodate a plurality of micro-well containers. The micro-well array container of the present invention may also be combined with the multi-well plate 710.

Referring to FIG. 7, the microwell array container 700 may further include a multi-well plate 710. The body 210 is inserted into a plurality of open grooves 711 of the multi-well plate 710, respectively, and the lid 730 is an open groove of the multi-well plate 710 instead of being directly coupled to the body 210 In the form of covering 711, the inside of the main body 210 is sealed. The multi-well plate 710 and the body 210 in FIG. 7 become a new body and thus correspond to the body 210 of FIGS. 2 and 4, and in the process of removing air bubbles as in FIG. 2 and 4 may perform the same function as the body 210 of FIG.

In the above, preferred embodiments of the present invention have been illustrated and described, but the present invention is not limited to the specific embodiments described above, and is generally used in the technical field to which the present invention belongs without departing from the gist of the present invention claimed in the claims. Various modifications are possible by those skilled in the art of course, and these modifications should not be individually understood from the technical idea or perspective of the present invention.

200, 400, 700: microwell array vessel
210: main body 211: bottom
213: sidewall 215: microwell array
217: microwell 219: space
221: jaw 230, 410, 730: lid
231: upper plate 233: discharge hole
234: insertion unit 235: guide unit
237: edge (guide part) 411: vertical space part
cf: culture solution 710: multi-well plate
711: groove of the multi-well plate

Claims (13)

In the microwell array vessel,
A main body that forms a space to receive a culture solution and has a microwell array formed at the bottom of the space; And
And a lid having a discharge hole for providing a temporary negative pressure to the space part (the negative pressure is used to remove air bubbles trapped in the micro-well when the culture solution is poured into the main body) Microwell array vessel made of.
The method of claim 1,
The lid is,
It is provided under the lid and inserted into the space of the main body, and the edge is formed to be inclined toward the discharge hole in a funnel shape from the edge to the inner surface of the main body, so that the main body is turned over to recover the cell mass. Further comprising a guide for guiding the cell mass to the discharge hole,
The diameter of the discharge hole is larger than the size of the cell mass, the microwell array container, characterized in that formed to a size capable of at least delaying the discharge of the culture medium in the recovery process by the surface tension formed between the culture medium.
The method of claim 2,
In the guide,
A microwell array container, characterized in that a vertical space portion provided at a portion in contact with the discharge hole to provide a space for collecting the cell mass during the recovery process.
The method of claim 3,
The vertical space portion,
Microwell array container, characterized in that the shape of a concave groove formed in a concave and in which the discharge hole is located in the center.
The method of claim 4,
The concave groove,
Microwell array container, characterized in that the cross section cut into a plane including the central axis of the discharge hole is U-shaped.
The method according to any one of claims 2 to 5,
The body is a microwell array container, characterized in that the body is implemented with an elastic material that can be deformed by an external pressure applied to the floor, so that the culture solution can be discharged through the discharge hole under the external pressure during the recovery process. .
The method of claim 1,
In the main body
Microwell array container, characterized in that the indicator portion for indicating the amount of the culture medium to be poured primarily into the main body to remove the air bubbles trapped in the microwell by the negative pressure.
The method of claim 1,
Further comprising a multi-well plate each accommodating the main body in a plurality of grooves with an open top surface,
The lid is a microwell array container, characterized in that by covering the open groove of the multi-well plate to seal the space.
As a lid covering and sealing the inner space of the microwell array container (the microwell array container forms the space to receive the culture solution, and a microwell array is formed at the bottom of the space),
A lid for a microwell array container, characterized in that a discharge hole for providing a temporary negative pressure is formed in the space part (the negative pressure is used to remove air bubbles trapped in the microwell when the culture solution is poured into the main body).
The method of claim 9,
It is inserted into the space of the main body, the edge is in contact with the inner surface of the main body and is formed inclined toward the discharge hole in a funnel shape from the edge, so that the cell mass is transferred to the discharge hole in the recovery process of overturning the main body to recover the cell mass Further comprising a guide to guide to,
For a microwell array container, characterized in that the diameter of the discharge hole is larger than the size of the cell mass, and is formed to have a size capable of at least delaying the discharge of the culture solution during the recovery process by a surface tension formed between the culture solution Lid.
The method of claim 10,
In the guide,
A lid for a microwell array container, characterized in that a vertical space part provided at a portion in contact with the discharge hole to provide a space for collecting the cell mass during the recovery process.
The method of claim 11,
The vertical space portion,
A lid for a microwell array container, characterized in that it has a concave shape and a concave groove in which the discharge hole is located in the center.
The method of claim 12,
The concave groove,
A lid for a microwell array container, characterized in that the cross section cut into a plane including a central axis of the discharge hole is U-shaped.

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