3D organoid analysis cell chip

Abstract

The present disclosure provides a 3D organoid analysis cell chip adapted to perform non-destructive electrical analysis on a 3D organoid in real time, whereby the chip allows for multiple experiments through safe handling of organoids and can acquire research outcomes such as real-time drug response monitoring, evaluation of growth depending on size changes, metabolic analysis, etc., more rapidly compared to conventional technologies, through non-destructive real-time electrical analysis, reduce variables for failure risk factors during culture and analysis, and find technical applications in various technical fields with high measurement sensitivity.

Description

3D organoid analysis cell chip

[0001] The present invention relates to a 3D organoid analysis cell chip capable of performing real-time, non-destructive electrical analysis on 3D organoids.

[0002] 3D spheroids / organoids (hereinafter collectively referred to as "3D organoids") and tissue analysis are currently being attempted in various ways, from culture methods to analysis technologies, for stem cell differentiation, drug screening, disease modeling, and in-depth genetic screening analysis. Various analysis technologies are required to correspond to the manufacturing form that varies depending on the culture method.

[0003] Representative 3D organoid culture methods include culture in a 3D suspension state using a gel type such as hydrogel or Matrigel, culture in a 2D / 3D structure through coating with culture medium and extracellular matrix protein (ECM), or production and culture using a 3D bioprinting method.

[0004] The fabricated 3D organoids are being analyzed using various technologies, including optical microscopy analysis using fluorescent dye marker labeling, image analysis using micro-CT, 3D imaging analysis, analysis of organ-on-a-chip platforms made of microfluidic flow devices and biocompatible polymer structures, and electrical analysis technology using the combination of 3D culture culture structures and electrode plates.

[0005] However, despite the advancement of these analysis technologies, there is no standardization for whether the actually produced 3D organoids of the brain, liver, lung, intestine, etc. are properly differentiated from stem cells, whether the size or function is properly implemented, and whether they are produced with a structure suitable for disease modeling. To this end, various analysis technologies are being researched to introduce technologies optimized for analysis.

[0006] 3D organoids, such as those of the brain, liver, lung, and intestines, used in various analyses, require extensive production time, advanced culture technology, and the dedicated efforts of researchers. Therefore, they require reuse and reproducibility, not just one-time analysis. However, optical analysis using fluorescent staining, the use of contrast agents for tomography, and invasive and destructive analytical methods for internal analysis of 3D organoids are limited to single-use. Furthermore, 3D organoids produced using gel-based culture methods can be damaged during harvest. Furthermore, suspended 3D organoids cultured in culture media and on 2D / 3D structures are difficult to handle and are therefore more prone to damage.

[0007] Thus, in 3D organoid experiments, the importance of a single experiment is extremely high, and failure can lead to a significant burden due to the subsequent months required from 3D organoid preparation to analysis. Therefore, there is a strong need for new methods that can address the shortcomings of conventional analysis techniques.

[0008] The present invention provides a 3D organoid analysis cell chip that enables multi-experimentation through safe organoid handling by performing non-destructive electrical analysis on 3D organoids in real time, and can produce research results faster than existing technologies, such as real-time drug response, growth according to size change, and metabolite analysis through non-destructive real-time electrical analysis, and can reduce variables related to risk factors for failure during culture and analysis, and can be applied to various fields with high measurement sensitivity.

[0009] The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0010] The present invention relates to a 3D organoid analysis cell chip including a measuring unit and a control unit, and in one embodiment, the measuring unit includes a cultureware having a receiving unit for containing a culture medium and a 3D organoid and having at least one first electrode unit on a bottom surface; a top lid disposed above the cultureware, having a penetrating portion in the center, and having at least one second electrode unit disposed along an outer edge of the penetrating portion so as to extend into the receiving unit of the cultureware; and an upper electrode unit having a third electrode unit; wherein the third electrode unit is capable of moving up and down through the penetrating portion of the top lid and includes at least one microinjection needle electrode extending into the receiving unit of the cultureware.

[0011] In one embodiment, the control unit is electrically connected to the first to third electrode units, and can control to perform at least one measurement mode among an impedance measurement mode, an LFP (Local Field Potential) measurement mode, a DPV (Differential Pulse Voltametry) measurement mode, and a TEER (Trans Epithelial Electrical Resistance) measurement mode through electrode settings for the first to third electrode units.

[0012] In one embodiment, the control unit, when performing the impedance measurement mode, sets the third electrode unit as a working electrode while setting the first electrode unit as a counter electrode, and, as the upper electrode unit moves downward, can measure in real time an impedance change in a process in which the third electrode unit transitions from a state in which the third electrode unit is in contact with the culture medium to a state in which the third electrode unit is in contact with the 3D organoid, and then to a state in which the 3D organoid is fixed to the first electrode unit.

[0013] And, the control unit, when performing the LFP measurement mode, may set the third electrode unit as a recording electrode or a recording electrode and a stimulation electrode, while setting the second electrode unit as a reference electrode or a reference electrode and a stimulation electrode, or may set the first electrode unit as a stimulation electrode together with the first electrode unit.

[0014] And, the control unit may set the third electrode unit as a working electrode and the second electrode unit as a reference electrode while setting the first electrode unit as a counter electrode when performing the DPV measurement mode.

[0015] And, the control unit, when performing the TEER measurement mode, may set the third electrode unit as a working electrode while setting the first electrode unit as a counter electrode, or may set the four micro-needle electrodes of the third electrode unit as chopstick electrodes.

[0016] Meanwhile, the control unit can control to perform a composite mode in which at least two measurement modes among the impedance measurement mode, the LFP measurement mode, the DPV measurement mode, and the TEER measurement mode are performed simultaneously or sequentially.

[0017] In one embodiment, the composite mode may include an impedance / LFP measurement mode, an impedance / DPV measurement mode, an impedance / LFP / DPV measurement mode, and a TEER / DPV measurement mode.

[0018] Meanwhile, the microneedle electrode forming the third electrode section can be used as a working electrode, a drug delivery electrode, or a stimulation electrode for electrical stimulation.

[0019] According to the 3D organoid analysis cell chip of the present invention, which comprises the above-described configuration, it is possible to confirm through impedance measurement whether 3D organoid cells floating in a culture medium contained within the cultureware container are in proper contact with the electrodes and thus in a state where electrical analysis is possible. Accordingly, safe handling of 3D organoids is ensured, enabling multiple experiments, and non-destructive electrical analysis of 3D organoids can be performed repeatedly in real time.

[0020] Furthermore, the 3D organoid analysis cell chip of the present invention can perform various types of non-destructive electrical analyses, either alone or in parallel, depending on the configuration of the first to third electrode sections. Accordingly, by comprehensively performing a wide range of non-destructive electrical analyses on various types of 3D organoids and tailored to various experimental purposes, research results can be derived more quickly than with existing technologies. Furthermore, the technology can be applied in various fields with high measurement sensitivity while reducing variables related to failure risk factors during culture and analysis.

[0021] The effects of the present invention are not limited to those mentioned above, and also include other effects that are not explicitly mentioned, although they can be clearly understood by those skilled in the art from the description throughout the specification.

[0022] FIG. 1 is a perspective view of a measurement unit of a 3D organoid analysis cell chip according to one embodiment of the present invention.

[0023] Figure 2 is an exploded perspective view of the measuring unit of Figure 1.

[0024] Figure 3 is a diagram illustrating one embodiment of the impedance measurement mode.

[0025] Fig. 4 is a diagram illustrating the lowering process of the third electrode part in the impedance measurement mode of Fig. 3.

[0026] Figure 5 is a diagram illustrating one embodiment of the LFP measurement mode.

[0027] Figure 6 is a drawing illustrating one embodiment of the DPV measurement mode.

[0028] Figure 7 is a diagram illustrating one embodiment of the TEER measurement mode.

[0029] Figure 8 is a diagram showing an example of an impedance / LFP measurement mode among composite modes.

[0030] Figure 9 is a perspective view of the measurement section of a conventional 3D organoid analysis cell chip.

[0031] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. The advantages and features of the present invention, and methods for achieving them, will become clear with reference to the embodiments described in detail below together with the attached drawings. However, the present invention is not limited to the embodiments disclosed below, but can 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. Like reference numerals refer to like elements throughout the specification.

[0032] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, singular forms also include plural forms, unless specifically stated otherwise.

[0033] The terms "comprises" and / or "comprising" as used in the specification do not exclude the presence or addition of one or more other components, steps, operations and / or elements.

[0034]

[0035] FIG. 1 is a perspective view of a measurement unit (100) of a 3D organoid analysis cell chip (10) according to one embodiment of the present invention, FIG. 2 is an exploded perspective view of the measurement unit (100) of FIG. 1, and FIG. 3 is a diagram illustrating one embodiment of an impedance measurement mode. Here, FIGS. 1 and 2 illustrate the configuration of the measurement unit (100), and FIG. 3 illustrates a control unit (200) electrically connected to the first to third electrode units (116, 124, 132) provided in the measurement unit (100). Various non-destructive electrical analyses performed by the control unit (200) will be described in detail after the description of the measurement unit (100).

[0036] Referring to FIGS. 1 and 2, the measurement unit (100) of the 3D organoid analysis cell chip (10) includes a cultureware (110), a top lid (120), and an upper electrode unit (130).

[0037] The cultureware (110) forms a well, i.e., a receiving portion (112) that contains a culture medium and 3D organoids. In addition, at least one first electrode portion (116) is provided on the bottom surface of the cultureware (110). Here, as illustrated, the cultureware (110) may include a truncated cone shape with a narrow bottom surface so that the 3D organoid contained in the receiving portion (112) can make good contact with the first electrode portion (116). However, the present invention is not limited thereto, and the cultureware (110) may be modified in various ways as long as the 3D organoid contained in the receiving portion (112) can make good contact with the first electrode portion (116).

[0038] The first electrode portion (116) must be exposed to the receiving portion (112). The first electrode portion (116) can be formed directly on the inner bottom surface of the cultureware (110), but for improved manufacturing convenience and economy, it can be manufactured as a two-piece structure in which the cultureware (110) with a hole in the bottom is combined on the substrate (114) on which the first electrode portion (116) is formed.

[0039] The top lid (120) is an upper structure placed above the cultureware (110), and the top lid (120) covers the receiving portion (112) of the cultureware (110), thereby preventing external contaminants from penetrating into the receiving portion (112) of the cultureware (110) or performing a function of exchanging gases.

[0040] The top lead (120) has a central penetrating portion (122) and at least one second electrode portion (124) arranged along the outer edge of the penetrating portion (122). The second electrode portion (124) extends through the top lead (120) into the receiving portion (112) of the cultureware (110). For example, the second electrode portion (124) may have four electrodes evenly arranged along the outer edge of the penetrating portion (122), but is not limited thereto.

[0041] Meanwhile, in another embodiment of the present invention, the second electrode portion (124) may be configured as a separate type. At this time, one of the second electrode portions (124) may be arranged at the upper end along the outer edge of the penetration portion (122), and the other of the second electrode portions (124) may be arranged at the lower end along the outer edge of the penetration portion (122). A separate signal line may be inserted into the penetration portion (122) to electrically connect the second electrode portions (124) arranged at the upper and lower ends of the penetration portion (122), thereby allowing the top lead (120) to be configured.

[0042] In another embodiment, one of the second electrode parts (124) may be placed on the upper side of the top lead (120) and the other of the second electrode parts (124) may be placed on the lower side along the outer edge of the through-hole (122), and a separate signal line may be inserted inside the top lead (120) so that the second electrode parts (124) placed respectively are electrically connected.

[0043] The upper electrode unit (130) includes a third electrode unit (132). At this time, at least a portion of the third electrode unit (132) can be moved up and down through the penetration portion of the top lid (120). In other words, by moving up and down the upper electrode unit (130), the third electrode unit (132) can approach the first electrode unit (116) of the cultureware (110) or move away from the first electrode unit (116). The driving unit (not shown) for moving up and down the upper electrode unit (130) is not limited to any particular structure. For example, various linear movement mechanisms such as screw-type, gear-type, or pneumatic-type can be applied as the driving unit. However, the driving unit for the upper electrode unit (130) may preferably have a structure capable of an appropriate level of micro-actuating in consideration of the safe handling of the 3D organoid contained in the receiving unit (112) of the cultureware (110).

[0044] Meanwhile, in another embodiment of the present invention, the upper electrode part (130) can be configured in a form in which only the third electrode part (132) moves up and down while being fixed, and by the up and down movement of the third electrode part (132), the third electrode part (132) can approach the first electrode part (116) of the cultureware (110) or move away from the first electrode part (116). At this time, the driving part (not shown) that moves the third electrode part (132) up and down is not limited to any special structure.

[0045] In addition, the third electrode unit (132) may include at least one micro-needle electrode (133) extending into the receiving portion (112) of the cultureware (110). As the third electrode unit (132) includes the micro-needle electrode (133), the third electrode unit (132) can perform various functions, such as an electrode for electrical measurement, a micro-needle for drug delivery, or a stimulation electrode (Stim) for electrical stimulation.

[0046] Meanwhile, the cultureware (110), top lead (120), and upper electrode portion (130) are preferably made of a biocompatible material. Although not particularly limited, they may be made of biocompatible plastics such as PDMS, PMMA, PET, PC, etc.

[0047] In addition, it is preferable that the first electrode portion (116), the second electrode portion (124), and the third electrode portion (132) be made of a biocompatible conductive metal. Although not particularly limited, for example, they may be made of a biocompatible conductive metal such as Au, Ti, Pt, ITO, PEDOT:PSS, or a composite material thereof.

[0048] Here, the specific shape, arrangement, number, etc. of the first to third electrode parts (116, 124, 132) provided in the cultureware (110), top lead (120), and upper electrode part (130) are not limited to the contents exemplarily illustrated in the drawing. The design of each electrode part can be freely made as long as it conforms to the operating methods of the various measurement modes described below.

[0049] And, the 3D organoid analysis cell chip (10) of the present invention includes a control unit (200) that controls to perform various non-destructive electrical analyses. The control unit (200) is electrically connected to the first to third electrode units (116, 124, 132) provided in the measurement unit (100). And, the control unit (200) can select any electrode unit among the first to third electrode units (116, 124, 132) to suit a specific measurement mode to be performed and set it as a required electrode. For example, the 3D organoid analysis cell chip (10) of the present invention can perform at least one measurement mode among an impedance measurement mode, an LFP (Local Field Potential) measurement mode, a DPV (Differential Pulse Voltametry) measurement mode, and a TEER (Trans Epithelial Electrical Resistance) measurement mode, and the control unit (200) is provided with a circuit for performing all of the above measurement modes.

[0050] The control unit (200) selects an electrode unit suitable for the corresponding measurement mode and performs the corresponding measurement mode using the selected electrode unit. Circuits for the impedance measurement mode, LFP measurement mode, DPV measurement mode, and TEER measurement mode are known, so their description will be omitted. Below, a description will be given of which electrode unit is selected and which electrical analysis is performed in each measurement mode.

[0051] For reference, in order to help understand the present invention, the 3D organoid analysis cell chip (10) having the most basic single well structure has been described above, but it is of course possible to expand the 3D organoid analysis cell chip (10) to 24 wells, 48 ​​wells, etc., in which such single wells are arranged in a grid pattern vertically and horizontally.

[0052]

[0053] 1. Impedance measurement mode

[0054] Impedance measurements are used to analyze cell growth and toxicity of 3D organoids. Furthermore, impedance measurements are also used to determine whether 3D organoid cells floating in a culture medium (CM) within the receiving compartment (112) of the cultureware (110) are in proper contact with the electrodes, enabling electrical analysis, i.e., are in an adsorbed / fixed state to the electrodes.

[0055] FIG. 3 is a diagram illustrating one embodiment of an impedance measurement mode. Referring to FIG. 3, the control unit (200) sets the third electrode unit (132) as a working electrode (WE) while setting the first electrode unit (116) as a counter electrode (CE) when performing the impedance measurement mode. The third electrode unit (132) and the first electrode unit (116) are in vertically opposite contact with the 3D organoid, thereby allowing the impedance of the 3D organoid to be measured.

[0056] In addition, the third electrode part (132), which is the working electrode (WE), can contact the 3D organoid by the downward movement of the upper electrode part (130), and further adsorb / fix the 3D organoid to the first electrode part (116).

[0057] Whether the 3D organoid has electrode contact and is capable of electrical analysis can be determined by measuring impedance in real time while the upper electrode part (130) is lowered.

[0058] Meanwhile, referring to FIG. 9, in the past, a pattern for performing the functions of a working electrode (WE) and a counter electrode (CE) was formed on a substrate (A), and when an electrostatic discharge medium (ECM) was coated on the working electrode (WE) and the counter electrode (CE), the 3D organoid was adsorbed / contacted only from the bottom to measure impedance. In contrast, the present invention configures the working electrode (WE) and the counter electrode (CE) to be separated from each other vertically so that they are in vertically opposite contact with the 3D organoid, thereby having the effect of accurately determining whether the 3D organoid is in an adsorbed / fixed state regardless of whether or not a separate electrostatic discharge medium (ECM) is coated on the electrode. FIG. 4 is a diagram illustrating the lowering process of the third electrode part (132) in the impedance measurement mode of FIG. 3.

[0059] Referring to FIG. 4, as the upper electrode part (130) moves downward, the third electrode part (132) transitions from a state in which it is in contact with the culture medium (CM) {see (a) of FIG. 4}, to a state in which it is in contact with the 3D organoid (Org) {see (b) of FIG. 4}, and to a state in which the 3D organoid (Org) is adsorbed and fixed to the first electrode part (116) {see (c) of FIG. 4}, and the control part (200) measures the impedance change during this transition process in real time. At this time, since a distinct difference occurs in the impedance measurement value at each stage, each stage can be distinguished.

[0060] In this way, since the impedance change according to the lowering of the upper electrode part (130) can be measured in real time to distinguish each step, the micro needle electrode (133) forming the third electrode part (132) can be brought into contact so that the 3D organoid (Org) can be in a state in which drug injection is possible while minimizing damage to the organoid (Org). In addition, since the impedance change according to the lowering of the upper electrode part (130) can be measured in real time to distinguish each step, it is possible to confirm whether the floating 3D organoid (Org) has moved and been absorbed / fixed toward the first electrode part (116) on the bottom surface. In other words, it is possible to verify whether the 3D organoid (Org) is in a state in which electrical analysis is possible through real-time impedance measurement.

[0061] In this way, the micro-needle electrode (133) forming the third electrode section (132) can be utilized as a working electrode (WE) or a micro-needle for drug delivery. In addition, the impedance measurement mode can be utilized for non-destructive electrical analysis on its own, and can also be used in parallel with the LFP measurement mode and DPV measurement mode described later.

[0062]

[0063] 2. LFP measurement mode

[0064] LFP (Local Field Potential) measurement refers to the measurement of local field potential, and can be performed after confirming whether the 3D organoid is absorbed and fixed to the first electrode part (116) through impedance measurement. The electrical signal change of the 3D organoid is measured by LFP measurement, and through this, signal excitation and synaptic block analysis according to electrical / drug stimulation for brain organoids, cardiovascular pharmacological evaluation analysis according to drug response for heart organoids, damage degree classification and inflammation degree analysis according to epithelial barrier damage for intestinal organoids, and analysis of cilia reproduction and damage degree, and auditory nerve cell connection degree according to electrical / chemical stimulation for inner ear organoids can be performed.

[0065] Although LFP measurement technology is specialized for brain and heart organoid analysis, there are increasing cases of analysis of the process by which neurons in sensory organs such as hearing, smell, taste, touch, and pain transmit signals to the brain. For example, in relation to olfaction, the activation of a group of vomeronasal neurons on the surface of the epithelium of the vomeronasal organ, a horn-shaped bone located in the nose, by ligands in response to pheromones and other chemical signals was analyzed using LFP measurement technology.

[0066] Figure 5 is a diagram illustrating one embodiment of the LFP measurement mode.

[0067] Referring to FIG. 5, the control unit (200) may set the third electrode unit (132) as a recording electrode (Rec) or a recording electrode (Rec) and a stimulation electrode (Stim) while setting the second electrode unit (124) as a reference electrode (RE) or a reference electrode (RE) and a stimulation electrode (Stim) when performing the LFP measurement mode. In addition, the control unit (200) may set the first electrode unit (116) as a stimulation electrode (Stim) as needed.

[0068] Meanwhile, referring to FIG. 9, there was a problem in that electrical stimulation was transmitted to a limited extent to the 3D organoid by placing a recording electrode (Rec), a reference electrode (RE), and a stimulation electrode (Stim) on a substrate (A).

[0069] In contrast, in the LFP measurement mode of the present invention, the third electrode unit (132) can be basically used as multiple recording electrodes (Rec) for internal / external measurement of a 3D organoid, and can also be used as a stimulation electrode (Stim) for electrical stimulation, and the first electrode unit (116) can also be used as a stimulation electrode (Stim) for electrical stimulation of the lower absorption surface of the 3D organoid. Through this, there is an effect that allows for more diverse experiments to be performed by delivering electrical stimulation to various areas of the 3D organoid.

[0070] At this time, a 3D organoid (Org) may be positioned in the area between the recording electrode (Rec) and the reference electrode (RE) of the second electrode unit (124). In addition, in order to reduce noise in measuring the potential difference between the recording electrode (Rec) and the reference electrode (RE), it is preferable that the shape and position of the second electrode unit (124) be determined so that the 3D organoid (Org) does not touch the reference electrode (RE).

[0071]

[0072] 3. DPV measurement mode

[0073] DPV (Differential Pulse Voltametry) measurement refers to differential pulse voltage measurement, and can be performed after it has been confirmed through impedance measurement that the 3D organoid is adsorbed and fixed to the first electrode section (116).

[0074] DPV measurement technology is an electrochemical measurement technique that allows for the determination of protein concentrations and the differentiation of 3D organoid types by metabolite through electrochemical redox signal analysis. It can also be used to analyze tumor and cancer cell activity, target protein concentration, and anticancer drug efficacy.

[0075] For example, applications of 3D organoid analysis using DPV measurement technology include analysis of histomorphological, functional, and biochemical changes in atopic dermatitis in skin organoids, analysis of cytokine and chemokine induction under in vitro bronchial culture conditions in airway organoids, analysis of hepatic stem cell activity and fibrosis according to hepatic fibrosis-inducing substances in liver organoids, and analysis of the interaction between intestinal Escherichia coli-secreted toxic substances and small intestinal organoids in intestinal organoids.

[0076] FIG. 6 is a drawing illustrating one embodiment of a DPV measurement mode, wherein the DPV measurement mode can be performed using a three-electrode method.

[0077] Referring to FIG. 6, the control unit (200) may set the third electrode unit (132) as a working electrode (WE) and the second electrode unit (124) as a reference electrode (RE), while setting the first electrode unit (116) as a counter electrode (CE), when performing the DPV measurement mode.

[0078] Meanwhile, referring to FIG. 9, in the past, a working electrode (WE) and a counter electrode (CE) were placed on a substrate (A), and a reference electrode (RE) in the form of a separate rod was placed in an electrochemical vessel to perform DPV electrode surface modification, which was inconvenient. In contrast, the present invention provides a conductive material (such as Ag / AgCl) necessary for forming a reference potential on the surface of the second electrode portion (124) through electrical deposition and / or surface modification, thereby enabling DPV measurement within a single cell chip without a separate additional device that serves as a reference electrode (RE), thereby improving convenience.

[0079]

[0080] 4. TEER measurement mode

[0081] Transepithelial Electrical Resistance (TEER) measurement refers to the measurement of transepithelial electrical resistance and is a derivative of impedance measurement. TEER measurement technology is a method for measuring cell membrane resistance in cell barriers and cell layer fusion, and is utilized in various fields, including the gastrointestinal tract, lung spleen, brainstem, tissues, and the blood-brain barrier (BBB).

[0082] FIG. 7 is a drawing illustrating one embodiment of a TEER measurement mode, wherein the TEER measurement mode can be performed using a two-electrode method {see (a) of FIG. 7} or a four-electrode method {see (b) of FIG. 7}.

[0083] The two-electrode method of TEER measurement mode is similar to the impedance measurement mode described above, because the TEER measurement technique is a derivative technique of impedance measurement.

[0084] Referring to FIG. 7, when performing the TEER measurement mode, the control unit (200) sets the third electrode unit (132) as a working electrode (WE) while setting the first electrode unit (116) as a counter electrode (CE).

[0085] In some cases, a unique cell membrane resistance measurement that eliminates electrode impedance may be required. For this purpose, a four-electrode method is utilized, and the measurement unit (200) is configured with a third electrode unit (132) composed of four micro-needle electrodes (133), and the four micro-needle electrodes (133) are set as chopstick electrodes (ChE).

[0086] In the four-electrode method, a chopstick electrode (ChE) applies a constant alternating current to two microneedle electrodes (I+, I-) for four microneedle electrodes (133), and measures the voltage drop output from the remaining two microneedle electrodes (V+, V-). By measuring the voltage drop, the total electrical resistance excluding the electrode impedance is calculated, and from this, the inherent cell membrane resistance is derived.

[0087]

[0088] 5. Composite mode

[0089] Composite mode refers to a measurement mode in which at least two of the aforementioned impedance measurement mode, LFP measurement mode, DPV measurement mode, and TEER measurement mode are performed in parallel. Each measurement mode is based on the electrode settings described above, and therefore, the composite mode can be understood as two or more measurement modes being performed in parallel, either simultaneously or sequentially.

[0090] In order to perform the composite mode, the number of first to third electrode sections (116, 124, 132) must be provided in such a number that each measurement mode can be performed simultaneously. In one embodiment of the present invention, the composite mode can be provided as an impedance / LFP measurement mode, an impedance / DPV measurement mode, an impedance / LFP / DPV measurement mode, and a TEER / DPV measurement mode.

[0091] Looking at the minimum number of electrodes required for each composite mode, the impedance / LFP measurement mode requires at least 5 electrodes, including a working electrode (WE) and a counter electrode (CE) for impedance measurement, and a stimulation electrode (Stim), a recording electrode (Rec), and a reference electrode (RE) for LFP measurement. The impedance / DPV measurement mode requires at least 5 electrodes, including a working electrode (WE) and a counter electrode (CE) for impedance measurement, and a working electrode (WE), a counter electrode (CE), and a reference electrode (RE) for DPV measurement. The impedance / LFP / DPV measurement mode requires at least 7 electrodes (excluding the stimulation electrode for LFP measurement). The TEER / DPV measurement mode requires at least 5 or 7 electrodes, depending on whether the TEER measurement mode is a 2-electrode method or a 4-electrode method.

[0092] Figure 8 is a diagram showing an example of an impedance / LFP measurement mode among composite modes.

[0093] Referring to Fig. 8, the control unit (200) selects a working electrode (WE) and a counter electrode (CE) for impedance measurement, and a stimulation electrode (Stim), a recording electrode (Rec), and a reference electrode (RE) for LFP measurement, and can perform impedance measurement and LFP measurement simultaneously. Other composite modes also operate similarly to Fig. 8, and the details thereof will be clearly understood without a separate drawing through the series of measurement modes described above.

[0094] Referring to FIG. 9, conventionally, electrodes are arranged on a substrate (A), but in this case, there was a problem that impedance or LFP measurements could not be accurately performed on cells or tissues located in an area that did not contact the substrate (A). In addition, DPV and TEER measurements for 3D organoids could not be performed using only electrodes implemented on the conventional substrate (A), or there were difficulties in handling the 3D organoids. However, according to the embodiments of the present invention described above, impedance, LFP, DPV, and TEER measurements for 3D organoids can be performed efficiently and stably.

[0095] Although embodiments of the present invention have been described with reference to the attached drawings, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without altering the technical concept or essential features thereof. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

[0096] 10: 3D Organoid Analysis Cell Chip

[0097] 100: Measurement section

[0098] 110: Cultureware

[0099] 112: Reception area

[0100] 114: Substrate

[0101] 116: First electrode section

[0102] 120: Top Lead

[0103] 122: Penetration

[0104] 124: Second electrode section

[0105] 130: Upper electrode section

[0106] 132: Third electrode section

[0107] 133: Microneedle electrode

[0108] 200: Control Unit

[0109] WE: Working electrode

[0110] CE: Counter electrode

[0111] RE: Reference electrode

[0112] Rec: recording electrode

[0113] Stim: Stimulating electrode

[0114] ChE: Chopstick Electrode

[0115] CM: culture medium

[0116] Org: 3D Organoids

Claims

1. In a 3D organoid analysis cell chip including a measurement unit and a control unit, The above measuring part, A cultureware having a container for containing a culture medium and a 3D organoid, and having at least one first electrode portion on the bottom surface; A top lead having a penetrating portion in the center and at least one second electrode portion arranged along the outer edge of the penetrating portion so as to extend into the receiving portion of the cultureware; and An upper electrode section having a third electrode section; Including, but not limited to, A 3D organoid analysis cell chip characterized in that the third electrode section is capable of moving up and down through the penetration section of the top lead and includes at least one micro-injection needle electrode extending into the receiving section of the cultureware.

2. In claim 1, The above control unit, A 3D organoid analysis cell chip characterized in that it is electrically connected to the first to third electrode sections and controls to perform at least one measurement mode among an impedance measurement mode, an LFP (Local Field Potential) measurement mode, a DPV (Differential Pulse Voltametry) measurement mode, and a TEER (Trans Epithelial Electrical Resistance) measurement mode through electrode settings for the first to third electrode sections.

3. In claim 2, The above control unit, In performing the above impedance measurement mode, the third electrode part is set as a working electrode, while the first electrode part is set as a counter electrode. A 3D organoid analysis cell chip characterized in that, according to the downward movement of the upper electrode part, the impedance change in the process of transitioning from a state in which the third electrode part is in contact with the culture medium to a state in which the third electrode part is in contact with the 3D organoid to a state in which the 3D organoid is fixed to the first electrode part is measured in real time.

4. In claim 2, The above control unit, A 3D organoid analysis cell chip characterized in that, in performing the above LFP measurement mode, the third electrode unit is set as a recording electrode or a recording electrode and a stimulation electrode, while the second electrode unit is set as a reference electrode or a reference electrode and a stimulation electrode, or the first electrode unit is set as a stimulation electrode together therewith.

5. In claim 2, The above control unit, A 3D organoid analysis cell chip characterized in that, when performing the above DPV measurement mode, the third electrode part is set as a working electrode, the second electrode part is set as a reference electrode, and the first electrode part is set as a counter electrode.

6. In claim 2, The above control unit, A 3D organoid analysis cell chip characterized in that, when performing the above TEER measurement mode, the third electrode part is set as a working electrode while the first electrode part is set as a counter electrode, or the four micro-injection needle electrodes of the third electrode part are set as chopstick electrodes.

7. In claim 2, The above control unit, A 3D organoid analysis cell chip characterized by controlling to perform a composite mode in which at least two or more measurement modes among an impedance measurement mode, an LFP measurement mode, a DPV measurement mode, and a TEER measurement mode are performed simultaneously or sequentially.

8. In claim 7, The above composite mode is, A 3D organoid analysis cell chip characterized by including an impedance / LFP measurement mode, an impedance / DPV measurement mode, an impedance / LFP / DPV measurement mode, and a TEER / DPV measurement mode.

9. In claim 1, The micro needle electrode forming the third electrode section is A 3D organoid analysis cell chip characterized by being used as an operating electrode, a drug delivery electrode, or a stimulation electrode for electrical stimulation.

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