KR102187633B1 - Microwell plate for real-time monitoring - Google Patents

Abstract

The present invention relates to a microwell plate including a plurality of microwells, to correctly analyze an organoid or a spheroid in real-time. According to the present invention, the microwell plate comprises: a plate main body including a plurality of microwell sets formed on first to third microwells arranged in parallel in one direction, wherein the bottom surfaces of the first to third microwells forming the microwell set have first to third holes, a flow path communicating with the first to third holes is disposed on the lower part of the first to third microwells, and the bottom surface of the flow path is in an open state; and a substrate coupled to the lower part of the plate main body to form the bottom surface of the flow path, wherein a cultivation groove is formed at a position corresponding to the second microwell in a portion forming the bottom surface of the flow path.

Description

Microwell plate for real-time monitoring {MICROWELL PLATE FOR REAL-TIME MONITORING}

The present invention relates to a microwell plate, and more particularly, to a microwell plate for cultivation and analysis of spheroids/organoids.

In recent years, screening of drug efficacy evaluation and studies of differentiation inducing factors have been actively conducted. In these studies, various cell aggregates such as organoids and spheroids are used for evaluation. Accordingly, there is a need for a culture method for efficiently forming cell aggregates such as organoids and spheroids.

In addition, in evaluation such as screening, it is necessary to rapidly process multiple samples at once. Accordingly, a culture vessel for the formation and analysis of a large amount of cell aggregates is required.

For this purpose, a microwell plate in which a plurality of microwells are formed is used. In microwell plates, for example, 96 wells, 384 wells, and 1,536 wells are commercially available, and a large number of specimens can be handled.

1 is a view for explaining a conventional microwell plate. Referring to FIG. 1, each of a plurality of microwells formed in a conventional microwell plate is provided in the same shape as the inside of a test tube. The user can inject the cells and the culture medium into the microwell to cultivate the cells in the culture medium to form a cell aggregated mass, and add a reagent to analyze the efficacy of the drug against the aggregated mass.

However, in the case of a conventional microwell plate, since the organoid/spheroid is not supplied with nutrients through blood flowing through the blood vessel and is cultured in a state accommodated in the microwell plate together with the culture medium, an accurate analysis result may not be derived.

In addition, in the case of a conventional microwell plate, since the state of organoids/spheroids is analyzed using a separate analysis device rather than the microwell plate itself, real-time analysis of organoids/spheroids is not easy.

Japanese Laid-Open Patent No. 5954422

In order to solve the above problems, an object of the present invention is to provide a microwell plate capable of accurate and real-time analysis of organoids or spheroids.

In order to solve the above problems, the microwell plate of the present invention includes a plurality of microwell plates composed of first to third microwells arranged side by side in one direction, and the first to the first to third microwells constituting the microwell set First to third holes are formed in the bottom surface of the third microwell, a flow path communicating with the first to third holes is provided under the first to third microwells, and the bottom surface of the flow path is open A plate body provided in a state of being; And a substrate coupled to a lower portion of the plate body to form a bottom surface of the flow path, and having a culture groove provided at a position corresponding to the second microwell in a portion forming the bottom surface of the flow path.

In addition, in an embodiment, a culture solution is injected into at least one of the first microwell and the third microwell, and any one of cells, organoids, and spheroids is injected into the second microwell.

In addition, in an embodiment, the flow path functions as a micro channel.

In addition, in an embodiment, a culture solution injected into at least one of the first microwell and the third microwell is supplied into the culture groove according to the slope of the microplate.

In addition, in an embodiment, the first and third holes formed on the bottom surfaces of the first and third microwells have a diameter size larger than that of the second holes formed on the bottom surfaces of the second microwells.

In addition, in an embodiment, a fluid flow control membrane is provided at each of the lower ends of the first hole and the third hole to control a supply rate of the culture solution supplied into the flow path through the first hole and the third hole.

In addition, in an embodiment, the fluid flow control membrane includes a porous membrane material.

In addition, in an embodiment, it further includes an evaporation prevention film provided on the plate body to prevent evaporation of the culture medium in the microwell.

In addition, in an embodiment, a biosensor is provided on a portion of the upper surface of the substrate that forms the bottom surface of the flow path.

In addition, in an embodiment, the biosensor is provided on the upper surface of the substrate at a fourth position between a first position corresponding to the first microwell and a second position corresponding to the second microwell, or It is provided at a fifth position between the second position corresponding to the second microwell and the third position corresponding to the third microwell.

According to the present invention, since the flow path formed in the microplate performs the function of a microchannel, it is possible to analyze an organoid or spheroid in an environment similar to that of an actual human body.

According to the present invention, by providing the biosensor in the flow path formed in the microplate, it is possible to analyze the organoid or spheroid in real time.

1 is a view for explaining a conventional microwell plate.
2 is an exploded perspective view of a microwell plate viewed from a downward direction according to an embodiment of the present invention.
FIG. 3A is a top view of the plate body shown in FIG. 2 from above, FIG. 3B is a rear view of the plate body shown in FIG. 2 from below, and FIG. 3C is a top view of the substrate shown in FIG. 2 from above. , FIG. 3D is a perspective view for explaining a flow path.
4 is a cross-sectional view of a microplate for explaining a cross-section in the direction A-A' in the microwell plate of FIG. 2.
5 is a cross-sectional view showing various shapes of the bottom surface of the culture groove shown in FIG. 4.
6 is a diagram for explaining a driving principle of the microwell plate shown in FIG. 2.
7 shows the results of culturing organoids or spheroids using the microplate shown in FIG. 4.
FIG. 8 is a diagram for explaining a monitoring system including a microwell plate shown in FIG. 4.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. However, the embodiments introduced herein are provided so that the disclosed contents may be thorough and complete, and the spirit of the present invention may be sufficiently conveyed to those skilled in the art. In the drawings, in order to clearly express the constituent elements of each device, the size of the constituent elements, such as width or thickness, is slightly enlarged. Overall, when the drawing was described from the observer's point of view, when an element is mentioned as being positioned on another element, this means that the element is positioned directly on another element or that an additional element may be interposed between the elements. Include. In addition, those of ordinary skill in the relevant field will be able to implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in the plurality of drawings refer to substantially the same elements.

Meanwhile, the meanings of terms described in the present invention should be understood as follows. Terms such as “first” or “second” are used to distinguish one element from other elements, and the scope of the rights is not limited by these terms. For example, a first component may be referred to as a second component, and similarly, a second component may be referred to as a first component.

In addition, expressions in the singular should be understood as including plural expressions unless clearly defined otherwise in the context, and terms such as “include” or “have” are described features, numbers, steps, actions, components, and parts. It is to be understood that it is intended to designate the existence of a combination of these and not to preclude the possibility of the presence or addition of one or more other features or numbers, steps, actions, components, parts, or combinations thereof. In addition, in performing the method or manufacturing method, each of the processes constituting the method may occur differently from the specified order unless a specific order is clearly stated in the context. That is, each process may occur in the same order as the specified order, may be performed substantially simultaneously, or may be performed in the reverse order.

2 is an exploded perspective view of the microwell plate according to an embodiment of the present invention as viewed from below, FIG. 3A is a top view of the plate body shown in FIG. 2, and FIG. 3B is a bottom view of the plate body shown in FIG. It is a rear view as viewed from, FIG. 3C is a top view of the substrate shown in FIG. 2, and FIG. 3D is a perspective view for explaining a flow path. And FIG. 4 is a cross-sectional view of a microplate for explaining a cross section in the direction A-A' in the microwell plate of FIG. 2.

Hereinafter, a microwell plate will be described with reference to FIGS. 2 to 4.

2 to 4, the microwell plate 10 of the present invention includes a plate body 100 and a substrate 200 coupled to a lower portion of the plate body 100.

The plate body 100 is provided with a plurality of microwell sets 112 composed of first to third microwells 111a, 111b, and 111c arranged side by side in one direction, and the first to second microwell sets constituting the microwell set. 3 First to third holes H1, H2, H3 are formed on the bottom surface of the microwells 111a, 111b, and 111c, and holes formed at the bottom of each of the first to third microwells 111a, 111b, and 111c The flow path communicating with is provided with the bottom surface open. The bottom surface of the flow path 121 provided with the bottom surface open is formed by the substrate 200 coupled to the plate body 100.

Hereinafter, the plate body 100 is divided into an upper portion 110 and a lower portion 120 positioned below the upper portion 110 and described in detail. The upper portion 110 and the lower portion 120 may be formed integrally or separately.

3A and 4, the upper portion 110 of the plate body 100 includes a microwell 111 having a groove having a predetermined depth. The groove formed in the microwell 111 may be provided in various shapes such as a polygonal groove shape and a cylindrical groove shape. Meanwhile, the first and third microwells 111a and 111c to be described later are provided in any one of the various shapes, and the second microwell 111b is different from the first and third microwells 111a and 111c. It may be provided in a shape.

A plurality of microwells such as 96, 384 and 1536 may be provided.

At this time, the first to third microwells 111a, 111b, and 111c arranged side by side in one direction among the plurality of microwells 111 constitute one microwell set 112. The microwell set 112 is provided on the upper portion 110 of the plate body 100.

For example, when the number of microwells 111 formed in the upper portion 110 of the plate body 100 is 96, the number of microwell sets 112 is 32, and the number of first to third microwells There are 32 each.

The first microwell 111a and the third microwell 111c are microwells for a culture medium. A culture solution may be injected into one of the first microwell 111a and the third microwell 111c, or the culture solution may be injected into both the first microwell 111a and the third microwell 111c. Depending on the tilting angle of the microwell plate, the culture liquid in the first microwell 111a and the third microwell 111c flows through the flow path 121, and the culture liquid flowing through the flow path 121 flows through cells, organoids, or spores. It is supplied to the culture groove 220 in which the Lloyd is located.

The second microwell 111b is a microwell into which any one of cells, organoids, and spheroids is injected, and at least one of cells, organoids, and spheroids may be injected therein.

First to third holes H1, H2, and H3 are formed in each of the bottom surfaces of the three first to third microwells 111a, 111b, and 111c constituting one microwell set 112.

The diameter of the first and third holes H1 and H3 formed on the bottom surfaces of the first and third microwells 111a and 111c is a hole H2 formed on the bottom surface of the second microwell 111b It can be formed larger than the diameter size of. For example, the diameter size of the holes H1 and H3 formed on the bottom surfaces of the first and third microwells 111a and 111c may be formed to be approximately 2mm, and are formed on the bottom surface of the second microwell 111b. The diameter size of the formed hole H2 may be approximately 1 mm.

By reducing the diameter size of the hole H2 formed on the bottom surface of the second microwell 111b, when the microwell plate is tilted and the culture solution flows in one direction, the culture solution leaks through the second hole H2. The degree can be minimized.

By forming the diameter size of the holes H1 and H3 formed on the bottom surfaces of the first and third microwells 111a and 111c to be large, the feed rate of the culture solution may be increased. In addition, in order to control the supply rate of the culture medium, a fluid flow control film 320 may be provided at each of the lower ends of the holes H1 and H3 formed on the bottom surfaces of the first and third microwells 111a and 111c. . The fluid flow control membrane 320 controls the supply rate of the culture solution through which the culture solution in the first and third microwells 111a and 111c flows into the flow path 121. The fluid flow control membrane 320 may be formed of a porous membrane material through which the culture medium can pass.

3B and 4, a flow path 121 is formed in the lower end 120 of the plate body 100. Specifically, the flow path 121 is formed one by one for each microwell set 112 composed of the first to third microwells 111a, 111b, and 111c. The flow path 121 includes first to third holes H1, H2, H3 formed on the bottom surfaces of the first to third microwells 111a, 111b, and 111c constituting one microwell set 112, and Communicate. At this time, the flow path 121 functions as a micro channel. The bottom surface of the flow path 121 is in an open state, and the substrate 200 coupled to the lower portion of the plate body 110 forms the bottom surface of the flow path 121 so that a fluid can flow. That is, when viewed from the rear surface of the plate body 100, the flow path 121 may be seen in the form of a groove.

The shape of the flow path 121 will be described in detail with reference to FIG. 3D.

In FIG. 3D, the first position P1, the second position P2, and the third position P3 are on the bottom surface of the flow path 121. The first microwell 111a, the second microwell 111b, and the third These are positions respectively corresponding to the microwells 111c. And the fourth position (P4) is a position between the first position (P1) and the second position (P2) on the bottom surface of the flow path, and the fifth position (P5) is the second position (P2) on the bottom surface of the flow path. It is a position between the third positions P3.

The cross-sectional area of the flow path 121 in the fourth position P4 and the fifth position P5 is smaller than the cross-sectional area of the flow path 121 in the first position P1, the second position P2, and the third position P3. Can be formed. When the cross-sectional area of the flow path 121 is adjusted, the shear stress of the fluid flowing in the flow path 121 is changed, thereby changing the fluid velocity. The cross-sectional area of the flow path 121 in the fourth position P4 and the fifth position P5 so that the culture medium can be supplied at a speed similar to the speed of the fluid supplied when the cells, organoids, and the body to be analyzed are actually in the human body. May be designed differently depending on the cell to be measured. The microwell plate 10 of the present invention may provide a culture environment similar to the environment in which the actual cells are located.

The first to fifth positions P1, P2, P3, P4, and P5 in FIGS. 3C and 4 are as described with reference to FIG. 3D.

The plate body 100 is a polyolefin resin, such as a polypropylene resin, a polyethylene resin, an ethylene-propylene copolymer, or a cyclic polyolefin resin, a polystyrene resin such as a polystyrene, acrylonitrile-butadiene-styrene resin, a polycarbonate resin, Methacrylic resins such as polyethylene terephthalate resin and polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, poly It may be provided with at least one of an etherimide resin, a fluorine resin such as polytetrafluoroethylene, an acrylic resin such as polymethylpentene resin, and polyacrylonitrile, a propionate resin, and an elastomer. Particularly, in the plate body 100, the flow path 121 is preferably made of a biocompatible elastomer material in order to perform a function as a microchannel. The hole manufacturing of the plate body 100 may be manufactured through an NC processing machine, and the flow path portion may be manufactured using Material Jet 3D printing technology.

The substrate 200 is coupled to the lower portion of the plate body 110 and forms a bottom surface of the flow path 121 formed in the lower end portion 120 of the plate body 110. A culture groove 220 in which organoids or spheroids are cultured is located in a second position P2 corresponding to the second microwell 111b in a portion of the upper surface of the substrate 200 forming the bottom surface of the flow path 121 It is equipped. Organoid refers to a three-dimensional mass of cells that has a structure, cellular components, and functions similar to an organ in a living body (human). And spheroid means that the structure and function are matured by making various differentiated cells constituting each organ of a living body take on a three-dimensional structure outside the body.

The biosensor 210 may be provided on a portion of the upper surface of the substrate 200 that forms the bottom surface of the flow path 121. Specifically, the biosensor 210 is provided on the upper surface of the substrate at a fourth position between a first position corresponding to the first microwell 111a and a second position corresponding to the second microwell 111b, or It may be provided at a fifth position P5 between the second position P2 corresponding to the second microwell 111b and the third position P3 corresponding to the third microwell 111c. The biosensors 210 may be provided in more or less numbers.

The biosensor 210 detects in real time a specific substance secreted from the organoid or spheroid in the culture medium when the culture medium passes through the organoid or spheroid. For example, the biosensor 210 may detect specific components such as acidity (pH), demand oxygen (DO), reactive oxygen species (ROS), and lactic acid in the culture medium.

The biosensor 210 may be provided in the form of an embedded sensor on the substrate 200 using a printed electronic technology (EHD, inkjet printing, etc.) based on gold and silver ink. Therefore, only by combining the substrate and the plate body, it is possible to implement a microwell plate having a biosensor in the flow path.

The culture groove 220 is provided at a second position P2 corresponding to the second microwell 111b. A space in which organoids or spheroids are cultivated in the culture groove 220 and may be provided in various shapes.

5 is a cross-sectional view showing various shapes of the bottom surface of the culture groove shown in FIG. 4.

In the case of the V-type in which the cross-sectional shape of the bottom surface of the culture groove 220 is'V' as shown in FIG. 5A, it is a form suitable for sedimentation analysis and sample storage. As shown in (b) of FIG. 5, the F-type in which the cross-sectional shape of the bottom surface of the culture groove 220 is flat is suitable for optical measurement through a flat bottom surface. As shown in (c) of FIG. 5, in the case of the C type in which the bottom surface of the culture groove 220 is generally flat but the edge portion of the bottom surface is rounded, the sample can be easily mixed by the rounded edge portion, and It is a form that makes light measurement easy. In the case of the U-type in which the bottom surface of the culture groove 220 is rounded as shown in FIG. 5(d), it is a form that is easy to stir or agglomerate the sample.

The bottom surface of the culture groove 220 of the present invention may be provided in any one of V type, F type, C type, and U type. In particular, the bottom surface of the culture groove 220 of the present invention is preferably provided in a U-type shape that facilitates the formation or culture of a three-dimensional organoid or spheroid by cell-cell interaction.

A coating layer 230 for facilitating cell aggregation may be provided on the surface of the culture groove 220. The coating layer 230 may include at least one of Pluronic and Poly-Hema. When cells, organoids, or spheroids are injected through the second microwell 111b, the cells, organoids, or spheroids injected into the second microwell 111b are cultured groove 220 through the second hole H2. ) And is cultured in the culture groove 220.

3A and 4, an evaporation prevention film 310 for preventing evaporation of the culture solution may be provided on the upper portion of the plate body 100. The evaporation prevention film 310 is made of a porous membrane material to prevent evaporation of the culture solution. In addition, a ring-shaped contact member 330 may be provided for contact between the upper portions of the first to third microwells 111a, 111b, and 111c and the evaporation prevention film 320. The contact member 330 may be formed of a rubber material.

6 is a view for explaining the operating principle of the microwell plate shown in FIG.

Referring to FIG. 6, a culture solution is injected into the first microwell 110a and the third microwell 110c, and the culture solution injected by tilting the microwell plate 10 is selectively supplied into the culture groove 220 Can be.

Unlike this, the culture medium may be injected into only one of the first microwell 110a and the third microwell 110c. In this case, the microwell into which the culture solution is injected becomes a culture solution supply unit that supplies the culture solution into the culture groove 220 by tilting the microwell plate, and the microwell to which the culture solution is not injected may be a culture solution recovery unit.

A separate microwell plate 10 support device for tilting the microwell plate 10 may be used. While the microwell plate 10 is culturing organoids or spheroids, the biosensor can measure the components in the culture medium in real time.

According to the present invention, it is possible to analyze organoids or spheroids in real time by providing a biosensor in the flow path formed in the microplate. In addition, according to the present invention, the flow path formed in the microplate performs the function of the microchannel, thereby providing an environment similar to the actual human body environment, thereby enabling accurate analysis.

7 shows the results of culturing organoids or spheroids using the microplate shown in FIG. 4.

Referring to FIG. 7, as a result of culturing A549 cells, CaCo-2 cells, HaCat cells, and HK-2 cells, it can be seen that the cells are not killed and that the cells are well cultured in an aggregated state.

8 is a diagram for describing a monitoring system including a microwell plate shown in FIG. 4.

Referring to FIG. 8, the monitoring system 60 includes a microwell plate 10, a sensor board 20, a support device 30, a data collection device 40, and a monitoring device 50.

The microwell plate 10 is as described in FIGS. 2 to 4.

The sensor board 20 is connected to a terminal of at least one biosensor 121 provided in the microwell plate 10 to receive sensor data from the biosensor 121, and is connected to the data collection device 40 to provide data The sensor data is transmitted to the collection device 40.

The support device 30 adjusts the tilting state of the microwell plate 10, that is, the tilt angle and rotation speed of the microwell plate 10, and supports the microwell plate 10. According to the tilting state of the microwell plate 10, the fluid direction and the fluid flow rate of the microwell plate 10 are changed, thereby changing a shear stress transmitted to the cells.

The data collection device 40 transmits the sensor data received from the sensor board 20 to the monitoring device 50 by wire or wirelessly. The data acquisition device 40 may be provided with a DAQ (Data Aquisition) board. At this time, the data collection device 40 may also transmit information on the tilting state such as the tilting direction, tilting angle, and rotation speed of the support device 30 to the monitor device 50 together.

The monitoring device 50 receives sensor data from the data collection device 40, and displays a result such as a graph to the user using the sensor data display means. The monitoring device 50 may analyze the information on the received tilting state and sensor data, and change and control the tilting state of the microwell plate 10 of the support device 30.

10: microwell plate 100: plate body
110: upper part 111: microwell
111a, 111b, 111c: first to third microwells
120: lower part 121: euro
200: substrate 210: biosensor
220: culture groove 230: coating layer
310: evaporation prevention film 320: fluid flow control film
330: contact member
H1, H2, H3: first to third holes
P1, P2, P3, P4, P5: 1st to 5th positions

Claims (10)

In the microwell plate having a plurality of microwells,
A plurality of microwell sets consisting of first to third microwells arranged side by side in one direction are provided, and first to third holes are formed on each bottom surface of the first to third microwells constituting the microwell set A plate main body having flow paths communicating with the first to third holes provided under the first to third microwells, and having a bottom surface of the flow path open; And
A substrate coupled to a lower portion of the plate body to form a bottom surface of the flow path, and a substrate having a culture groove at a position corresponding to the second microwell at a portion forming the bottom surface of the flow path,
A fluid flow control membrane is provided at each lower end of the first hole and the third hole to control a supply rate of the culture solution supplied into the flow path through the first hole and the third hole,
Diameter sizes of the first and third holes formed on the bottom surfaces of the first and third microwells are larger than the diameter sizes of the second holes formed on the bottom surfaces of the second microwells,
A culture solution is injected into at least one of the first microwell and the third microwell, and any one of cells, organoids, and spheroids is injected into the second microwell,
Microwell plate, characterized in that the culture solution injected into at least one of the first microwell and the third microwell is supplied into the culture groove according to the slope of the microplate. delete The method of claim 1,
The flow path is a microwell plate, characterized in that the function of a micro channel. delete delete delete The method of claim 1,
The fluid flow control membrane is a microwell plate, characterized in that comprising a porous membrane material. The method of claim 1,
Microwell plate, characterized in that it further comprises an evaporation prevention film provided on the upper portion of the plate body to prevent evaporation of the culture medium in the microwell. The method of claim 1,
Microwell plate, characterized in that the biosensor is provided on a portion of the upper surface of the substrate that forms the bottom surface of the flow path. The method of claim 9,
The biosensor,
On the upper surface of the substrate, provided at a fourth position between a first position corresponding to the first microwell and a second position corresponding to the second microwell, or a second position corresponding to the second microwell Microwell plate, characterized in that provided in a fifth position between the third position corresponding to the third microwell.

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