KR102309955B1 - Well plate unit for spheroid culture and well plate unit for multilayer spheroid culture apparatus - Google Patents

Abstract

The well plate unit for culturing spheroids according to the present invention includes a base part forming a bottom in which the cell-medium mixture is accommodated, and is provided on the base part and forms a collection space inside, but the first direction is opened and not closed A first spheroid guard formed in a curved shape and a second formed in a shape surrounding a portion of the perimeter of the first spheroid guard, opened in a second direction opposite to the first direction and formed in a curved shape that is not closed Including a spheroid guard, the spheroids positioned in the collecting space do not escape to the outside of the collecting space, but the medium in liquid form forms a structure in which it is discharged to the outside of the collecting space.

Description

Well plate unit for spheroid culture and well plate unit for multi-layer spheroid culture apparatus {WELL PLATE UNIT FOR SPHEROID CULTURE AND WELL PLATE UNIT FOR MULTILAYER SPHEROID CULTURE APPARATUS}

The present invention relates to a well plate unit, and more particularly, it is formed in a form applied to a multi-layer spheroid culture apparatus that forms a hanging drop or independently to culture a spheroid composed of a single or various layers. It relates to a well plate unit for culturing roids.

Cell culture is a technique for culturing cells isolated from an individual outside an individual. In such cell culture, it is essential to consistently maintain a cultured environment similar to that of an individual's body.

With respect to these key points, various cell culture methods have been proposed as technology advances today.

Among these, the traditionally used cell culture method has a method of culturing single-layered cells spread thinly in a two-dimensional direction, but this has a problem in that the specific function that the cell originally has in the body cannot be maintained for a long time.

Therefore, in recent years, the culture of spheroids, which are three-dimensional tissues having the same function as in vivo, has been attracting attention, and various three-dimensional spheroid culture techniques are currently being developed.

However, even in such various three-dimensional spheroid culture techniques, since the spheroid culture is very small, when the medium is replaced in a state in a predetermined plate, there is a problem that there is a high probability of loss.

This is because the medium is generally removed using a vacuum pump, so it is very inconvenient and difficult to visually check very small spheroids and replace only the medium, as well as because the operator feels high fatigue, research There are additional issues such as delays in schedules and poor health of workers.

Accordingly, it is intended to solve the above-described problems and propose a technology suitable for a situation requiring development through the present invention.
[Related Prior Patent Literature]
Registered Patent Publication No. 10-1341572 (2013.12.13.)

The present invention is an invention devised to solve the problems of the prior art described above, and in the process of discharging or replacing the medium of the spheroid culture, a well plate to prevent the loss of spheroids and to easily discharge only the medium The purpose is to provide a unit.

In addition, it has the purpose of reducing the fatigue of the operator and enabling high-reliability research work.

The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The well plate unit for culturing spheroids for achieving the above object includes a base portion forming a bottom in which the cell-medium mixture is accommodated, a base portion provided on the base portion and forming a collection space inside, the first direction being an opening The first spheroid guard formed in a curved shape that is not closed is formed in a shape surrounding a portion of the circumference of the first spheroid guard, and is opened in a second direction opposite to the first direction and is formed in a curved shape that is not closed Including a second spheroid guard to be, the spheroids positioned in the collecting space do not escape to the outside of the collecting space, the medium in liquid form forms a structure that is discharged to the outside of the collecting space.

And the present invention may further include a side wall portion extending upwardly from the circumference of the base portion.

In addition, the first spheroid guard, the second spheroid guard and the side wall portion may be formed to have a height corresponding to each other.

And the well plate unit for a multi-layer spheroid culture apparatus of the present invention for achieving the above object is a well plate unit provided under the drop forming part of the multi-layer spheroid culture apparatus in which hanging drops of cells and medium are formed. In the following, a base portion forming a bottom from which the hanging drop falls, a first spheroid guard provided on the base portion and forming a collecting space inside, the first spheroid guard being formed in a curved shape that is not closed with an opening in the first direction, and the The first spheroid is formed to surround a portion of the perimeter of the guard, and includes a second spheroid guard that is opened in a second direction opposite to the first direction and is formed in a curved shape that is not closed, falling into the collecting space. The spheroids included in one hanging drop do not escape to the outside of the collecting space, but the medium in liquid form forms a structure in which it is discharged to the outside of the collecting space.

In this case, the base unit may be formed with a recessed groove in an area corresponding to the collecting space.

In addition, the recessed groove may be formed to gradually deepen from the outer portion of the collecting space toward the central portion.

And the width between the first spheroid guard and the second spheroid guard may be formed smaller than the diameter of the spheroid.

In addition, a medium suction space may be formed in the opening of the second spheroid guard.

And the present invention may further include a side wall portion extending upwardly from the circumference of the base portion.

At this time, the side wall portion may be provided with a medium suction unit for sucking the medium discharged to the outside of the collection space.

In addition, the side wall portion may be provided with a medium supply for supplying the medium to the base portion.

The well plate unit for spheroid culture and the well plate unit for multi-layer spheroid culture apparatus of the present invention for solving the above problems, the spheroids are located in the collecting space by the first spheroid guard and the second spheroid guard Since the state can be maintained and only the medium can be easily discharged or replaced, there is an advantage that can minimize the probability of loss of spheroids.

In addition, since it is possible to reduce the fatigue felt by the operator in discharging or replacing the medium according to this, it is possible to protect the health of the operator and to conduct a high-reliability research work.

And the present invention has the advantage that the spheroid does not leak to the outside and has a tendency to be located in the central part, so that research work, including image taking, is easy.

Effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

1 is a view showing a state of a multi-layer spheroid culture apparatus to which a well plate according to an embodiment of the present invention is applied;
Figure 2 is a view showing the state of the hanging drop culture unit of the well plate unit and multi-layer spheroid culture apparatus according to an embodiment of the present invention;
Figure 3 is a view showing a state in which a hanging drop is formed in the hanging drop culture unit of the multi-layer spheroid culture apparatus;
4 to 6 are views showing the structure of the well plate unit according to an embodiment of the present invention;
7 is a view showing a process of removing the medium using the well plate unit according to an embodiment of the present invention;
8 is a view showing a state of a well plate unit according to another embodiment of the present invention; and
9 is a view showing a state of a well plate unit according to another embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention in which the object of the present invention can be specifically realized will be described with reference to the accompanying drawings. In describing the present embodiment, the same names and the same reference numerals are used for the same components, and an additional description thereof will be omitted.

1 is a view showing a state of a multi-layer spheroid culture apparatus to which a well plate according to an embodiment of the present invention is applied.

As shown in FIG. 1 , the multi-layer spheroid culture apparatus according to an embodiment of the present invention includes a supply tube assembly 5 , a hanging drop culture unit 100 , and a well plate unit 200 .

The supply pipe assembly 5 is a component for supplying cells and a medium to the hanging drop culture unit 100 .

In this embodiment, the supply pipe assembly 5 includes a main supply unit 10 having a supply passage formed therein, a cell injection unit 20a for injecting cells into the supply passage, and a cell in the supply passage for culturing the cells. It has a form including a medium injection unit 20b for injecting a medium, and an air injection unit 30 for injecting air into the supply passage.

And a first valve 25 is provided at a connection point between the cell injection unit 20a, the medium injection unit 20b, and the main supply unit 10, and the air injection unit 30 and the main supply unit 10 ) and a second valve 35 is provided at the connection point, so that it is possible to control whether the injection path for each material is opened or closed, the amount of opening, and the like.

Accordingly, when air is injected from the air injection unit 30 , the cells and medium injected into the main flow path of the main supply unit 10 may flow toward the other end by the pressure of the air.

In addition, in this embodiment, the main supply unit 10 includes a first supply pipe 10a to which the cell injection unit 20a, the medium injection unit 20b, and the air injection unit 30 are connected, and the first supply pipe. It may include a second supply pipe (10b) coupled to the other end of (10b). The second supply pipe 10b has a shape formed in a downward direction from the other end in the direction of gravity.

In addition, the supply pipe assembly 5 may further include a distribution unit 15 provided to connect the main supply unit 10 and the hanging drop culture unit 100 to each other. In this embodiment, the distribution unit 15 is a component for dropping the cells and medium flowing through the main supply unit 10 to the drop path 120 to be described later of the hanging drop culture unit 100, and the It has a branched form from the other end of the second supply pipe (10b) of the main supply unit (10).

In particular, in the present embodiment, a plurality of the hanging drop culture unit 100 is provided in a state mounted on the cover plate 50, and the distribution unit 15 is provided with a plurality of pieces to correspond to each of the hanging drop culture unit 100. It is provided so as to distribute the cells and the medium flowing through the main supply unit 10 to each of the hanging drop culture units 100 .

The hanging drop culture unit 100 is a component provided at the lower portion of the distribution unit 15 to receive cells and a medium so that three-dimensional culture is performed.

Figure 2 is a view showing the state of the hanging drop culture unit 100 of the well plate unit 200 and the multi-layer spheroid culture apparatus according to an embodiment of the present invention, Figure 3 is a hanging of the multi-layer spheroid culture apparatus It is a view showing a state that the hanging drop (B) is formed in the drop culture unit (100).

As shown in FIGS. 2 and 3 , in this embodiment, the hanging drop culture unit 100 has a fall flow path 120 formed in the vertical direction to communicate with the main flow path of the main supply unit 10 , and the falling flow path It is provided at the lower part of the 120 and includes a drop forming unit 130 that allows the cells and medium flowing in the direction of gravity through the fall flow path 120 to form a water droplet-shaped hanging drop (B) by surface tension. .

In particular, the fall flow path 120 is formed to have the same diameter as a whole, and the drop forming part 130 is formed to have a shape corresponding to a partial shape of a sphere by gradually increasing its diameter from the top to the bottom. Accordingly, the cell-medium mixed solution flowing toward the drop forming unit 130 through the drop flow path 120 forms a hanging drop B in the form of a droplet by surface tension in the drop forming unit 130 . .

That is, the hanging drop (B) gradually grows in the form of water droplets as the cell-medium mixture continuously flows into the drop forming unit 130, and inside the hanging drop (B), the cells contained in the mixture By the spheroid (S) is formed.

Such a hanging drop (B) is separated from the drop forming unit 130 in situations such as when it grows to a certain weight or more, or when air is injected above a specific pressure through the air injection unit 30 described above. It falls on the well plate unit 200 according to the present invention.

And in this embodiment, the hanging drop culture unit 100 may further include an air circulation passage 140 and a medium discharge passage 160 .

The air circulation passage 140 is a component that extends to communicate the fall passage 120 and the outside with each other to form a flow path of air. At a position higher than the communication part 142, the first connection circulation part 144 connecting the lower end of the external communication part 142 and the fall flow path 120 to each other, and the first connection circulation part 144 and a second connection circulation unit 146 connecting a predetermined point of the external communication unit 142 and the fall flow path 120 to each other.

The air circulation passage 140 as described above allows the air injected by the air injection unit 30 to be discharged to the outside.

The medium discharge passage 150 is a component that extends to communicate the fall passage 120 and the outside with each other to form a discharge path of the medium accommodated in the fall passage 120 .

In this embodiment, the medium discharge flow path 150 has one side adjacent to the upper end of the drop forming part 130 and connected to the falling flow path 120 , and the other side protrudes to the outside of the hanging drop culture unit 100 . It extends to the inside of the exhaust unit 160 . In this case, each discharge unit 160 formed in the plurality of hanging drop culture units 100 may be connected to a pump connection unit 60 (refer to FIG. 1 ) connected to a separate suction pump.

Therefore, in the present embodiment, the medium accommodated in the drop passage 120 may be discharged to the outside through the medium discharge passage 150 by the suction power of the suction pump.

The reason for doing this is to discharge the medium accommodated in the drop passage 120 to the outside so that the cell-medium mixture remains only in the drop forming unit 130 to form a hanging drop (B).

And in this embodiment, the hanging drop culture unit 100 is provided in the falling flow path 120, and the cell-medium mixed solution flowing through the falling flow path 120 moves toward the inner wall side of the falling flow path 120. It further includes a buffer member 170 for guiding it to flow down.

That is, the buffer member 170 is to prevent the cell-medium mixed solution flowing through the falling flow path 120 from directly falling onto the already formed hanging drop (B) and applying a large drop energy, and the cell-medium mixed solution As the falling speed is reduced and the cell-medium mixed solution flows down through the inner wall of the falling flow path 120 , the falling energy can be minimized.

In particular, in this embodiment, the buffer member 170 includes a buffer part 172 provided at the center of the cross-section of the fall flow path 120 , and from the circumference of the buffer part 172 to the inner wall side of the fall flow path 120 . It extends and includes a fixing portion 174 for fixing the buffer portion (172).

Meanwhile, a well plate unit 200 receiving the hanging drop B falling from the drop forming unit 130 is provided at a lower portion of the hanging drop culture unit 100 .

Hereinafter, the well plate unit 200 will be described in detail.

4 to 6 are views showing the structure of the well plate unit 200 according to an embodiment of the present invention.

Referring to FIGS. 4 to 6 as well as FIG. 2 , the well plate unit 200 according to an embodiment of the present invention includes a base part 210 , a side wall part 220 , and a first spheroid guard 230 . and a second spheroid guard 240 .

The base part 210 is a component that forms a floor on which the hanging drop B generated in the drop forming part 130 of the hanging drop culture unit 100 directly falls, and the side wall part 220 is extended upwardly from the circumference of the base part 210 and has a shape surrounding a portion of the side surface of the hanging drop culture unit 100 .

That is, in this embodiment, the well plate unit 200 is formed in a cup shape, and as the height of the side wall part 220 is higher than the position of the lower end of the hanging drop culture unit 100, the drop forming part It is possible to prevent the hanging drop (B) falling from 130 from leaking to the outside.

And the first spheroid guard 230 and the second spheroid guard 240 are formed on the base portion 210 and are formed inside the sidewall portion 220 .

Specifically, the first spheroid guard 230 is formed on the base portion 210, and forms a collecting space (R ₁ ) on the inside, but is formed in a curved shape that is not closed with an opening in the first direction. That is, in this embodiment, the first spheroid guard 230 has the same shape as the alphabet 'C', and the opening faces the first direction.

And the second spheroid guard 240 is formed on the base portion 210, is formed in a shape surrounding a portion of the circumference of the first spheroid guard 230, the first direction opposite to the first direction It is opened in two directions and is formed in a curved shape that is not closed. That is, in this embodiment, the second spheroid guard 240 has a shape like the letter 'C' larger than that of the first spheroid guard 230, and the opening faces the second direction opposite to the first direction. .

Thus, Spanish of the first spheroid guard 230 and the second spheroid guard 240 is to form an opening in the opposite directions to each other, trapping space (R ₁₎ a hanging drop (B) fall in Lloyd ( S) is to prevent leakage to the outside of the collection space (R ₁ ), and only the medium in liquid form can be discharged to the outside of the _{collection space (R 1 ).}

Specifically, as shown in Figure 5, in this embodiment, the width w between the first spheroid guard 230 and the second spheroid guard 240 is included in the dropped hanging drop (B). It may be formed smaller than the diameter (d) of the spheroid (S).

Therefore, the spheroid (S) cannot move through the path formed between the first spheroid guard 230 and the second spheroid guard 240 even if the well plate unit 200 itself is tilted, and the collection A state remaining in the space (R _{1 ) is maintained.}

And unlike this, the medium in liquid form may flow to the outside of _{the collecting space (R 1} ) through a path formed between the first spheroid guard 230 and the second spheroid guard 240 .

At this time, in the present embodiment, a medium suction space (R ₂ ) having a predetermined width is formed in the opening of the second spheroid guard 240 , and the operator sucks and discharges the medium flowing into the medium suction space (R _{2 )} can do it

In addition, as shown in the longitudinal section of FIG. 6 , in the present embodiment, the base part 210 has a shape in which a recessed groove 212 is formed in an area corresponding to _{the collecting space R 1 .}

Such a recessed groove 212 induces the spheroid (S) to be located in the central portion of the base portion 210, so that the spheroid (S) does not easily flow to the outer side of _{the collecting space (R 1 ).}

To this end, in the present embodiment, the recessed groove 212 is formed to become deeper from the outer portion to the center portion _{of the collecting space R 1 , and the surface thereof is formed in a rounded shape.}

7 is a diagram illustrating a process of removing a medium using the well plate unit 200 according to an embodiment of the present invention.

First, as described above, the hanging drop B generated in the drop forming unit 130 of the hanging drop culture unit 100 falls into _{the collection space R 1 of the well plate unit 200 .}

At this time, in this embodiment, since the recessed groove 212 is formed to become deeper from the outer part to the central part, the spheroid (S) is located in the center of the base part 210 as shown on the left side of FIG. In particular, the spheroids (S) do not float on the medium and sink to the bottom.

Thereafter, as shown in the center of FIG. 7 , the well plate unit 200 itself is tilted so that the medium moves through the path formed between the first spheroid guard 230 and the second spheroid guard 240 . _{, the medium suction space (R 2} ) formed in the opening of the second spheroid guard 240 flows toward the side.

And the operator removes the medium located in _{the medium suction space (R 2} ) using a pipette (P) or a tip to which a vacuum pump is connected, as shown on the right side of FIG. 7 .

As such, the present invention has the advantage that only the medium can be easily discharged or replaced, and only the spheroid (S) can be easily separated.

8 is a view showing a state of the well plate unit 200 according to another embodiment of the present invention.

The well plate unit 200 according to another embodiment of the present invention shown in FIG. 8 is also the base part 210, the side wall part 220, the first spheroid guard 230 and the second spade similar to the above-described embodiment. Lloyd guard 240 is included. Since these components have already been described in the previous part, overlapping descriptions will be omitted.

And this embodiment has a form that further includes a medium suction unit 260 and a medium supply unit 250 provided on the side wall portion 220 .

The medium suction unit 260 _{is formed in a tube shape to suck the medium discharged to the outside of the collection space R 1} , and a separate metering pump is connected to generate suction power to the medium suction unit 260 . can do it

In the case of this embodiment the medium suction unit 260 is connected to the lower end side of the side wall portion 220 so as to be adjacent to the medium intake space (R _2), to facilitate the medium located in the medium intake area (R ₂₎ formed to be inhaled.

And the medium supply unit 250 is a component that can supply the medium to the side of the base 210, and is formed in the form of a tube through which the medium flows. In addition, the medium supply unit 250 may also be connected to a separate metering pump to supply the medium at a predetermined pressure.

In this embodiment, the medium supply unit 250 is connected to a higher position than the medium suction unit 260 in the side wall portion 220, but the connection position of the medium supply unit 250 may be variously made. Of course.

9 is a view showing a state of the well plate unit 200 according to another embodiment of the present invention.

The well plate unit 200 according to another embodiment shown in FIG. 9 is also the base part 210, the side wall part 220, the first spheroid guard 230 and the second spheroid guard like the above-described embodiment. (240).

At this time, in this embodiment, the height of the side wall part 220 is formed to have a height corresponding to the first spheroid guard 230 and the second spheroid guard 240 , and thus the well plate unit 200 is generally It is different from the other embodiments described above in that it is formed in the form of a chalet.

The well plate unit 200 of this type may be applied to the multi-layer spheroid culture apparatus shown in another embodiment, but separately from the multi-layer spheroid culture apparatus, a spheroid having a single layer as well as a multi-layer spheroid is cultured. It can also be used independently to

As described above, preferred embodiments according to the present invention have been described, and the fact that the present invention can be embodied in other specific forms without departing from the spirit or scope of the present invention in addition to the above-described embodiments is one of ordinary skill in the art. It is obvious to them. Therefore, the above-described embodiments are to be regarded as illustrative rather than restrictive, and accordingly, the present invention is not limited to the above description, but may be modified within the scope of the appended claims and their equivalents.

5: Supply pipe assembly
10: main supply
15: distribution unit
20a: cell injection unit
20b: medium injection unit
30: air inlet
100: hanging drop culture unit
120: fall flow path
130: drop forming part
140: air circulation path
150: medium discharge path
170: buffer member
200: well plate unit
210: base part
220: side wall
230: first spheroid guard
240: second spheroid guard

Claims (11)

a base portion forming a bottom in which the cell-medium mixture is accommodated;
a first spheroid guard provided on the base and forming a collecting space on the inside, the first spheroid guard being opened and not closed in a curved shape; and
A second spheroid guard formed in a shape surrounding a portion of the circumference of the first spheroid guard, which is opened in a second direction opposite to the first direction and is formed in a curved shape that is not closed; Including;
The spheroids positioned in the collecting space do not escape to the outside of the collecting space, but the medium in liquid form forms a structure in which the medium is discharged to the outside of the collecting space,
Well plate unit for spheroid culture.
According to claim 1,
Further comprising a side wall portion extending upwardly from the periphery of the base portion,
Well plate unit for spheroid culture.
3. The method of claim 2,
The first spheroid guard, the second spheroid guard and the side wall portion is formed to have a height corresponding to each other,
Well plate unit for spheroid culture.
In the well plate unit provided under the drop forming part of the multi-layer spheroid culture apparatus in which hanging drops of cells and medium are formed,
a base portion forming a floor on which the hanging drop falls;
a first spheroid guard provided on the base and forming a collecting space on the inside, the first spheroid guard being opened and not closed in a curved shape; and
A second spheroid guard formed in a shape surrounding a portion of the circumference of the first spheroid guard, which is opened in a second direction opposite to the first direction and is formed in a curved shape that is not closed; Including;
The spheroids included in the hanging drop that have fallen into the collecting space do not escape to the outside of the collecting space, and the medium in liquid form forms a structure in which it is discharged to the outside of the collecting space,
Well plate unit for multi-layer spheroid culture apparatus.
5. The method of claim 4,
In the base portion, a recessed groove is formed in an area corresponding to the collecting space,
Well plate unit for multi-layer spheroid culture apparatus.
6. The method of claim 5,
The recessed groove is formed to gradually deepen from the outer part of the collecting space to the central part,
Well plate unit for multi-layer spheroid culture apparatus.
5. The method of claim 4,
The width between the first spheroid guard and the second spheroid guard is formed smaller than the diameter of the spheroid,
Well plate unit for multi-layer spheroid culture apparatus.
5. The method of claim 4,
A medium suction space is formed in the opening of the second spheroid guard,
Well plate unit for multi-layer spheroid culture apparatus.
5. The method of claim 4,
Further comprising a side wall portion extending upwardly from the periphery of the base portion,
Well plate unit for multi-layer spheroid culture apparatus.
10. The method of claim 9,
The side wall portion is provided with a medium suction unit for sucking the medium discharged to the outside of the collection space,
Well plate unit for multi-layer spheroid culture apparatus.
10. The method of claim 9,
The side wall portion is provided with a medium supply unit for supplying the medium to the base portion,
Well plate unit for multi-layer spheroid culture apparatus.

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