Automated system and method for anticancer drug sensitivity testing based on organoid cell culture

Abstract

An automated system for anticancer drug sensitivity testing, according to an embodiment of the present invention, may comprise: a chopper configured to mince patient tissue; a cell dissociation device configured to apply a cell dissociation enzyme to the patient tissue minced by the chopper; a centrifuge configured to centrifuge cells to which the cell dissociation enzyme has been applied by the cell dissociation device; a liquid handler configured to mix the centrifuged cells with extracellular matrix to thereby generate a mixed sample and to inject the mixed sample into a nozzle tip; a spotter configured to dispense the mixed sample onto a pillar by using the nozzle tip; an automatic incubator configured to culture the mixed sample to generate cell organoids; a scanner configured to generate an image by scanning the cell organoids at high speed; and a multi-joint robot configured to move at least one of the patient tissue, the cells, and the mixed sample between at least some of the chopper, the cell dissociation device, the centrifuge, the liquid handler, the spotter, the automatic incubator, and the scanner. Thus, the entire process of cell extraction from tissue isolated from a patient to organoid culture, drug testing, and scanning can be performed fully automatically.

Description

Automated system and method for anticancer drug sensitivity testing based on organoid cell culture

[0001] The present invention relates to an automated anticancer drug sensitivity test system and method, and more particularly, to an anticancer drug sensitivity test system and method in which the entire test process, including crushing, dissociation, centrifugation, organoid culture, and scanning, from tissue isolated from a patient is automatically performed within the system.

[0002] Organoid cell culture is an innovative technology that mimics the functions of actual human cells through three-dimensional cultivation of stem cells or patient-derived cells. It holds significant potential in diverse fields, including disease modeling, new drug development, regenerative medicine, and precision medicine. In particular, patient-specific disease models enable a deeper understanding of disease mechanisms, provide greater accuracy in evaluating the efficacy and safety of new drugs, and enable personalized treatment strategies in precision medicine.

[0003] However, organoid culture technology still has areas in need of improvement. Previous technology lacked automation for the entire process, from tissue preparation to organoid culture. Patient-derived tissue had to be crushed, then centrifuged and cultured separately in separate equipment. This crushing or transport process, coupled with the stress or fluid applied to cells, could adversely affect organoid production. Consequently, despite the limited amount of tissue available from patients, the number of usable organoids was limited, making it difficult to ensure accuracy and reliability in diagnostic applications such as drug screening.

[0004] Therefore, there is a growing need for an automated anticancer drug sensitivity testing system that can overcome the limitations of conventional organoid culture technology and increase the accuracy of drug response prediction by minimizing adverse effects associated with organoid production and improving accuracy and reliability in performing diagnosis through fully automating the process from tissue isolated from a patient to crushing, organoid culture, and diagnosis.

[0005] Therefore, the automated anticancer drug sensitivity test system and method according to one embodiment of the present invention aims to realize all stages of cell culture in a single system, and to perform the movement of samples in each stage by a multi-joint robot.

[0006] In addition, the present invention aims to form organoids homogeneously by dispensing and culturing a mixed sample of cells and extracellular matrix on a pillar, thereby increasing the accuracy and reliability of diagnosis during drug screening.

[0007] In addition, another object of the present invention is to improve the formation rate of organoids and the survival rate of cells during cell culture, and to increase the possibility of contact between organoids and drugs during drug screening, thereby significantly improving the accuracy of diagnosis.

[0008] In order to solve the above-described problem, an automated anticancer drug sensitivity test system according to an embodiment of the present invention comprises: a chopper configured to pulverize patient tissue, a cell dissociation device configured to apply a cell dissociation enzyme to patient tissue pulverized by the chopper, a centrifuge configured to centrifuge cells to which the cell dissociation enzyme has been applied by the cell dissociation device, a liquid handler configured to mix centrifuged cells and an extracellular matrix to create a mixed sample and inject the mixed sample into a nozzle tip, a spotter configured to dispense the mixed sample onto a pillar or an anticancer agent into a well plate using the nozzle tip, an automated incubator configured to culture the mixed sample to create cell organoids, a scanner configured to scan the cell organoids at high speed to create images, and a multi-joint device configured to move at least one of the patient tissue, the cell, the mixed sample, and the anticancer agent between at least some of the chopper, the cell dissociation device, the centrifuge, the liquid handler, the spotter, the automated incubator, and the scanner. May include robots.

[0009] Specific details of other embodiments are included in the detailed description and drawings.

[0010] According to the present invention as described above, the following effects are obtained.

[0011] The present invention has the effect that all steps of extracting cells from tissue isolated from a patient, culturing them, and testing anticancer drug sensitivity can be performed on a single system without manual intervention by the user.

[0012] In addition, the present invention has the effect of homogeneously forming organoids by dispensing and culturing a mixed sample of cells and extracellular matrix on a pillar, thereby increasing the accuracy and reliability of diagnosis during drug screening.

[0013] In addition, the present invention can significantly improve the accuracy of diagnosis by improving the formation rate of organoids and the survival rate of cells during cell culture and increasing the possibility of contact between organoids and drugs during drug screening.

[0014] In addition, the present invention can increase the concentration (distribution) of cells on the surface of the mixed sample by preventing cells from moving toward the end of the filter after the mixed sample of cells and extracellular matrix is ​​dispensed onto the filter, thereby improving the possibility of contact between the organoid and the culture medium material or the drug to be diagnosed.

[0015] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0016] FIG. 1 is a drawing for explaining an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0017] FIG. 2 is a flowchart illustrating an automated method for testing anticancer drug sensitivity according to one embodiment of the present invention.

[0018] FIG. 3 is a drawing for explaining a chopper of an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0019] FIG. 4 is a drawing for explaining a cutting component of a chopper of an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0020] FIG. 5 is a drawing for explaining an automatic incubator of an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0021] FIG. 6 is a drawing for explaining an automated method for testing anticancer drug sensitivity according to one embodiment of the present invention.

[0022] FIG. 7 is a drawing for explaining the effect of an automated method for testing anticancer drug sensitivity according to one embodiment of the present invention.

[0023] FIG. 8 is a drawing for explaining the effect of an automated anticancer drug sensitivity test method according to one embodiment of the present invention.

[0024] FIG. 9 is a drawing for explaining a head cooling module used in a spotter of an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0025] FIG. 10 is a drawing for explaining a workbench cooling unit used in a spotter of an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0026] FIG. 11 is a diagram illustrating an automated anticancer drug sensitivity test, in particular, an automated organoid culture preprocessing system, according to another embodiment of the present invention.

[0027] Hereinafter, various embodiments of the present invention will be described in detail with reference to the accompanying drawings. The advantages and features of the present invention, and methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0028] The shapes, sizes, ratios, angles, numbers, etc. disclosed in the drawings for explaining embodiments of the present invention are illustrative and are not limited to the matters illustrated in the drawings. Like reference numerals refer to like components throughout the specification. In addition, in describing the present invention, if a detailed description of a related known technology is judged to unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. When the terms “includes,” “has,” and “consists of” are used in this specification, other parts may be added unless “only” is used. When a component is expressed in the singular, it includes a case where the plural is included unless there is a specifically explicit description.

[0029] When interpreting a component, it is interpreted as including the error range even if there is no separate explicit description.

[0030] When describing a positional relationship, for example, when the positional relationship between two parts is described as 'on top of', 'upper part of', 'lower part of', 'next to', etc., one or more other parts may be located between the two parts, unless 'right away' or 'directly' is used.

[0031] Although terms like "first" and "second" are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, a "first" component referred to below may also be a "second" component within the technical scope of the present invention.

[0032] Identical reference numerals throughout the specification refer to identical components.

[0033] The size and thickness of each component shown in the drawing are shown for convenience of explanation, and the present invention is not necessarily limited to the size and thickness of the component shown.

[0034] It should be understood that all conditional terms and embodiments listed in this specification are, in principle, expressly intended only for the purpose of making the concept of the invention understood, and are not limited to the specifically listed embodiments and conditions.

[0035] Additionally, in the following description, ordinal expressions such as first, second, etc. are intended to describe objects that are equal and independent of each other, and should be understood as having no meaning in terms of main / sub or master / slave.

[0036] The above-described purposes, features and advantages will become clearer through the following detailed description with reference to the attached drawings, so that a person having ordinary skill in the art to which the invention pertains can easily practice the technical idea of ​​the invention.

[0037] The individual features of the various embodiments of the present invention can be partially or wholly combined or combined with each other, and as can be fully understood by those skilled in the art, various technical connections and operations are possible, and each embodiment can be implemented independently of each other or can be implemented together in a related relationship.

[0038] An automated anticancer drug sensitivity test system according to various embodiments of the present invention may include a chopper configured to pulverize patient tissue, a cell dissociation device configured to apply a cell dissociation enzyme to the patient tissue pulverized by the chopper, a centrifuge configured to centrifuge cells to which the cell dissociation enzyme has been applied by the cell dissociation device, a liquid handler configured to mix the centrifuged cells and an extracellular matrix to create a mixed sample and inject the mixed sample into a nozzle tip, a spotter configured to dispense the mixed sample onto a pillar using the nozzle tip, an automated incubator configured to culture the mixed sample to create cell organoids, a scanner configured to scan the cell organoids at high speed to create images, and a multi-joint robot configured to move at least one of the patient tissue, the cells, and the mixed sample between at least some of the chopper, the cell dissociation device, the centrifuge, the liquid handler, the spotter, the automated incubator, and the scanner.

[0039] According to another feature of the present invention, the chopper may include a cutting component, the cutting component may include a rotating blade configured to pulverize patient tissue, and a disposable tube surrounding the rotating blade and configured to receive the patient tissue.

[0040] According to another feature of the present invention, the automatic incubator includes a main door and a plate door smaller than the main door, and the plate transfer drive unit can move a pillar into which a mixed sample is dispensed into the automatic incubator through the plate door excluding the main door.

[0041] According to another feature of the present invention, the automatic incubator further includes a cooler for cooling the mixed sample located at the bottom of the pillar while the pillar plate is turned over so that the mixed sample is located at the bottom of the pillar, and the automatic incubator can be configured to gel the mixed sample located at the bottom of the pillar and culture the gelled mixed sample.

[0042] According to another feature of the present invention, cells of a mixed sample cooled inside a cooler with the pillar plate turned over can move in a direction away from the end of the pillar within the mixed sample.

[0043] An automated anticancer drug sensitivity test method according to various embodiments of the present invention comprises the steps of (a) crushing patient tissue by a chopper, (b) applying a cell dissociation enzyme to the patient tissue by a cell dissociation device, (c) filtering cells to which the cell dissociation enzyme has been applied by a liquid handler, (d) centrifuging the filtered cells by a centrifuge, (e) mixing the centrifuged cells and an extracellular matrix by the liquid handler to create a mixed sample and injecting the mixed sample into a nozzle tip, (f) dispensing the mixed sample in the nozzle tip onto a pillar by a spotter, (g) culturing the mixed sample by an automatic incubator to create cell organoids, (h) injecting an anticancer agent into the nozzle tip by the liquid handler, (i) dispensing the anticancer agent in the nozzle tip onto a well plate by the spotter, (j) combining the pillar plate and the well plate by a multi-joint robot, and (k) culturing the combined plate by an automatic incubator. And (l) a step of scanning the cell organoid at high speed by a scanner to generate an image, wherein in at least some of steps (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k) and (l), at least one of the patient tissue, cell, and mixed sample can be moved by a multi-joint robot.

[0044] Hereinafter, embodiments of the present invention will be described in more detail with reference to the drawings.

[0045] FIG. 1 is a diagram illustrating an automated anticancer drug susceptibility testing system according to an embodiment of the present invention. FIG. 2 is a flowchart illustrating an automated anticancer drug susceptibility testing method according to an embodiment of the present invention. FIG. 3 is a diagram illustrating a chopper of an automated anticancer drug susceptibility testing system according to an embodiment of the present invention. FIG. 4 is a diagram illustrating a cutting component of a chopper of an automated anticancer drug susceptibility testing system according to an embodiment of the present invention. FIG. 5 is a diagram illustrating an automatic incubator of an automated anticancer drug susceptibility testing system according to an embodiment of the present invention. FIG. 6 is a diagram illustrating an automated anticancer drug susceptibility testing method according to an embodiment of the present invention. FIG. 7 is a diagram illustrating an effect of an automated anticancer drug susceptibility testing method according to an embodiment of the present invention. FIG. 8 is a diagram illustrating an effect of an automated anticancer drug susceptibility testing method according to an embodiment of the present invention. FIG. 9 is a diagram illustrating a head cooling module used in a spotter of an automated anticancer drug susceptibility testing system according to an embodiment of the present invention. FIG. 10 is a drawing for explaining a workbench cooling unit used in a spotter of an automated anticancer drug sensitivity test system according to one embodiment of the present invention.

[0046] Referring to FIGS. 1 to 10, an automated anticancer drug susceptibility test system (1000) and method according to an embodiment of the present invention are a system and method that can automatically perform an anticancer drug susceptibility test without separate user intervention, in which tissue separated from a patient is introduced into the system, and a crushing process, a dissociation process, a centrifugation process, and a culturing process are all performed within the automated anticancer drug susceptibility test system (1000), and a high-speed scan image of the cultured cell organoid is generated so that the image analysis result can be provided to the user. The automated anticancer drug susceptibility test system (1000) of the present invention can screen more than 32,000 drugs per day, and can be a cell organoid-based High Throughput / Content Screening (HTS / HCS) integrated research equipment.

[0047] In the case of existing anticancer drug susceptibility testing devices, the user may have to cut the patient tissue into small pieces and insert them into the device, or the user may have to crush the patient tissue and then insert it into the device. However, the automated anticancer drug susceptibility testing system (1000) of the present invention has a technical feature in that it can process both cell organoid culture and high-speed scan image analysis without separate manual work by the user by introducing tissue separated from the patient into the automated anticancer drug susceptibility testing system (1000) without crushing the patient tissue.

[0048] More specifically, the automated anticancer drug susceptibility test system (1000) and method of the present invention may include a chopper (100), a cell dissociation device (shaker) (200), a centrifuge (300), a liquid handler (500), a spotter (600), an automated incubator (700), a scanner (800), and an image analyzer (900).

[0049] The automated anticancer drug susceptibility test method implemented by the automated anticancer drug susceptibility test system (1000) first grinds patient tissue using a chopper (100) (S110). The chopper (100) of the automated anticancer drug susceptibility test system (1000) may be configured to grind patient-derived tissue. After tissue, for example, cancer tissue, is extracted through surgery or biopsy, it is placed in a transfer medium and transported to a laboratory while being refrigerated. At this time, the user may directly cut the patient tissue into small pieces or, rather than cutting the patient tissue using a separate milling device and applying pressure to crush it and injecting the resulting product into the system, may introduce tissue of a size that can be introduced into the cutting component (110) of the chopper (100) into the chopper (100), and the automated anticancer drug susceptibility test system (1000) may automatically perform a grinding step of cutting the patient tissue. The obtained tissue may be cut (pulverized) into pieces of 0.5 mm to 1 mm.

[0050] Referring to FIGS. 3 and 4, the chopper (100) may include a cutting component (110) mounted on a joint portion (121) of the main body of the chopper (100). The cutting component (110) may be configured to allow patient tissue to be introduced therein and crush the patient tissue into small pieces (e.g., 1 mm in size). The cutting component (110) may include a tube (111), and the patient tissue may be introduced into the tube (111) and coupled with the remaining portion of the cutting component (110), thereby containing the patient tissue within the cutting component (110). The tube (111) may be used for a disposable purpose, but is not limited thereto. A rotating blade (112) may be arranged at the center of the cutting component (110). The rotary blade (112) may include a rotation axis in the extended direction of the cutting component (110) and may include a blade extending perpendicularly to the rotation axis around the rotation axis. The cutting component (110), which is driven in a manner similar to a blender, may cause patient tissue existing in a tube (111) to be pulverized by the blade by the rotation of the rotary blade (112). A sealing portion (113) and a moisture absorbing portion (114) may be arranged at the end of the rotary blade (112) of the cutting component (110), and the sealing portion (113) may prevent the patient tissue in the tube (111) from leaking out. In addition, the moisture absorbing portion (114) may absorb moisture inside the cutting component (110) to prevent the solution inside from leaking out. A cap (115) can be placed on the other end opposite to the end where the tube (111) of the cutting part (110) is placed, and the cap (115) can be combined with the connecting portion (121) so that the cutting part (110) can be connected to the main body of the chopper (100).

[0051] A display unit (122) may be arranged on the surface of the main body of the chopper (100), and the user may obtain information on the crushing stage of the patient tissue through the display unit. Referring to FIG. 3(b) which allows the interior of the main body of the chopper (100) to be confirmed, a solenoid gripper (123) connected to each of the connecting portions (121) on which the cutting component (110) is mounted may be arranged inside the main body of the chopper (100), and a motor (124) that rotates the rotary blade (112) of the cutting component (110) may be arranged. In addition, the chopper (100) may include an SMPS (125) and an electrical board (126). The arrangement of the shape and configuration of the chopper (100) is not limited to FIGS. 3 and 4.

[0052] In the past, extracted tissues were pulverized into very small sizes by a pulverizer, for example, by being cut into small pieces and then compressed and crushed by applying pressure. However, in the case of the automated anticancer drug sensitivity test system (1000) of the present invention, patient tissues can be pulverized into small pieces by a chopper (100). Therefore, according to the conventional technology, when extracting cells from tissue, the pressure and stress applied lowered the cell viability during culture, making it difficult to form homogeneous organoids. However, according to an embodiment of the present invention, by improving the cell separation process, homogeneous formation of organoids is possible, and the cell viability during culture and the organoid formation rate can be significantly improved.

[0053] Next, a cell dissociation enzyme is applied to the patient tissue by a cell dissociation device (shaker) (200) (S120). The small-sized patient tissue resulting from the patient tissue crushing step (S110) can be moved from the chopper (100) to the cell dissociation device (200) by a multi-joint robot (50).

[0054] Patient tissues moved to a cell dissociation device (200) by a multi-joint robot (50) can be subjected to cell dissociation enzymes within the cell dissociation device (200), and the patient tissues can be dissociated into single cells. For example, the pulverized patient tissues can be dissociated into single cells while shaking at a constant temperature of 37°C for 1 hour with 10 ml of Dissociation Buffer.

[0055] Afterwards, the cells to which the cell dissociation enzyme has been applied in the liquid handler (500) can be filtered with a 100-μm stainer and placed in a 50-mL tube containing 8 mL of STI (S130). The 8 mL of filtered cell suspension can be washed.

[0056] Next, the filtered cells are centrifuged by a centrifuge (300) (S140). The cells filtered in step (S130) by a 100-μm stainer can be transferred to the centrifuge (300) by a multi-joint robot (50). In the centrifuge (300), the cells can be centrifuged to extract cancer cells. For example, the cells can be centrifuged at 188 Х g for 5 minutes at 4°C.

[0057] Next, the centrifuged cells are mixed with the extracellular matrix by the liquid handler (500) to produce a mixed sample (A) (S150). The centrifuged cells can be moved from the centrifuge (300) to the liquid handler (500) by the multi-joint robot (50). The liquid handler (500) can mix the cells and the extracellular matrix with each other to produce a mixed sample (A) which is an output material of the spotter (600). The centrifuged cells can be, for example, 2 Х 10 4 Cells are dispensed onto Matrigel and mixed by a liquid handler (500).

[0058] Meanwhile, while mixing of cells and extracellular matrix by the liquid handler (500) is in progress, the mixed sample (A) is maintained at approximately 4°C in a cooling device and can be sufficiently resuspended 10 to 20 times. At this time, the concentration of the extracellular matrix can be maintained at 50% to 80%. This is because if the concentration of the extracellular matrix is ​​lower than 50%, the progress of the gelation step performed later may be slowed, and if the concentration of the extracellular matrix is ​​higher than 80%, the possibility of a problem of reduced drug permeability may increase.

[0059] In addition, the concentration of cells introduced into the liquid handler (500) may be a final concentration of 3,000 cells / μL to 7,000 cells / μL based on 10 days of culture. This is because, if the concentration of cells is lower than 3,000 cells / μL, the possibility of a problem in which the production of cell organoids is reduced increases, and if the concentration of cells is higher than 7,000 cells / μL, the possibility of a mixed sample (A) of cells and extracellular matrix falling off from the pillar (11) increases.

[0060] Next, the mixed sample (A) is injected into the nozzle tip of the spotter (600) by a liquid handler (500) (S150). The nozzle tip may be made of an elastic material and may be a disposable nozzle tip that is discarded after the mounted mixed sample (A) is dispensed. One end of the nozzle tip may be open as an inlet into which the mixed sample (A) can be mounted, and the other end may be provided with a discharge portion through which the mixed sample (A) can be discharged to the outside by air pressure. At this time, there is no bubble at the end of the disposable nozzle tip, and the mixed sample (A) can be injected into the disposable nozzle tip so that the height of the surface of the mixed sample (A) does not tilt. The amount of the mixed sample (A) injected into the disposable nozzle tip may be injected with an additional amount of 3 μL to 10 μL more than the appropriate amount. In addition, after the mixed sample (A) is injected into the disposable nozzle tip, the next step (S160) can be quickly dispensed without a separate waiting time.

[0061] Next, the mixed sample (A) inside the nozzle tip is dispensed onto the upper part of the pillar (11) by the spotter (600) (S160). The multi-joint robot (50) can move the nozzle tip on which the mixed sample (A) is mounted to the spotter (600). The spotter (600) can include a plurality of nozzles, and one end of the nozzle tip can be fitted to each nozzle. The spotter (600) can apply air pressure to the nozzle tip to discharge the mixed sample (A) inside the nozzle tip to the outside through the discharge portion at the other end of the nozzle tip. The mixed sample (A) discharged from the nozzle tip by the spotter (600) can be dispensed onto the upper part of the pillar (11) of the pillar plate (10).

[0062] Referring to FIG. 9, a spotter (600) according to an embodiment of the present invention may include a head cooling module (601) capable of maintaining a constant temperature of a mounted mixed sample (A) contained in a disposable nozzle tip while the mixed sample (A) is dispensed onto a pillar (11). The spotter (600) may include a head portion including a nozzle through which air is discharged, and a disposable nozzle tip may be detachably connected to the nozzle. The head cooling module (601) may be mounted in a manner connected to the head portion and the nozzle tip at the bottom of the nozzle tip. The spotter (600) may include a nozzle tip contact portion (603) and a head cooling portion (602) arranged to be adjacent to one surface of the head cooling module (601). The nozzle tip contact portion (603) is configured to be in contact with a disposable nozzle tip on which a mixed sample (A) is mounted, and the head cooling portion (602) includes a fan and can maintain a cooling temperature of 4°C of the mixed sample (A) mounted on the disposable nozzle tip in contact with the nozzle tip contact portion (603) until it is dispensed.

[0063] Referring to FIG. 10, a spotter (600) according to one embodiment of the present invention may include a workbench cooling unit (60). The workbench cooling unit (60) is configured to maintain a cooling state of a mixed sample (A) dispensed onto a pillar (11) in a dispensing step (S160), and is located below the pillar (11) to continuously provide a low-temperature environment to the mixed sample (A) dispensed onto the pillar (11). The workbench cooling unit (60) may include a cooling water channel (61), and water at 4°C may flow along the cooling water channel (61).

[0064] More specifically, the worktable cooling unit (60) may include a plurality of cooling channels (61), and some of the plurality of cooling channels (61) (for example, three cooling channels (61)) may be configured to cool the mixed sample (A) dispensed on the pillar plate (10) by having the pillar plate (10) seated thereon. In addition, some of the other cooling channels (61) may be configured to cool the well plate (20) by having the well plate (20) seated thereon. In addition, the remainder of the plurality of cooling channels (61) may be configured to cool the mixed sample (A) within the disposable nozzle tip by having the disposable nozzle tip seated thereon.

[0065] In this way, the head cooling module (601) and the worktable cooling unit (60) can continuously maintain the cooling state of the mixed sample (A) from before the dispensing step (S160) until the dispensing step (S160) is performed and until the gelation step of the mixed sample (A) thereafter.

[0066] According to one embodiment of the present invention, the pillar plate (10) can then be flipped over so that the mixed sample (A) is positioned at the bottom of the pillar (11). Referring to FIG. 6(b) and FIG. 7(a), in a state where the mixed sample (A) is dispensed and placed at the end of the pillar (11), the pillar plate (10) can be flipped over so that the mixed sample (A) is positioned lower than the end of the pillar (11), that is, so that the mixed sample (A) faces downward. For example, the multi-joint robot (50) can flip the pillar plate (10) as in the state of FIG. 6(a) as in the state of FIG. 6(b).

[0067] In this way, when the pillar plate (10) is turned over as in the state of FIG. 6(b) and FIG. 7(a), the cells can move in a direction away from the end of the pillar (11) within the mixed sample (A). Specifically, when the mixed sample (A) is maintained in a state where it is dispensed on the end of the pillar (11) as in FIG. 6(a) and FIG. 7(b), the cells included in the mixed sample (A) can move adjacent to the end of the pillar (11) due to gravity as in FIG. 7(b), and thus, the portion of the mixed sample (A) adjacent to the end of the pillar (11) can have a higher cell concentration than the remaining portion. In contrast, the automated anticancer drug sensitivity test method according to one embodiment of the present invention can flip the pillar plate (10) as shown in FIG. 6(b) and FIG. 7(a), and thus, the cells included in the mixed sample (A) can move toward the direction away from the end of the pillar (11), i.e., toward the ground, by gravity as shown in FIG. 7(a). Accordingly, a portion of the mixed sample (A) adjacent to the pillar (11) may have a lower cell concentration than the remaining portion, i.e., the cell concentration may be higher in the remaining portion of the mixed sample (A) that is away from the pillar (11).

[0068] Next, the mixed sample (A) located at the bottom of the pillar (11) can be cooled. The anticancer drug susceptibility test automation system (1000) of the present invention may further include a cooler (950), and as shown in FIG. 6(b), the pillar plate (10) with the pillar positioned downward may be introduced into the cooler (950) in a state combined with a well plate (20) that does not contain a culture medium (21), and the mixed sample (A) may be cooled to a pre-designated temperature for a pre-designated time. For example, the mixed sample (A) may be cooled at a temperature of 6 degrees or less, preferably 4°C, for about 15 to 25 minutes, most preferably 20 minutes, in the cooler (950). If the cooling step of the mixed sample (A) is performed for a time longer than 25 minutes, the degree of damage to cells in the mixed sample (A) may increase, and therefore, the cooling of the mixed sample (A) may be performed for only 25 minutes or less. In addition, the process of introducing the pillar plate (10) into the cooler (950) may be performed by a multi-joint robot (50) of an anticancer drug sensitivity test automation system (1000). In addition, the present applicants have discovered a phenomenon in which the production of organoids in the mixed sample (A) is significantly reduced when the cooling step of the mixed sample (A) is performed for a time shorter than 15 minutes.

[0069] Next, the mixed sample (A) located at the bottom of the pillar (11) can be gelled inside the incubator. Specifically, the low-temperature well plate (20) that was combined with the pillar plate (10) in the cooling step and introduced into the cooler (950) can be replaced with a well plate (20) of, for example, 35°C or higher, preferably 36°C, and combined with the pillar plate (10). This may be to prevent a decrease in the effect of the gelling step due to condensation.

[0070] The pillar plate (10) combined with the high temperature well plate (20) in an inverted state can be introduced into an automatic incubator (700), and the cooled mixed sample (A) hanging from the lower end of the pillar (11) of the pillar plate (10) can undergo gelation for about 1 hour. The temperature of the incubator during the gelation stage can be 36°C, and the humidity can be 90%, and these temperature and humidity can be maintained constantly.

[0071] Next, the mixed sample (A) is cultured by an automated incubator (700) to generate cell organoids (S170). The mixed sample (A) that has completed the gelation step can be combined with a well plate (20) containing a culture medium (21) by a multi-joint robot (50) and moved to the automated incubator (700). The automated incubator (700) can be configured to culture cells and generate cell organoids by an automated anticancer drug sensitivity test system (1000) without user intervention. The mixed sample (A) can be placed on a well plate (20) containing a culture medium (21) by the multi-joint robot (50) as shown in FIG. 6(c), and then, as shown in FIG. 6(d), the mixed sample (A) can be immersed in the culture medium (21) by combining the pillar plate (10) and the well plate (20). The mixed sample (A) immersed in the culture solution (21) can be introduced into an automatic incubator (700) by being combined in an incubation chamber as shown in Fig. 6(d), and culture can be performed with sterilized distillation filled at the edge of the culture chamber. The culture step (S170) may not be performed with the pillar plate (10) turned over as shown in Fig. 6(d), and for example, after the gelation step is performed, the pillar plate (10) may be turned over again so that the mixed sample (A) is positioned on the pillar (11) as shown in Fig. 6(a), and then immersed in the culture solution (21) and cultured.

[0072] Referring to FIG. 5, the automatic incubator (700) may include a main door (711) and an inner door (712), and may further include a plate door (761) smaller in size than the main door (711). When a pillar plate (10) is loaded and unloaded through the main door (711) and the inner door (712), a constant temperature required for cell culture may not be maintained due to temperature changes inside the automatic incubator (700), and it may be difficult to achieve stable culture conditions due to vibration and shock caused by opening and closing the main door (711) and the inner door (712). Accordingly, the automatic incubator (700) of the automated anticancer drug sensitivity test system (1000) of the present invention may include a plate door (761), and a pillar plate (10) on which a mixed sample (A) is dispensed may be brought into and out of the automatic incubator (700) through the plate door (761) by a plate transfer drive unit (732) inside the automatic incubator (700). In this case, the temperature inside the automatic incubator (700) can be maintained within the culture conditions, and shock and vibration can also be effectively prevented, so that organoid generation of other cells being cultured can be effectively performed.

[0073] An automatic incubator (700) may include a display unit (721, 722) for checking the cell culture environment on the outside of the main body, a plate storage unit (731) for storing a pillar plate (10), a plate transfer drive unit (732) for moving the pillar plate (10) inside the automatic incubator (700), a water tray (740) for containing water, and an ultraviolet lamp (750).

[0074] Next, referring to FIG. 6(e), after the culture step (S170) of the mixed sample (A) is completed, the cultured mixed sample (A) located at the end of the pillar (11) of the pillar plate (10) can be exposed to radiation (771) generated from the radiation irradiation unit (770), and the cell organoids included in the mixed sample (A) can react by the radiation (771).

[0075] Next, a detailed description will be given of the process of treating the cultured organoid with an anticancer agent. The anticancer agent can be injected into the nozzle tip by a liquid handler (500) (S180). Then, the anticancer agent in the nozzle tip is dispensed into the well plate (20) by a spotter (600) (S190). Next, the pillar plate (10) and the well plate (20) are joined together by a multi-joint robot (50) (S200). Then, the joined plates (10, 20) can be cultured by an automatic incubator (700) (S210).

[0076] Next, the cell organoid is scanned at high speed by a scanner (800) to generate an image (S220). The cell organoid, which has been cultured in an automatic incubator (700), can be moved from the plate door (761) of the automatic incubator (700) to the scanner (800) by a multi-joint robot (50). The scanner (800) can check changes in cells through methods such as image scanning, and can generate an image by scanning the cell organoid at high speed.

[0077] Next, the image generated by the scanner (800) can be transmitted to an image analyzer (900), and the image analyzer (900) can calculate indices such as IC50 and AUC and provide them to the user.

[0078] According to one embodiment of the present invention, the automated anticancer drug sensitivity test system (1000) can flip the pillar plate (10) so that the mixed sample (A) is positioned lower than the pillar (11) after the mixed sample (A) is dispensed on the upper part of the pillar (11) of the pillar plate (10). In this way, the cooling and gelation steps can be performed with the pillar plate (10) flipped over, and in this case, the cells in the mixed sample (A) can move by gravity in the direction away from the end of the pillar (11), that is, toward the ground. Accordingly, the concentration of cells in the part of the mixed sample (A) that is away from the pillar (11), rather than the surface of the end of the pillar (11), can be greater than the concentration of cells in the remaining part. In this case, compared to the case where the pillar plate (10) is flipped over and the cooling step is not performed, the formation of cell organoids after the culturing step can be increased by 50% or more.

[0079] Specifically, Fig. 8(a) shows a case where cooling is performed by flipping the pillar plate (10) over so that the mixed sample (A) is positioned at the bottom of the pillar (11) as in the automated anticancer drug sensitivity test method of the present invention, and in contrast, Fig. 8(b) shows a case where the step of cooling by flipping the pillar plate (10) over is not performed. Referring to Fig. 8(a), the formation area of ​​the cell organoid in the rightmost drawing after 7 days of culture may be 378.14, and referring to Fig. 8(b), the formation area of ​​the cell organoid in the rightmost drawing after 7 days of culture may be 251.09.

[0080] Accordingly, the automated anticancer drug sensitivity test system (1000) and method according to one embodiment of the present invention performs a cooling step by flipping the pillar plate (10) so that the mixed sample (A) is positioned at the bottom of the pillar (11) after dispensing the mixed sample (A), thereby allowing cells in the mixed sample (A) to be densely packed on the surface of the mixed sample (A) spaced apart from the end of the pillar (11), thereby effectively increasing the production rate of cell organoids after the culturing step.

[0081] In addition, in the prior art, tissues obtained from patients had to be crushed and then centrifuged and cultured separately in a separate device. This crushing or the stress or flow applied to the cells during transport by the user could have a negative impact on the production of organoids. As a result, even though the amount of tissue that can be obtained from patients is limited, the number of organoids produced for use is limited, and it is difficult to ensure accuracy or reliability when performing diagnoses such as drug screening. However, the automated anticancer drug sensitivity test system (1000) according to one embodiment of the present invention performs a cutting (crushing) step using a chopper, thereby homogeneously forming organoids, thereby having the effect of increasing the accuracy and reliability of diagnosis during drug screening.

[0082]

[0083] It goes without saying that one or more of the components of the present invention can be combined to form a new embodiment. For example, referring to FIG. 11, a combination of the components of the present invention, such as FIG. 11, that performs only the preprocessing necessary for organoid culture is also possible.

[0084] Referring to FIG. 11 in more detail, an organoid culture preprocessing system (2000) and method according to another embodiment of the present invention are systems and methods that can automatically perform the crushing process, dissociation process, and centrifugation process all within the organoid culture preprocessing system (2000) by introducing tissue separated from a patient into the system without separate user intervention.

[0085] In the case of existing organoid culture preprocessing devices, the user may have to directly cut the patient tissue into small sizes and introduce it into the device, or the user may have to crush the patient tissue and then introduce it into the device. However, the organoid culture preprocessing system (2000) of the present invention has a technical feature in that the tissue separated from the patient is introduced into the organoid culture preprocessing system (2000) without crushing the patient tissue by the user, thereby performing preprocessing for the culture of cell organoids without separate manual work by the user.

[0086] More specifically, the organoid culture preprocessing system (2000) and method of the present invention will be described. The organoid culture preprocessing system (2000) of the present invention according to FIG. 11 may include a chopper (100), a cell dissociation device (200), a centrifuge (300), a liquid handler (500), and a multi-joint robot (50). For the sake of brevity, descriptions of overlapping components that are the same as those in other embodiments will be omitted.

[0087] In addition, the organoid culture preprocessing method implemented by the organoid culture preprocessing system (2000) includes crushing patient tissue by a chopper (100) (S110). Subsequently, a cell dissociation enzyme is applied to the patient tissue by a cell dissociation device (shaker) (200) (S120).

[0088] Afterwards, the cells to which the cell dissociation enzyme was applied in the liquid handler (500) can be filtered with a 100-μm stainer and placed in a 50-mL tube containing 8 mL of STI (S130).

[0089] Next, the filtered cells are centrifuged by a centrifuge (300) (S140). The cells filtered in step (S130) by a 100-μm stainer can be transferred to the centrifuge (300) by a multi-joint robot (50). The cells are centrifuged within the centrifuge (300) to extract cancer cells. For the sake of brevity, descriptions of steps that are identical to those in other embodiments and that are redundant will be omitted.

[0090]

[0091] Alternatively, the components of the present invention may be combined with one another to form one product, for example, including a liquid handler (500), a spotter (600), an automatic incubator (700), and an image analyzer (900), or may form a product for various purposes, including a centrifuge (300), a liquid handler (500), and an automatic incubator (700).

[0092]

[0093] In addition, the chopper (100), cell dissociation device (200), centrifuge (300), liquid handler (500), spotter (600), automatic incubator (700), scanner (800), and image analyzer (900) of the present invention may also be configured as individual products.

[0094]

[0095] In addition, when configuring the spotter (600) of the present invention as an individual product, it is also possible to configure the product to have a separate cooling unit including a head cooling module (601) and a cooling water channel (602).

[0096]

[0097] Therefore, according to the present invention, the following effects are obtained.

[0098] The present invention has the effect that all steps of extracting cells from tissue isolated from a patient, culturing them, and testing anticancer drug sensitivity can be performed on a single system without manual intervention by the user.

[0099] In addition, the present invention has the effect of homogeneously forming organoids by dispensing and culturing a mixed sample of cells and extracellular matrix on a pillar, thereby increasing the accuracy and reliability of diagnosis during drug screening.

[0100] In addition, the present invention can significantly improve the accuracy of diagnosis by improving the formation rate of organoids and the survival rate of cells during cell culture and increasing the possibility of contact between organoids and drugs during drug screening.

[0101] In addition, the present invention can increase the concentration (distribution) of cells on the surface of the mixed sample by preventing cells from moving toward the end of the filter after the mixed sample of cells and extracellular matrix is ​​dispensed onto the filter, thereby improving the possibility of contact between the organoid and the culture medium material or the drug to be diagnosed.

[0102] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0103]

[0104] Although the embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be implemented without departing from the technical spirit of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain it, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The protection scope of the present invention should be interpreted by the claims below, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. A chopper configured to crush patient tissue; A cell dissociation device (shaker) configured to apply a cell dissociation enzyme to the patient tissue pulverized by the chopper; A centrifuge configured to centrifuge cells to which the cell dissociation enzyme has been applied by the cell dissociation device; A liquid handler configured to mix the centrifuged cells and extracellular matrix to create a mixed sample and inject the mixed sample into a nozzle tip; A spotter configured to dispense the mixed sample onto the upper part of the pillar using the nozzle tip; An automated incubator configured to culture the above mixed sample to produce cell organoids; a scanner configured to scan the above cell organoids at high speed to generate images; and An automated anticancer drug susceptibility test system comprising a multi-joint robot configured to move at least one of the patient tissue, the cell, and the mixed sample between at least some of the chopper, the cell dissociation device, the centrifuge, the liquid handler, the spotter, the automatic incubator, and the scanner.

2. In paragraph 1, The above chopper includes a cutting part, The above cutting parts are, a rotating blade configured to crush the patient tissue; and An automated anticancer drug susceptibility test system comprising a disposable tube configured to surround the rotating blade and accommodate the patient tissue.

3. In paragraph 1, The above automatic incubator comprises a main door and a plate door smaller than the main door, An automated anticancer drug sensitivity test system in which the plate transfer drive unit moves the pillar into which the mixed sample is dispensed through the plate door excluding the main door into the automatic incubator.

4. In paragraph 1, Further comprising a cooler for cooling the mixed sample located at the bottom of the pillar while the pillar plate is turned over so that the mixed sample is located at the bottom of the pillar, The above automatic incubator is an automated anticancer drug sensitivity test system configured to gel the mixed sample located at the bottom of the pillar and culture the gelled mixed sample.

5. In paragraph 4, An automated anticancer drug sensitivity test system configured such that the cells of the mixed sample cooled inside the cooler move in a direction away from the end of the pillar within the mixed sample while the pillar plate is turned over. 6.(a) A step in which patient tissue is crushed by a chopper; (b) a step of applying a cell dissociation enzyme to the patient tissue by a cell dissociation device; (c) a step in which cells to which the cell dissociation enzyme has been applied are filtered by a liquid handler; (d) a step of centrifuging the filtered cells using a centrifuge; (e) A step in which cells and extracellular matrix separated by centrifugation are mixed by a liquid handler to create a mixed sample and injected into a nozzle tip; (f) a step in which the mixed sample inside the nozzle tip is dispensed onto the upper part of the pillar by a spotter; (g) a step of culturing the mixed sample using an automatic incubator to produce cell organoids; (h) a step in which an anticancer agent is injected into a nozzle tip by a liquid handler; (i) a step in which an anticancer agent is dispensed into a well plate by a spotter within a nozzle tip; (j) a step of combining a pillar plate and a well plate by a multi-joint robot; (k) a step of culturing the combined plates by an automatic incubator; and (l) a step of scanning the cell organoid at high speed by a scanner to generate an image; An automated method for testing anticancer drug sensitivity, wherein at least one of the patient tissue, the cell, and the mixed sample is moved by a multi-joint robot in at least some of the steps (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), and (l). 7.(a) A step in which patient tissue is crushed by a chopper; (b) a step of applying a cell dissociation enzyme to the patient tissue by a cell dissociation device; (c) a step of filtering cells to which the cell dissociation enzyme has been applied by a liquid handler; and (d) a step of centrifuging the filtered cells using a centrifuge, An organoid culture pretreatment method, wherein at least one of the patient tissue and the cells is moved by a multi-joint robot in at least some of the steps (a), (b), (c), and (d).

8. In paragraph 7, The above chopper includes a cutting part, The above cutting parts are, a rotating blade configured to crush the patient tissue; and An organoid culture preprocessing method comprising a disposable tube configured to surround the rotating blade and accommodate the patient tissue.

9. In paragraph 7, (e) a step of mixing the cells and extracellular matrix separated by centrifugation using a liquid handler to create a mixed sample and injecting the mixed sample into a nozzle tip; and (f) An organoid culture pretreatment method further comprising a step of dispensing the mixed sample within the nozzle tip onto the upper part of the pillar by a spotter.

10. In paragraph 7, A step of flipping the pillar plate so that the mixed sample containing the cells and extracellular matrix is ​​positioned at the bottom of the pillar while the mixed sample is dispensed at the top of the pillar; A step of cooling (icing) the mixed sample located at the bottom of the above pillar; and An organoid culture pretreatment method further comprising a step of gelling the mixed sample located at the lower portion of the pillar inside an incubator.

11. A chopper configured to crush patient tissue; A cell dissociation device (shaker) configured to apply a cell dissociation enzyme to the patient tissue pulverized by the chopper; A liquid handler configured to filter the cell to which the cell dissociation enzyme is applied by the cell dissociation device; A centrifuge configured to centrifuge the filtered cells by the liquid handler; and An organoid culture preprocessing system comprising a multi-joint robot configured to move at least one of the patient tissue and the cells between at least some of the chopper, the cell dissociation device, the liquid handler, and the centrifuge.

12. In paragraph 11, A liquid handler having an additional function of mixing the centrifuged cells and extracellular matrix to create a mixed sample and injecting the mixed sample into a nozzle tip; and An organoid culture pretreatment system further comprising a spotter configured to dispense the mixed sample onto the upper part of the pillar using the nozzle tip.

13. In paragraph 11, The above chopper includes a cutting part, The above cutting parts are, a rotating blade configured to crush the patient tissue; and An organoid culture preprocessing system comprising a disposable tube configured to surround the rotating blade and accommodate the patient tissue.

14. In paragraph 11, An organoid culture pretreatment system further comprising a cooler in which the mixed sample is cooled while the pillar plate is inverted.

15. A liquid handler configured to mix cells and extracellular matrix to create a mixed sample and inject the mixed sample into a disposable nozzle tip; A spotter that dispenses the mixed sample onto the top of the pillar using the disposable nozzle tip; An incubator for culturing the above mixed sample to create cell organoids; a scanner configured to scan the above cell organoids at high speed to generate images; and An anticancer drug sensitivity test system comprising a multi-joint robot configured to move the mixed sample and the pillar onto which the mixed sample is dispensed between the liquid handler, the spotter, the automatic incubator, and the scanner.

16. In paragraph 15, An anticancer drug sensitivity test system further comprising a cooler for cooling the filter plate on which the mixed sample is dispensed while in an inverted state, wherein the mixed sample is configured to be cultured by gelation.

17. In paragraph 15, The above spotter comprises a head cooling module that maintains a constant temperature of the mixed sample mounted on the disposable nozzle tip; and An anticancer drug sensitivity test system comprising a cooling unit including a plurality of cooling water channels positioned at the lower portion of the pillar and cooling a mixed sample dispensed to the pillar.

18. In paragraph 17, An anticancer drug sensitivity test system, wherein the head cooling module comprises: a nozzle tip contact portion that contacts the nozzle tip; and a head cooling portion that maintains the temperature of the nozzle tip that contacts the nozzle tip contact portion.

19. Includes a spotter that dispenses the mixed sample onto the top of the pillar using a disposable nozzle tip injected with the mixed sample created by mixing cells and extracellular matrix, An anticancer drug sensitivity test system, wherein the spotter comprises a head cooling module for maintaining a constant temperature of a mixed sample mounted on the disposable nozzle tip; and a cooling unit including a plurality of cooling water channels located at the lower portion of the pillar for cooling the mixed sample dispensed to the pillar.

20. In paragraph 19, An anticancer drug sensitivity test system, wherein the head cooling module comprises: a nozzle tip contact portion that contacts the nozzle tip; and a head cooling portion that maintains the temperature of the nozzle tip that contacts the nozzle tip contact portion.

Images