KR20210053010A - Disk for cell distribution and culture, real-time monitoring system, and method for distribution and culture of cells - Google Patents

Abstract

A disk for cell division and culture of the present disclosure, as a disk having an inlet and an outlet, comprises: a cell supply region connected to the inlet; a cell distribution region comprising a cell transport channel extending in a radial direction of the disk; a cell trapping unit having a trapping groove close to the end of the cell transport channel; and a cell culture unit having a space connected to the trapping groove. An object of the present invention is to provide a real-time monitoring system that can perform cell division and culture while monitoring the same in real time by mounting a disk for cell division and culture, and a cell division and culture method using the same.

Description

Disk for cell dispensing and cultivation, real-time monitoring system and cell dispensing and culturing method {DISK FOR CELL DISTRIBUTION AND CULTURE, REAL-TIME MONITORING SYSTEM, AND METHOD FOR DISTRIBUTION AND CULTURE OF CELLS}

The present disclosure relates to a disk for dispensing and culturing cells, a real-time monitoring system, and a method for distributing and culturing cells using the same.

Protein production using cells is the most important part of the pharmaceutical industry. In order to produce a specific protein drug using cells, CHO (Chinese hamster ovary) cells are genetically engineered to overexpress and secrete a specific protein. After that, cells with the best secretion of the target protein and good growth are selected, introduced into the bioreactor, and the protein is collected by repeating the replication process numerous times.

When genetically engineered to overexpress the target protein, the gene is generally introduced into cells through a transfection process in tens of thousands of cells or more. However, since the degree to which genes are introduced and expressed varies greatly for each cell, selecting the best-growing cells that produce the best protein at the level of a single cell is the most important part of protein restriction.

Currently, at least hundreds of cells per well are distributed to a plate having dozens of wells, and the amount of the target protein produced in each well is measured, and cells of the most suitable well are introduced into the bioreactor. According to this method, the yield is lowered by tens to hundreds of times compared to cultured single cells that make the most protein, resulting in many problems such as poor productivity, time consuming, and high cost.

In order to solve this problem, it can be separated into single cells and tested after dispensing, but with the current technology, a lot of time, effort, and cost are required for the separation process. In addition, since there is no technology to put isolated single cells into microscopic spaces, it is necessary to experiment by putting them in a well plate having dozens of wells. Each well of the well plate is at least several thousand times larger than a single cell, and these are imaged through a microscope. It is impossible to monitor cell growth in real time.

In addition, for the development of a new protein drug, confirming the binding between a protein secreted by a single cell and a target reactant (antigen) is a key part of the new drug development. However, it is very difficult to find a single cell that produces a protein that reacts with the target reactant because the types of proteins secreted by the same genetically engineered cells are different and there are many similar subtypes. Currently, genetically engineered cell lines and cell lines with target reactants are mixed and cultured in well plates, etc., and potent cell groups are found through imaging or molecular biological analysis. In spite of the high cost and effort, this method cannot be tested in thousands of wells, and even after the experiment, a single cell cannot be specified, making it difficult to find an accurate new drug protein.

One aspect of the embodiment is that at least thousands of single cells can be dispensed at the same time, and each isolated single cell is placed in a culture space, and then cultured, and a cell dispensing and culture disk that can be monitored in real time through imaging. I want to provide.

Another aspect of the embodiment is to provide a real-time monitoring system capable of performing cell dispensing and culturing while performing real-time monitoring by mounting a cell dispensing and culturing disk, and a cell dispensing and culturing method using the same.

However, the problems to be solved by the embodiments of the present invention are not limited to the above-described problems and may be variously expanded within the scope of the technical idea included in the present invention.

A disk for dispensing and culturing cells according to an embodiment is a disk having an inlet and an outlet, the cell distribution region including a cell supply region connected to the inlet, a cell transfer channel extending in a radial direction of the disk, and the cells It includes a cell capture portion having a capture groove close to the end of the transfer channel, and a cell culture portion having a space connected to the capture groove.

The space of the cell culture unit may be located in different layers separated from the cell transfer channel.

It may further include a cell recovery region in communication with the cell distribution region and connected to the outlet.

At the height measured in the thickness direction of the disk, the height of the cell distribution region may be lower than the height of the cell supply region.

The cell transfer channel may be narrower in width toward the outer side in the radial direction of the disk.

The capture groove may be formed to be concave in a direction toward the center of the disk.

The trapping groove may have a radius of curvature corresponding to the radius of a single cell.

The cell culture unit may be connected to a cell discharge unit opened to the outside of the disk.

A disk for dispensing and culturing cells according to another embodiment is a disk having an inlet and an outlet connected to an inner space on a surface, a cell supply area located in the inner space and connected to the injection port, and the cell supply area in the inner space And a cell distribution region including a plurality of cell transfer channels each extending in a radial direction of the disk, a cell capture portion having a capture groove close to an end of the cell transfer channel, and a culture space connected to the capture groove. It may include a cell culture unit, and a cell recovery region connected to the outlet and communicated with the cell transfer channel.

The injection port and the discharge port may be spaced apart from each other in a radial direction of the disk, the injection port may be closer to a center of the disk, and the discharge port may be closer to an edge of the disk.

The cell supply region may include a diffusion portion connected to the injection port, and a transition portion extending from the diffusion portion and having a portion lower than the height measured in the disk thickness direction of the diffusion portion and connected to the cell transfer channel. .

The cell distribution region may include a plurality of partitions extending in a radial direction of the disk, and the partition may form a boundary between the cell transfer channel.

Each of the cell transfer channels may be narrower in width toward the outer side in the radial direction of the disk.

The capture groove may be formed to be concave in a direction toward the center of the disk.

The trapping groove may have a radius of curvature corresponding to the radius of a single cell.

The cell trapping portion has a pointed attachment in a direction toward the center of the disk, and the trapping grooves may be located at one end of the attachment.

Includes a plurality of partitions forming the boundary of the cell transfer channel, each of the partitions has an attachment at a distal end facing outward in the radial direction of the disk, and the attachment of the cell trapping portion and the attachment of the partition portion are alternately arranged. I can.

The cell trapping unit is located downstream of the cell transfer channel and may be provided as many as a number corresponding to each of the plurality of cell transfer channels.

The culture space of the cell culture unit may be located in a second layer separated from the first layer in which the cell transfer channel is located.

The cell culture unit may further include a cell collecting unit in communication with the capturing groove, and the cell collecting unit may be disposed to overlap the capturing groove when viewed from a plane perpendicular to the rotational axis of the disk.

The cell culture part may be connected to a cell discharge part opened to the outside of the disk, and the cell discharge part may be connected to a part spaced apart from the cell collection part.

The cell discharge part may include a first discharge pipe communicating with the culture space of the cell culture part, and a second discharge pipe having a larger diameter than the first discharge pipe and opening to the outside of the disk.

The cell recovery region may further include a re-diffusion unit positioned between the cell distribution region and the outlet.

A real-time monitoring system for cell dispensing and cultivation according to another embodiment is a monitoring system for distributing and culturing cells by carrying in a disk for cell dispensing and culturing, and rotating by mounting the disc for dispensing and culturing cells. And a fluorescent image pickup unit positioned at a lower portion of the cell dispensing and culturing disk to correspond to the cell culture unit to irradiate light and measure reflected fluorescence.

The real-time monitoring system for cell dispensing and culturing according to another embodiment may further include an incubating chamber provided to surround the cell dispensing and culturing disk separated from the fluorescence image pickup unit and outside air.

A cell dispensing and culturing method according to another embodiment includes preparing a cell dispensing and culturing disk having an inlet and an outlet connected to the inner space on the surface, injecting cells into the inlet, and for distributing and culturing the cells. Rotating a disk and distributing and transferring the injected cells for each of a plurality of cell transfer channels, rotating the cell dispensing and culturing disk, and from the transferred cells to a capture groove of a cell trap located downstream of the cell transfer channel It may include capturing a single cell, and moving the captured single cell to a cell culture unit.

The step of moving the captured single cells to the cell culture unit includes the process of inverting the cell dispensing and culturing disk to move the captured single cells to the cell collection unit by gravity, and rotating the cell dispensing and culturing disk. It may further include a process of moving a single cell of the cell collection unit to the cell culture unit.

The method of distributing and culturing cells according to another embodiment may further include culturing the single cells in the cell culture unit, and extracting the cultured cells from the cell culture unit.

A cell dispensing and culturing method according to another embodiment includes measuring the growth of cells cultured in the cell culture unit through real-time imaging, and measuring the amount of protein produced in the cell culture unit using a fluorescent reagent. It may further include.

The cell dispensing and culturing method according to another embodiment may further include the step of recovering the transferred cells excluding the captured single cells from the outlet of the cell dispensing and culturing disk.

According to the cell dispensing and culturing disk according to the embodiment, the real-time monitoring system, and the cell dispensing and culturing method, at least thousands of single cells can be dispensed at the same time, and the isolated single cells are placed into the culture space, respectively, It can be cultured and monitored in real time through imaging.

In addition, through the design of the disk according to the embodiment, by culturing thousands of cells separated into single cells in each culture room and monitoring in real time, it is possible to obtain a single cell with the best growth and a single cell that produces the most target protein. have.

1 is a perspective view showing a disk for dispensing and culturing cells according to an embodiment of the present invention.
2 is a plan view and an enlarged plan view showing a disk for dispensing and culturing cells according to an embodiment of the present invention.
3 is a partially cut-away perspective view showing a part of a disk for dispensing and culturing cells according to an embodiment of the present invention.
4 is a perspective view showing an enlarged part of a disk for cell dispensing and culture according to an embodiment of the present invention.
5 is a perspective view showing a real-time monitoring system for dispensing and culturing cells according to another embodiment of the present invention.
6 is an enlarged perspective view illustrating a disk driving unit of the monitoring system shown in FIG. 5.
7 is a flow chart showing a cell dispensing and culturing method according to another embodiment of the present invention.
8 to 12 are views for explaining a cell dispensing and culturing method according to another embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings will be described in detail so that those of ordinary skill in the art can easily implement the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and the same reference numerals are assigned to the same or similar components throughout the specification. In addition, the accompanying drawings are for easy understanding of the embodiments disclosed in the present specification, and the technical idea disclosed in the present specification is not limited by the accompanying drawings, and all changes included in the spirit and scope of the present invention It should be understood to include equivalents or substitutes.

In addition, throughout the specification, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

1 is a perspective view showing a disk for dispensing and culturing cells according to an embodiment of the present invention.

Referring to FIG. 1, the disk 100 for dispensing and culturing cells according to the present embodiment (hereinafter, also referred to as “disc”) has a disk-shaped appearance and has an approximately central region 110 along a radial direction from the center to the edge. ), a dummy area 115, a cell supply area 120, a cell distribution area 130, and a cell recovery area 160.

The central region 110 includes the center of the disk 100 and may be formed as a circular region having a predetermined radius. The disk 100 may be fixed and used in a device that provides a rotational driving force, and for this purpose, a groove or a protrusion may be provided in the central region 110 so as to be fixed to the device. The dummy area 115 may be formed of an annular area extending radially outward from the boundary of the center area 110 by a predetermined distance.

The cell supply region 120, the cell distribution region 130, and the cell recovery region 160 may extend from the boundary of the dummy region 115 to the outer edge in the radial direction. In these areas, a process of supplying and distributing cells to the inner space of the disk 100 from the outside, capturing each single cell, and recovering the remaining cells may be performed.

The disk 100 may be entirely made of a transparent plastic material, and for example, may be made of transparent polypropylene (PP) or polystyrene (PS).

2 is a plan view and an enlarged plan view showing a disk for cell dispensing and culturing according to an embodiment of the present invention, in which the cell supply area 120, the cell distribution area 130, and the cell recovery area 160 are enlarged. Shown.

Referring to FIG. 2, the disk 100 for dispensing and culturing cells according to the present embodiment may include an inlet 121 and an outlet 161 connected to an inner space. The injection port 121 and the discharge port 161 may be opened from the inner space to the outside, and are spaced apart from each other in the radial direction of the disk 100. In this case, the injection hole 121 may be located closer to the center of the disk 100 and the discharge hole 161 may be located closer to the edge of the disk 100. In addition, the cell supply area 120 is an area connected to the injection port 121 in the inner space of the disk 100, and the cell recovery area 160 is an area connected to the discharge port 161 in the inner space of the disk 100. .

The cell distribution region 130 may distribute and capture the supplied cells by providing a plurality of cell transfer channels 132 between the cell supply region 120 and the cell recovery region 160. That is, a plurality of partitions 134 extending in the radial direction of the disk 100 may be positioned in the inner space of the disk 100 to form a plurality of cell transfer channels 132.

A cell capture unit 141 may be positioned at a boundary between the cell distribution region 130 and the cell recovery region 160. The cell capture portions 141 may be provided as many as the number corresponding to each of the plurality of cell transfer channels 132, and may be disposed close to the distal end of the cell transfer channel 132. Each cell capture part 141 has one capture groove 143, and the capture groove 143 is located downstream of the cell transfer channel 132 and may be formed concave in a direction toward the center of the disk 100. have. In addition, the trapping groove 143 may have a radius of curvature corresponding to the radius of a single cell, thereby enabling a single cell to be captured.

Meanwhile, the cell capture part 141 may be formed of a block having a pointed attachment in a direction toward the center of the disk 100, and a capture groove 143 may be formed at the end of the attachment. The partition portion 134 may have an attachment at a distal end facing radially outward of the disk 100, and attachment of the cell trapping portion 141 and the attachment of the partition portion 134 may be alternately disposed. Accordingly, the cell solution flowing through the cell transfer channel 132 defined by the partition portion 134 may be branched into two branches while meeting the cell trapping portion 141 and transferred to the cell recovery region 160.

The cell recovery area 160 communicates with the plurality of cell transfer channels 132 in the inner space of the disk 100 and may be connected to the discharge port 161.

3 is a partially cut-away perspective view showing a partial cut-away view of a cell dispensing and culturing disk according to an embodiment of the present invention, showing a cell supply area 120, a cell distribution area 130, and a cell recovery area 160 A three-dimensional perspective view is shown.

Referring to FIG. 3, the cell supply region 120 may include a diffusion part 123 and a transient part 125. The diffusion part 123 is connected to the injection hole 121 and may have a wide integrated annular area. The transition part 125 may extend from the diffusion part 123 and be fluidly connected to the cell transfer channel 132 of the cell distribution region 130. The height of the cell transfer channel 132 measured in the thickness direction of the disk may be formed to be lower than the height of the diffusion part 123, and the transient part 125 starts from the height of the diffusion part 123 and the cell transfer channel 132 It can be gradually lowered to the height of.

The cell transfer channel 132 of the cell distribution region 130 may be formed by positioning a plurality of partitions 134 in the inner space of the disk 100. That is, the partition portion 134 is provided as a partition block formed to contact the upper surface and the bottom surface of the inner space of the disk 100 to form a boundary between the cell transfer channel 132 and separate neighboring ones from each other. The partition portion 134 extends long in the radial direction of the disk 100, and the width in the circumferential direction of the disk 100 may gradually expand as it goes outward in the radial direction. Accordingly, the width of the cell transfer channel 132 in the circumferential direction may gradually decrease as it goes outward in the radial direction.

The cell recovery region 160 may include a re-diffusion unit 163, and the re-diffusion unit 163 may be connected to an outlet 161 adjacent to an edge of the disk 100. The re-diffusion unit 163 is fluidly connected to the cell transfer channel 132 and may provide a space in which the remaining cells other than the cells captured by the cell capture unit 141 gather again.

4 is an enlarged perspective view of a part of a cell dispensing and culturing disk according to an embodiment of the present invention, showing an enlarged view of the cell capture unit 141 and the cell culture unit 152.

Referring to FIG. 4, the capture groove 143 of the cell capture part 141 may be fluidly connected to the cell culture part 152, and the cell culture part 152 is formed of a cell transfer channel 132. A culture space 154 may be provided on a second layer separated from the first floor.

At this time, the cell culture unit 152 may include a cell collection unit 156 communicating with the capture groove 143, and the cell collection unit 156 is connected to the culture space 154 of the cell culture unit 152. I can. That is, the cell culture unit 152 provides a culture space 154 adjacent to the capture block of the cell capture unit 141, and this culture space 154 is the capture groove 143 through the cell collection unit 156. Can be connected to. When viewed from a plane perpendicular to the axis of rotation of the disk 100, the cell collection unit 156 is disposed to overlap the capture groove 143, whereby the first layer and the cell culture unit ( It may serve as a passage connecting the second floor where 152 is located.

Meanwhile, the cell culture unit 152 may be connected to the cell discharge unit 171 opened to the outside of the disk 100. The cell discharge unit 171 may be connected to a portion spaced apart from the cell collection unit 156 in the cell culture unit 152, and the opening of the cell collection unit 156 from the culture space 154 of the cell culture unit 152 It can extend in a direction opposite to the direction.

The cell discharge part 171 may include a first discharge pipe 172 communicating with the culture space 154 of the cell culture part 152 and a second discharge pipe 174 opened to the outside of the disk 100 therefrom. I can. In this case, the second discharge pipe 174 may have a funnel shape whose diameter increases toward the surface of the disk 100. The second discharge tube 174 may be closed by attaching an adhesive film to the surface of the disk 100 during the process of dispensing and culturing cells.

5 is a perspective view showing a real-time monitoring system for cell dispensing and culturing according to another embodiment of the present invention, and FIG. 6 is a perspective view showing an enlarged disk drive unit of the monitoring system shown in FIG. 5.

Referring to FIG. 5, the monitoring system 300 according to the present embodiment may include a disk driving unit 320, a fluorescence image capturing unit 340, and a control unit 360 in a housing 310. The housing 310 is made of a container having a substantially rectangular parallelepiped shape, and its upper surface may be opened to allow the carrying of the disk 100 in and out.

The disk driving unit 320 is a part that rotates by fixing the disk 100 for cell dispensing and cultivation, and referring to FIG. 6, a motor that generates a rotational force and a central region of the disk 100 are connected to a rotation shaft of the motor. It may include a fixing member 321 fitted to (110). The motor may include a step motor or a servo motor, and the fixing member 321 may be formed of a convex portion that can be fitted according to the shape of the central region 110 of the disk 100.

The fluorescence image capture unit 340 is a portion for capturing an image while observing fluorescence emitted by cells cultured and processed in the cell culture unit 152 of the disk 100 and may include a light source and a fluorescence microscope. The light source may include an LED light source, and the fluorescence microscope may be provided with an inverted microscope in which the objective lens is positioned below the sample. That is, the fluorescence image pickup unit 340 may observe cells in the cell culture unit 152 with a fluorescence microscope located under the disk 100. That is, the growth of cells can be measured through real-time imaging, and the amount of protein produced in each culture section can be measured using a fluorescent reagent.

In this case, an incubating chamber 350 surrounding and protecting the disk 100 may be provided in a region in which the disk 100 is fixed and rotated. The incubating chamber 350 is made of a container having an outer shape of a substantially rectangular parallelepiped and may be mounted on the stage 315 located at the inner upper end of the housing 310. In the stage 315, the fixing member 321 of the disk driving unit 320 may penetrate and protrude, and the disk 100 may be seated thereon. In addition, the stage 315 may be in contact with the objective lens of the fluorescence microscope, through which the observation window 352 made of transparent glass at a portion corresponding to the objective lens so that the cell culture portion 152 of the disk 100 can be observed. It may include. That is, the incubating chamber 350 may be separated from the fluorescence image pickup unit 340 and outside air to protect the disk 100 and maintain an environment suitable for cell culture.

A sample introduction door 354 through which the disk 100 can be carried in and out may be installed on the upper surface of the incubating chamber 350. The sample introduction door 354 may be slidably opened and closed, and may be formed at least a size sufficient to carry in/out the disk 100.

The control unit 360 is a part for controlling the operation of components included in the housing 310 of the monitoring system 300, and may include a temperature controller, a gas controller, an optical controller, and a disk drive controller.

The temperature controller and the gas controller may be controlled to maintain a constant temperature and humidity inside the incubating chamber 350. To this end, a heater may be installed around the incubating chamber 350 and a gas injection pipe may be installed to be connected to the connector 362. In addition, a sensor capable of detecting the temperature and humidity in the incubating chamber 350 may be installed to provide the sensed temperature and humidity information to the temperature controller and the gas controller.

The optical controller controls the operation of the light source and the glare microscope, and the disk drive controller may control the rotation of the motor driving the disk 100. That is, the disk drive controller can rotate the disk 100 at a predetermined speed for separation of cells, or stop the disk 100 for observing the cells inside the disk 100 and capturing images, and such disk drive control Synchronized with, the optical controller can open and close the light source and control the imaging of the glare microscope.

The monitoring system 300 may include a GPU (Graphic Processing Unit) computer inside the housing 310, and may store and analyze an image captured by a fluorescence microscope using this. In addition, a display is attached to the front of the monitoring system 300 to display the stored and analyzed images, and a shelf is provided on the lower front of the housing 310 to accommodate input means such as a keyboard and a mouse. have.

7 is a flowchart illustrating a method of distributing and culturing cells according to another embodiment of the present invention, and FIGS. 8 to 12 are views for explaining a method of distributing and culturing cells according to another embodiment of the present invention.

First, a disk 100 for dispensing and culturing cells is prepared (S110). The disk 100 for dispensing and culturing cells may be prepared as a disk 100 as described with reference to FIGS. 1 to 4.

Next, cells are injected into the injection port 121 of the disk 100 (S120). As shown in FIG. 8, a cell solution containing cells to be dispensed may be injected in an appropriate amount into each injection port 121 of the disk 100 using a pipette 20. The disk 100 is rotated while the disk 100 is fixed to the monitoring system 300 described with reference to FIGS. 5 and 6, and a cell solution can be injected into each injection port 121. Alternatively, the cell solution may be injected into the disk 100 through the injection port 121 and then fixed to the monitoring system 300.

Next, the cells injected by rotating the disk 100 may be distributed and transferred for each cell transfer channel 132 (S130). The disk 100 may be mounted on the fixing member 321 of the monitoring system 300 and rotated. As shown in FIG. 9, as the disk 100 rotates, a centrifugal force acts on the injected cell solution to move the cells CL outward in the radial direction of the disk 100. That is, the injected cell solution diffuses from the cell supply region 120 and moves the cells to the cell distribution region 130, and in the cell distribution region 130, the cells may be dispersed and moved for each cell transfer channel 132. The cell transfer channel 132 has a height and width in micrometers, and when a cell solution moves through it, a capillary force acts to promote the movement of cells.

Next, the transferred cells are captured in the capture groove 143 (S140). Since the capturing groove 143 formed in the cell capturing portion 141 of the disk 100 has a radius of curvature of approximately a size corresponding to the radius of a single cell, as shown in FIG. 10, the cell transfer channel 132 is formed. Cells (CL) moving along may be captured one by one in the capture groove (143). At this time, cells that are not captured in the capture groove 143 may flow together as the cell solution flows out to the cell recovery region 160, and the flowed out cells may be recovered through the outlet 161.

Next, the captured cells are moved to the cell collection unit 156 (S150). The cells captured in the capture groove 143 may be moved to the cell collection unit 156 by turning the disk 100 over and leaving it for a predetermined time. In this case, as shown in FIG. 11, the cells captured by gravity may be moved to the cell collection unit 156 in the process of moving downward without providing a separate driving force.

Next, the disk 100 is rotated to move the cells of the cell collection unit 156 to the cell culture unit 152 (S160). Since the cell collection unit 156 is located at the beginning of the cell culture unit 152, the disk 100 may be rotated so that the cells CL located therein enter the inside of the cell culture unit 152. That is, the plurality of capture grooves 143 each capture a single cell, and the cell collection units 156 connected to each capture groove 143 move the single cells CL to each cell culture unit. Can be stored in 152. Then, the cell separation can be completed by turning the disk 100 over again. Therefore, in a state in which cell separation is completed, one cell may be separated and stored in each cell culture unit 152.

Next, the separated cells are cultured in the cell culture unit 152 (S170). While maintaining the cell culture conditions in the incubating chamber 350 of the monitoring system 300 of the disk 100, as shown in FIG. 12, cells (CL) stored in the culture space 154 of the cell culture unit 152 ) Can be cultivated.

Next, cell growth is measured through real-time imaging (S180). For example, cell growth may be measured through real-time imaging using the monitoring system 300 shown in FIG. 5. In this case, the cell culture unit 152 of the disk 100 may be monitored through the fluorescence image pickup unit 340 while slowly rotating the disk 100.

Next, the amount of protein produced in each culture section is measured using a fluorescent reagent (S190). When a fluorescent reagent is introduced into the cell culture unit 152, it reacts with proteins generated by the cultured cells to emit fluorescence. The emitted fluorescence image may be observed by the fluorescence image capture unit 340 to measure the amount of generated protein.

Next, the cultured cells are extracted from the cell culture unit 152 (S200). Cells cultured in the cell culture unit 152 may be selected, and the selected cells may be extracted through the cell discharge unit 171 using a pipette. At this time, the cell discharge part 171 of the disk 100 may be closed by attaching a film to the surface of the disk 100 during the cell separation and culture process, and the cell discharge part 171 is removed after the cell separation and culture are completed. By removing the blocking film, you can open it and extract the cells.

According to the cell dispensing and culturing disk according to the embodiment described above, the real-time monitoring system, and the cell dispensing and culturing method, at least thousands of single cells can be simultaneously dispensed, and each of the separated single cells is transferred to the culture space. After positioning, it can be incubated and monitored in real time through imaging.

That is, through the design of the disk according to the embodiment, by culturing thousands of cells separated into single cells in each culture chamber and monitoring in real time, it is possible to obtain a single cell with the best growth and a single cell that produces the most target protein. have.

In addition, thousands of single cells are introduced into each culture chamber, and then a cell line (adherent cell) having the target reactant is introduced into a space outside the culture chamber and then cultured. As a specific protein secreted by each single cell diffuses out of the culture chamber, it has a reactant. It can be induced to bind to the cell line. After that, a fluorescent antibody that reacts with a specific protein is introduced to react with a specific protein, and then confirmed through real-time fluorescence imaging, the cells in which the specific protein and the target reactant have reacted can be identified. It can be seen that it is a target new drug protein.

Although the preferred embodiments of the present invention have been described above, the present invention is not limited thereto, and various modifications can be made within the scope of the claims, the detailed description of the invention, and the accompanying drawings, and this is also the present invention. It is natural to fall within the scope of the invention.

100: disk for dispensing cells, disk 110: central area
115: dummy region 120: cell supply region
121: injection port 123: diffusion
125: transient 130: cell distribution region
132: cell transfer channel 134: partition
141: cell capture unit 143: capture groove
152: cell culture unit 154: culture space
156: cell collection unit 160: cell recovery region
161: outlet 163: re-diffusion unit
171: cell discharge part
300: monitoring system 310: housing
315: stage 320: disk drive
321: fixing member 340: fluorescence imaging imaging unit
350: incubating chamber 352: observation window
354: sample introduction statement 360: control unit
362: connector

Claims (30)

As a disk having an inlet and an outlet,
A cell supply region connected to the injection port;
A cell distribution region including a cell transfer channel extending in a radial direction of the disk;
A cell trapping portion having a trapping groove close to an end of the cell transfer channel; And
Cell culture unit having a space connected to the capture groove
Cell dispensing and culture disk comprising a. The method of claim 1,
The space of the cell culture unit is located in different layers separated from the cell transfer channel, cell dispensing and culture disk. The method of claim 1,
The cell dispensing and culturing disk further comprises a cell recovery region in communication with the cell distribution region and connected to the outlet. The method of claim 1,
At a height measured in the thickness direction of the disk, a height of the cell distribution region is lower than a height of the cell supply region. The method of claim 1,
The cell transfer channel becomes narrower as it goes outward in the radial direction of the disk, the disk for cell dispensing and culture. The method of claim 1,
The capture groove is formed concave in a direction toward the center of the disk, a disk for cell dispensing and culture. The method of claim 1,
The capture groove has a radius of curvature corresponding to the radius of a single cell, cell dispensing and culture disk. The method of claim 1,
The cell culture unit is connected to the cell discharge unit that is opened to the outside of the disk, cell dispensing and culture disk. A disk having an inlet and an outlet connected to the inner space on its surface,
A cell supply region located in the inner space and connected to the injection port;
A cell distribution region connected to the cell supply region in the inner space and including a plurality of cell transfer channels each extending in a radial direction of the disk;
A cell trapping portion having a trapping groove close to an end of the cell transfer channel;
A cell culture unit having a culture space connected to the capture groove; And
Cell recovery area in communication with the cell transfer channel and connected to the outlet
Cell dispensing and culture disk comprising a. The method of claim 9,
The inlet and outlet are spaced apart from each other in the radial direction of the disk,
The inlet is closer to the center of the disc, and the outlet is closer to the edge of the disc. The method of claim 9,
The cell supply region,
A diffusion unit connected to the injection port; And
Transient portion extending from the diffusion portion and having a portion lower than the height measured in the thickness direction of the disk of the diffusion portion and connected to the cell transfer channel
Containing, cell dispensing and culture disk. The method of claim 9,
The cell distribution area includes a plurality of partitions extending in a radial direction of the disk, and the partitions form a boundary between the cell transfer channel. The method of claim 9,
Each of the cell transfer channels is narrower in width toward the outer side in the radial direction of the disk, a disk for cell dispensing and culture. The method of claim 9,
The capture groove is formed concave in a direction toward the center of the disk, a disk for cell dispensing and culture. The method of claim 9,
The capture groove has a radius of curvature corresponding to the radius of a single cell, cell dispensing and culture disk. The method of claim 9,
The cell capture portion has a pointed attachment in a direction toward the center of the disk, and the capture grooves are located at one end of the attachment, for cell dispensing and culture. The method of claim 16,
Including a plurality of partitions forming the boundary of the cell transfer channel,
Each of the partitions has an attachment to a distal end facing radially outward of the disk,
The attachment of the cell capture portion and the attachment of the partition portion are arranged alternately with each other, and a disk for cell dispensing and culture. The method of claim 9,
The cell capture unit is located downstream of the cell transfer channel and is provided by a number corresponding to each of the plurality of cell transfer channels. The method of claim 9,
The culture space of the cell culture unit is located in a second layer separated from the first layer in which the cell transfer channel is located, and a disk for cell dispensing and culture. The method of claim 9,
The cell culture unit further includes a cell collecting unit in communication with the capturing groove, and the cell collecting unit is disposed to overlap the capturing groove when viewed from a plane perpendicular to the rotation axis of the disk. The method of claim 20,
The cell culture part is connected to the cell discharge part opened to the outside of the disk,
The cell discharging portion is connected to a portion spaced apart from the cell collecting portion, cell dispensing and culture disk. The method of claim 21,
The cell discharging unit comprises a first discharge tube in communication with the culture space of the cell culture unit, and a second discharge tube having a larger diameter than the first discharge tube and opening to the outside of the disk, a disk for cell dispensing and culture . The method of claim 9,
The cell recovery region further comprises a re-diffusion unit positioned between the cell distribution region and the outlet. In a real-time monitoring system for dispensing and culturing cells by carrying in a disk for cell dispensing and culturing and driving it,
A disk drive unit capable of rotating by mounting the disk for dispensing and culturing the cells; And
Fluorescence image pickup unit located at the bottom of the cell dispensing and culture disk so as to correspond to the cell culture unit to irradiate light and measure the reflected fluorescence
Monitoring system comprising a. The method of claim 24,
The monitoring system further comprises an incubating chamber separated from the fluorescence image pickup unit and outside air and provided to surround the cell dispensing and culture disk. Preparing a cell dispensing and culturing disk having an inlet and an outlet connected to the inner space on the surface;
Injecting cells into the injection port;
Rotating the disk for dispensing and culturing the cells, dispersing and transferring the injected cells for each of a plurality of cell transfer channels;
Rotating the cell dispensing and culturing disk and capturing single cells from the transferred cells into a capture groove of a cell capture unit located downstream of the cell transfer channel; And
Moving the captured single cells to a cell culture unit
Cell dispensing and culture method comprising a. The method of claim 26,
The step of moving the captured single cells to the cell culture unit,
The process of inverting the cell dispensing and culture disk to move the captured single cells to the cell collection unit by gravity, and
Cell dispensing and culturing method further comprising the step of moving the single cells of the cell collection unit to the cell culture unit by rotating the cell dispensing and culturing disk. The method of claim 26,
Culturing the single cell in the cell culture unit; And
Extracting the cultured cells from the cell culture unit
Cell dispensing and culture method comprising a further. The method of claim 28,
Measuring the growth of cells cultured in the cell culture unit through real-time imaging; And
Measuring the amount of protein produced in the cell culture part using a fluorescent reagent
Cell dispensing and culture method comprising a further. The method of claim 26,
Recovering the transferred cells except for the captured single cells from the outlet of the cell dispensing and culture disk
Cell dispensing and culture method comprising a further.

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