Apparatus and method for forming chondroprogenitor cell aggregates

Abstract

The apparatus for forming chondroprogenitor cell aggregates in this embodiment includes a plurality of wells each having a micromass disposed therein, an electrical stimulation unit that provides electrical stimulation to the micromass, an image acquisition unit that photographs the micromass disposed in the plurality of wells at predetermined time intervals to acquire images, and a stimulus provision determination unit that determines the provision of the electrical stimulation from the images, wherein the stimulus provision determination unit determines the provision of the electrical stimulation based on the degree of aggregation of the micromass to which the electrical stimulation is provided.

Description

[Technical Field]

[0001] The present invention relates to an apparatus and method for forming chondroprogenitor cell aggregates. [Background technology]

[0002] Heavy use of the knees causes cartilage to wear down, and inflammation of the knee joints and ligaments can lead to degenerative arthritis, which affects eight in ten people aged 65 and over in Korea. While degenerative arthritis was once known as a geriatric disease, in recent years, poor lifestyle habits and an increase in traumatic arthritis due to the exercise boom have led to an accelerated onset of arthritis among younger people. Research is being conducted into the use of artificial cartilage containing chondrocytes isolated from rib cartilage to treat cartilage-related diseases. Summary of the Invention [Problem to be solved by the invention]

[0003] Knee cartilage is thin, approximately 2-3mm thick, but it distributes impact to surrounding tissues, allowing smooth joint movement and preventing joint damage. Knee cartilage is a typical non-regenerative tissue, and can be reduced regardless of age due to external factors such as overuse and damage from external impacts. Pathological factors such as rheumatoid arthritis can also cause cartilage damage.

[0004] Degenerative arthritis occurs as cartilage tissue decreases with age. Damaged cartilage cannot function normally, exposing the underlying bone. This results in inflammation and pain, restricting movement and accelerating the loss of cartilage tissue.

[0005] Conventional techniques for treating these joint-related disorders involve the use of stem cells, which are differentiated into chondrocytes mainly through the addition of expensive growth factors, but cost issues must be resolved before they can be used clinically. These issues, such as cost and immunity, have yet to be resolved.

[0006] The present invention has been made in view of the above circumstances, and its purpose is to overcome the drawbacks of the prior art. That is, one of the problems to be solved by the present invention is to provide an apparatus and method for producing a therapeutic agent for joint-related diseases such as degenerative arthritis as well as rheumatoid arthritis by forming chondrocytes and / or chondrocyte totipotent cell aggregates using mesenchymal stem cells and fibroblasts. [Means for solving the problem]

[0007] The apparatus for forming chondroprogenitor cell aggregates according to this embodiment includes a plurality of wells each containing a micromass, an electrical stimulation unit that provides electrical stimulation to the micromass, an image acquisition unit that photographs the micromass placed in the plurality of wells at predetermined time intervals to acquire images, and a stimulation provision determination unit that determines the provision of the electrical stimulation from the images, wherein the stimulation provision determination unit determines the provision of the electrical stimulation based on the degree of aggregation of the micromass to which the electrical stimulation is provided.

[0008] In one aspect of the device according to this embodiment, the micromass is one of mesenchymal stem cells and fibroblasts.

[0009] In one aspect of the device according to this embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0010] According to one aspect of the device of this embodiment, the electrical stimulation is a pulsed electrical stimulation having a frequency greater than 0 and less than 20 Hz, an amplitude greater than -20 V and less than 20 V, and a duty ratio greater than 0 and less than 80%.

[0011] In one aspect of the device according to this embodiment, the electrical stimulus is at least one of a voltage signal and a current signal.

[0012] In one aspect of the device according to this embodiment, the electrical stimulus is provided to the micromass continuously or intermittently for a period of six days or less.

[0013] According to one aspect of the device of this embodiment, the stimulus provision determination unit includes a trained artificial neural network, and the stimulus provision determination unit receives the image, estimates when the micromass has maximally aggregated, and cuts off the electrical stimulation.

[0014] According to one aspect of the device of this embodiment, when the micromass aggregates to a maximum extent, the chondroprogenitor cells aggregate into a spheroid shape.

[0015] According to one aspect of the device of this embodiment, the device further includes a well in which the micromass stained with a fluorescent substance is located, a light source that provides light so that the stained micromass emits fluorescence, and a fluorescent image acquisition unit that photographs the micromass stained with the fluorescent substance to acquire an image, and the electrical stimulation unit further provides an electrical stimulation to the micromass stained with the fluorescent substance.

[0016] According to one aspect of the device of this embodiment, the fluorescent image acquisition unit acquires an image of calcium oscillations occurring in the micromass stained with the fluorescent substance within 6 hours to 40 hours after the electrical stimulus is applied.

[0017] The method for forming chondroprogenitor cell aggregates according to this embodiment includes an electrical stimulation providing step of providing electrical stimulation to micromasses arranged in a plurality of wells, an image acquiring step of photographing the micromasses at predetermined time intervals to acquire images, and a stimulation provision determining step of determining whether to provide the electrical stimulation based on the images, wherein in the stimulation provision determining step, the provision of the electrical stimulation is determined based on the degree of aggregation of the micromasses to which the electrical stimulation has been provided.

[0018] In one aspect of the method according to this embodiment, the micromass is one of mesenchymal stem cells and fibroblasts.

[0019] In one aspect of the method according to this embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0020] According to one aspect of the method of this embodiment, the electrical stimulation providing step is performed by providing pulsed electrical stimulation having a frequency greater than 0 and less than 20 Hz, an amplitude greater than -20 V and less than 20 V, and a duty ratio greater than 0 and less than 80%.

[0021] In one aspect of the method according to this embodiment, the electrical stimulus is at least one of a voltage signal and a current signal.

[0022] According to one aspect of the method of this embodiment, in the step of providing the electrical stimulation, the electrical stimulation is provided to the micromass continuously or intermittently for six days or less.

[0023] According to one aspect of the method of this embodiment, the stimulus provision decision step is performed using a trained artificial neural network, and in the stimulus provision decision step, the image is received, the time when the micromass has maximally aggregated is estimated, and the electrical stimulation is stopped.

[0024] According to one aspect of the method of this embodiment, when the micromass aggregates to a maximum extent, the chondroprogenitor cells aggregate into a spheroid shape.

[0025] According to one aspect of the method of this embodiment, the method further includes the steps of providing light to a well in which the micromass stained with a fluorescent substance is located so that the stained micromass emits fluorescence, and photographing the micromass stained with the fluorescent substance to obtain a fluorescent image, and an electrical stimulus is provided to the micromass stained with the fluorescent substance.

[0026] According to one aspect of the method of this embodiment, in the fluorescent image acquisition step, a calcium oscillation image occurring in the micromass stained with the fluorescent substance is acquired within 6 to 40 hours after the electrical stimulus is applied.

[0027] According to one aspect of the method of this embodiment, the image acquisition step is performed in parallel with the electrical stimulation application step. [Effects of the Invention]

[0028] According to the present embodiment, an apparatus and method are provided that can generate chondroprogenitor cells by providing electrical stimulation to micromasses. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram showing an overview of a cartilage totipotent cell aggregate-forming device according to the present embodiment. [Figure 2] FIG. 1 is a procedural diagram showing an outline of a method for forming a cartilage totipotent cell aggregate according to the present embodiment. [Figure 3] 1A and 1B are diagrams illustrating an example of electrical stimulation provided by an electrical stimulation unit through electrodes. [Figure 4] FIG. 10 is a diagram showing modeling of an example of an image observed over time when an electrical stimulus is applied to a micromass in a culture solution contained in a well. [Figure 5] FIG. 10 is a diagram illustrating a method for determining an inner closed curve in an arbitrary image. [Figure 6] 10 is an example of a graph showing values ​​output by an estimation unit of an electrical stimulation system according to an embodiment of the present invention over time. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present embodiment will be described below with reference to the accompanying drawings.

[0031] Fig. 1 is a schematic diagram showing an overview of a chondroprogenitor cell aggregate-forming device 10 according to this embodiment, and Fig. 2 is a procedural diagram showing an overview of a method for forming totipotent chondroprogenitor cell aggregates according to this embodiment. Referring to Figs. 1 and 2, the chondroprogenitor cell aggregate-forming device 10 includes a plurality of wells (W) in which micromasses (M) are respectively arranged, an electrical stimulation unit 100 that provides electrical stimulation to the micromasses M, an image acquisition unit 200 that photographs the micromasses M arranged in the plurality of wells W at predetermined time intervals to acquire images, and a stimulus provision determination unit 300 that determines the provision of electrical stimulation based on the images. The stimulus provision determination unit 300 determines the provision of electrical stimulation based on the degree of aggregation of the micromasses M to which the electrode stimulation is applied.

[0032] The method for forming chondroprogenitor cell aggregates according to this embodiment includes an electrical stimulation providing step (S100) of providing electrical stimulation to micromasses arranged in a plurality of wells, an image acquiring step (S200) of photographing the micromasses at predetermined time intervals to acquire images, and a stimulation provision determining step (S300) of determining the provision of electrical stimulation from the images, wherein the stimulation provision determining step (S300) is determined based on the degree of aggregation of the micromasses to which the electrical stimulation has been provided.

[0033] Micromasses (M) are placed in the plurality of wells (W). In a subsequent process, an electrical stimulus is applied to the micromasses, causing them to aggregate and form chondroprogenitor cells. In one embodiment, the micromasses M may be either mesenchymal stem cells or fibroblasts, and the mesenchymal stem cells may be adipose-derived mesenchymal stem cells.

[0034] Micromasses Mf stained with a fluorescent substance can be placed in any one or more wells Wf. The fluorescently stained micromasses Mf emit fluorescence when exposed to light of a specific wavelength provided by a light source (not shown). A fluorescent image acquisition unit (not shown) can detect calcium oscillations generated from the fluorescently stained micromasses Mf, from which it can be determined that the micromasses Mf are functioning without damage and the state of the remaining micromasses M can be inferred. The fluorescent image acquisition unit detects calcium oscillations by acquiring an image for light of a wavelength band different from that detected by the image acquisition unit 200.

[0035] In one embodiment, calcium oscillation detection can be performed at least once within 6 to 40 hours after the electrical stimulus is provided, and preferably periodically within 6 to 40 hours after the electrical stimulus is provided.

[0036] As an example, the micromass Mf can be stained with a staining agent, and the light source can be a light source that provides light of a wavelength that can be absorbed by the stained micromass Mf to produce fluorescence.

[0037] An electrode (E) is placed in the well W, and the electrode E applies an electrical stimulus provided by the electrical stimulation unit 100 to the micromass M located in the well W. In addition, the electrode E can also be placed in the well Wf where the micromass Mf stained with the dye is located, and an electrical stimulus can be applied to the micromass Mf stained with the dye.

[0038] The electrical stimulation unit 100 applies an electrical stimulation to the micromass M through the electrodes E. The micromass M to which the electrical stimulation is applied aggregates into spheroid shapes adjacent to spheres.

[0039] 3 is a diagram illustrating an example of electrical stimulation provided by the electrical stimulation unit 100 through the electrodes E. In the example illustrated in FIG. 3, the electrical stimulation may be a pulsed current signal and / or voltage signal. In the example illustrated, the electrical stimulation may be a pulsed signal oscillating with different polarities, but this is merely an example, and the electrical stimulation may be a pulse oscillating in a single direction, either positive or negative.

[0040] As an example, the electrical stimulation may have a frequency greater than 0 and 20 Hz, may oscillate between -20 V and 20 V, and may have a duty cycle greater than 0 and 80% or less. The electrical stimulation may be provided continuously or intermittently for up to six days. However, these are all examples, and the scope of the present invention is not limited thereto.

[0041] 1 and 2, the micromass M aggregates into a spheroid shape when the electrical stimulation unit 100 provides electrical stimulation. In functionally intact micromass, calcium oscillation occurs, in which calcium ions flow in and out of the cell membrane. Therefore, if calcium oscillation can be observed, it can be seen that the micromass has aggregated into chondroprogenitor cells without functional damage.

[0042] The image acquisition unit 200 acquires an image by photographing the micromass M placed in the well W. As an example, the image acquisition unit 200 may include an optical system (not shown) and an imaging element (not shown) that converts an image formed by the optical system into an electrical signal, and images the micromass M located inside the well W. As an example, the optical system may include a lens such as a convex lens or a concave lens, and the imaging element may include at least one of a charge coupled device (CCD) and a CMOS image sensor (CIS). The image photographed and formed by the image acquisition unit 200 is provided to the stimulus provision determination unit 300.

[0043] The image acquiring unit 200 photographs the micromass M placed in the well W at predetermined time intervals to form an image, and provides the formed image to the stimulus provision determining unit 300. The image acquired by the image acquiring unit 200 may be an image that indicates the degree to which the micromass M placed in the well W has aggregated over time.

[0044] The stimulus provision determination unit 300 includes a neural network circuit that is capable of learning and performs inference based on the results of learning. The stimulus provision determination unit 300, including the trained neural network circuit, determines whether the electrical stimulation unit 100 should provide electrical stimulation based on the image provided by the image acquisition unit 200.

[0045] A method for training a neural network circuit, including a method for determining labels to be used in training, will now be described.

[0046] For training of the neural network circuit included in the stimulus provision determination unit 300, an expert (person) may observe the multiple images output by the image acquisition unit 200 in chronological order and assign a reference label having a specific value to a first image (=reference image), which is the first image among the multiple images that is determined to have the maximum amount of chondroprogenitor cells generated. As an example, the specific value of the reference label may be 1. In other words, the reference label may be associated with the image acquired by the image acquisition unit 200 when the amount of chondroprogenitor cells generated by electrical stimulation reaches its maximum.

[0047] The captured images may have characteristics due to the phenomenon described below. That is, the micromass M contained in the well before the application of the electrical stimulus may be uniformly distributed in the culture medium contained in the well. When the electrical stimulus is applied, the micromass M aggregates and changes into chondroprogenitor cells, and the chondroprogenitor cells thus generated form aggregates.

[0048] However, as the amount of chondroprogenitor cells produced increases and electrical stimulation is continued, the aggregated chondroprogenitor cells scatter again, ultimately resulting in a decrease in the amount of chondroprogenitor cells. This phenomenon will be explained in more detail with reference to Figure 4.

[0049] FIG. 4 shows, by modeling, an example of an image observed over time when an electrical stimulus is applied to a micromass M in a culture solution contained in a well W. The large circle shown in FIGS. 4(a) to 4(h) represents the shape of the outer edge of the well W when viewed from above, assuming that the well W has a circular frame. From FIGS. 4(a) to 4(h), the results of further application of electrical stimulus over time are shown. FIG. 4(a) represents the point at which electrical stimulus was initiated. The small circles shown in FIGS. 4(b) to 4(h) represent the shape of the outer edge of a chondroprogenitor cell aggregate generated by electrical stimulus. In reality, the shape of a chondroprogenitor cell aggregate may not be an exact circle, but can be modeled as approximately circular. The small circles shown in FIGS. 4(b) to 4(h) may contain a mixture of generated chondroprogenitor cells and micromass M. However, since the chondroprogenitor cells are densely packed, they can be considered as an aggregated region of chondroprogenitor cells.

[0050] Referring to Figure 4, Figure 4(a) shows the point at which electrical stimulation is applied, so no separate aggregates of chondroprogenitor cells are observed. From Figure 4(a) to Figure 4(e), it can be seen that the area of ​​the chondroprogenitor cell clusters gradually decreases with electrical stimulation. Even though the area of ​​the chondroprogenitor cell aggregates decreases, the amount of chondroprogenitor cells actually present within these aggregates gradually increases over time. Once the amount of chondroprogenitor cells reaches a maximum, as shown in Figure 4(e), no further generation of chondroprogenitor cells occurs even with the application of electrical stimulation, maintaining the maximum amount as shown in Figure 4(f).

[0051] However, if electrical stimulation is continued without interruption thereafter, the area of ​​the chondroprogenitor cell aggregates begins to increase again, as shown in Figures 4(g) and 4(h), and the amount of chondroprogenitor cells actually produced decreases. Therefore, in order to maximize the production of chondroprogenitor cells from the micromass M, it is necessary to stop the electrical stimulation provided to the micromass M at an appropriate time. In the embodiment illustrated in Figure 4, it is preferable to stop the current at the state shown in Figures 4(e) and 4(f). For example, stopping the current at the stage shown in Figure 4(e) has the advantages of maximizing the number of chondroprogenitor cells obtained, minimizing the time required to obtain chondroprogenitor cells from the micromass M, and reducing current consumption.

[0052] 1 and 2, a method for setting the value of the reference label described above will be described. The value of the reference label assigned to the reference image captured when the amount (number) of chondroprogenitor cells contained in the chondroprogenitor cell aggregate is substantially at its maximum value can be set to a specific value, for example, 1.

[0053] Here, the entity that determines that the production amount of chondroprogenitor cells has reached a maximum value is an expert (person). The production amount of chondroprogenitor cells corresponding to the maximum value can be referred to as a first production amount, a reference production amount, or a maximum production amount, and the reference production amount can be normalized to a specific value as described above. For example, the reference production amount can be presented as 1.

[0054] The stimulus provision determination unit 300 provided according to an embodiment of the present invention may assign a unique label value to each of the remaining images, excluding the reference image, among the plurality of images provided by the image acquisition unit 200, using an algorithm. The label value assigned to each of the remaining images may be equal to or less than a specific value of the reference label. For example, the label value assigned to any second image among the remaining images may be equal to or less than 1.

[0055] The stimulus provision determination unit 300 can determine label values ​​to be assigned to each of the remaining images based on the value of the reference label, the reference photographed image, and each photographed image. For example, if the production amount of chondroprogenitor cells estimated from the reference photographed image is defined as the reference production amount, the production amount of chondroprogenitor cells estimated from the second image is defined as the second production amount, and the label to be assigned to the second image is defined as the second label, then the relationship of Equation 1 is established.

[0056]

number

[0057] In Equation 1, the reference label value is a value that has already been assigned by an expert. For example, the value of the reference label may be 1. The reference production amount may be a value normalized based on a preset value. In one embodiment, the production amount of chondroprogenitor cells at the maximum production point of chondroprogenitor cells determined by an expert may be defined as 1, regardless of the specific value of the total number of chondroprogenitor cells generated. In addition, the second production amount is a value that can be determined by the method described below. Therefore, the value of the second label to be assigned to the second image can be determined as shown in Equation 2 below.

[0058]

number

[0059] By generalizing Equation 2, the value of the k-th label for the k-th image can be determined as shown in Equation 3 below.

[0060]

number

[0061] A method for estimating the amount of chondrogenic progenitor cells generated from each image can be presented as follows. First, the stimulation-providing determination unit 300 can identify the edge of the well W from the image. The well W can be a circular or square dish. At this time, the edge of the culture well W can be a circular closed curve as shown in FIG. 4 as an example.

[0062] Subsequently, the stimulation-providing determination unit 300 can classify the captured image of the micromass M existing in the well W into two regions according to a predetermined criterion. The two regions are shown in FIG. 4. Among the two regions, the first region (outer region, 110) can be a donut-shaped region having an outer closed curve 111 and an inner closed curve 112. The outer closed curve 111 can be the outer edge of the well W. And, among the two regions, the second region (inner region, 120) can be the region inside the inner closed curve 112. In one embodiment, the inner closed curve 112 can also be defined by modeling it into a circle or an ellipse.

[0063] Hereinafter, referring to FIG. 5, a method for determining an inner closed curve in an arbitrary image according to an embodiment of the present invention will be described. The method for determining the inner closed curve 112 in an arbitrary image can include the following steps. First, the stimulation-providing determination unit 300 can determine a path P1 connecting the center point 1120 of the well W from the first contour point 1110 which is the edge point of the well W in an arbitrary captured image. The path P1 can be a straight line or a curve. When the length of the path P1 is L1, a second point that has moved from the first contour point to the center point by dL can be defined.

[0064] In this way, the (k + 1)-th point that has moved from the k-th point to the center point by dL can be continuously defined (k is a natural number of 1 or more, dL < L1). A difference value Dk can be defined between the image attribute value at the k-th point and the image attribute value at the (k + 1)-th point. The image attribute value can be, for example, an attribute value such as brightness or chroma at each point of an arbitrary image. The image attribute value is sufficient according to the criterion for classifying the attributes of the image in the field of image processing, and the present invention is not limited by the selection of its specific attributes.

[0065] The largest value can be selected from the difference values ​​Dk (k=1, 2, 3, ...) between the image attribute values ​​defined in this way. For example, if the difference value D3 is the largest, it is considered that the image attribute value has changed the most between the third point 1131 and the fourth point 1141 on the path P1. From this, it can be determined that the first inner point 1121 constituting the inner closed curve 112 exists between the third point 1131 and the fourth point 1141.

[0066] The process of determining the first inner point 1121 begins with the step of selecting a first outer boundary point on the edge of the well W. By performing the process of determining the first inner point starting from various other outer boundary points on the edge of the well W, it is possible to determine a plurality of other inner points that constitute the inner closed curve 112. By connecting the plurality of inner points determined in this way through interpolation, it is possible to complete the inner closed curve 112. As a result, it is possible to define a first region (outer region, 110) and a second region (inner region, 120).

[0067] If the largest difference value Dk (k=1, 2, 3, ...) is smaller than a predetermined value, the first inner point 1121 can be determined to be the first outer point 1110. In some cases, the second region (inner region 120) and the first region (outer region 110) may not exist. This situation can occur, for example, when no electrical stimulation is applied to the micromass M. For example, in FIG. 4(a), the inner region 120 and the outer region 110 cannot be defined.

[0068] The method for determining the first inner point described above is presented as one of various algorithms for dividing a second region (inner region, 120) that approximates a circle or ellipse, and a first region (outer region, 110) that exists outside the second region (inner region, 120). That is, the above description is merely an explanation of the feasibility of dividing an image of the micromass M culture solution present in the well W into the two regions described above, and the present invention is not limited to such an embodiment.

[0069] The process of dividing the captured image into two regions is performed for each of the multiple images captured over time as electrical stimuli are applied and for each of the micromasses M contained in the multiple wells W. Once the second region (inner region, 120) is defined in this manner, the area SB of the second region (inner region, 120) can be calculated.

[0070] If the kth generation amount of chondroprogenitor cells estimated from the kth image is the kth generation amount, and the area of ​​the second region (inner region, 120) calculated from the kth image is the kth area, the kth generation amount of chondroprogenitor cells estimated from the kth image can be defined as being inversely proportional to the kth area. However, depending on the embodiment, the kth generation amount can be estimated based on the kth area, and a rule can be established in which a nonlinear function is involved in the relationship between the kth generation amount and the kth area. According to one aspect of the present invention, the kth generation amount is estimated based on the kth area, but the present invention is not limited to this specific functional relationship. Once the kth generation amount is estimated from the kth image in this way, the value of the kth label can be determined using Equation 3.

[0071] Labels assigned to a series of images of the micromass M in the well, whose state changes as an electrical stimulus is applied through the above-described process, can be defined or calculated and obtained.

[0072] The stimulus provision determination unit 300 can input the kth image to the input layer of the neural network circuit and update the neural network circuit so that the error between the value output by the neural network circuit and the kth label corresponding to the kth image is reduced.

[0073] In one embodiment, the neural network circuit may be, for example, a convolutional neural network (CNN). The neural network circuit can be repeatedly trained using multiple images and the labels generated for them. Furthermore, the neural network can be repeatedly trained using multiple images captured while applying electrical stimuli to micromasses M placed in multiple wells W and the corresponding labels.

[0074] In the neural network learning method described above, the learning input data for the neural network is the photographic data, and one feature of the present invention is the method of creating labels corresponding to the correct branch corresponding to each learning input data.

[0075] It is possible to determine the time point at which to cut off the current supplied to the micromass M culture solution contained in the well W. First, the electrical stimulation unit 100 applies a current to the well W containing the micromass M culture solution using a pair of electrodes E (S100). Next, the image acquisition unit 200 photographs the micromass M culture solution at predetermined time intervals to acquire images (S200) and transmits them to the stimulus provision determination unit 300. The stimulus provision determination unit 300 inputs each of the transmitted photographed images to the input layer of the neural network circuit of the stimulus provision determination unit 300.

[0076] The stimulus provision determination unit 300 determines whether the electrical stimulation unit 100 should provide electrical stimulation (S300) and outputs a control signal to control the electrical stimulation unit 100. The electrical stimulation unit 100 provides electrical stimulation or terminates the provision of electrical stimulation in response to the control signal output by the stimulus provision determination unit 300. For example, the stimulus provision determination unit 300 may provide the control signal by terminating the application of current when it is determined that the label value determined from the provided image will no longer increase. To this end, for example, the stimulus provision determination unit 300 may monitor the moving average value of the label value output by the neural network circuit and provide a signal to terminate the provision of electrical stimulation when the moving average value begins to decrease or when it saturates for a certain period of time and no longer increases.

[0077] In another embodiment, the stimulus provision determination unit 300 can output a label value that is a scalar value. The electrical stimulation unit 100 can determine whether to terminate the provision of electrical stimulation based on a time history of a series of output values ​​output by the stimulus provision determination unit 300.

[0078] For example, the electrical stimulation unit 100 may determine to terminate application of current at the moment when it is determined that the output value output by the stimulation provision determination unit 300 will no longer increase. To this end, for example, the driving current control unit 30 may observe the moving average value of the output value output by the estimation unit 50 and determine to terminate application of current at the point when the moving average value begins to decrease or when it saturates for a certain period of time and no longer increases.

[0079] As described above, a method for cutting off the electrical stimulation provided to the micromass M culture solution by the drive current control unit 30 will be described with reference to FIG.

[0080] 6 is an example of a graph showing values ​​output by an estimation unit of an electrical stimulation system according to an embodiment of the present invention over time. The graph in FIG. 6 may directly show the original values ​​output by the estimation unit 50, or may show the original values ​​averaged over time.

[0081] In Figure 6, the label value calculated by the neural network circuit increases monotonically in the interval t0 to t1, remains constant in the interval t1 to t2, and decreases in the interval t2 onwards. In some cases, the time interval t1 to t2 may not actually exist, and the label value may begin to decrease immediately after t1.

[0082] When observing the curve shown in Figure 6 in real time over time, ideally the electrical stimulation should be commanded to end at time t1. However, since a certain time delay is required to observe the label value calculated by the neural network circuit and determine whether the amount of chondroprogenitor cells produced has reached its maximum, in reality the electrical stimulation may be commanded to end after time t1.

[0083] Although the present invention has been described with reference to the embodiments shown in the drawings to facilitate understanding of the present invention, these are merely illustrative examples for implementation, and those skilled in the art will recognize that various modifications and equivalent embodiments are possible. Therefore, the true technical scope of the present invention should be determined by the appended claims.

Claims

1. 1. A device for forming chondroprogenitor cell aggregates, comprising: The device comprises: a plurality of wells each having a micromass disposed therein; an electrical stimulation unit that provides electrical stimulation to the micromass; an image acquisition unit that acquires images by photographing the micromasses arranged in the plurality of wells at predetermined time intervals; a stimulus provision determination unit that determines provision of the electrical stimulus based on the image; Including, The stimulus provision determination unit is an apparatus that determines the provision of the electrical stimulus based on the degree of aggregation of the micromass to which the electrical stimulus is provided.

2. the micromass is one of mesenchymal stem cells and fibroblasts; The device according to claim 1 , wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

3. The electrical stimulation a frequency greater than 0 and less than or equal to 20 Hz; An amplitude of -20V or more and 20V or less; 2. The device of claim 1, wherein the duty ratio is greater than 0 and less than or equal to 80%.

4. The device comprises:

2. The device of claim 1, wherein the electrical stimulation is provided to the micromass continuously or intermittently for up to six days.

5. The device comprises: a well in which the micromass stained with a fluorescent substance is located; a light source that provides light so that the dyed micromass fluoresces; a fluorescent image acquisition unit that acquires an image by photographing the micromass stained with the fluorescent substance; Further comprising: The device according to claim 1 , wherein the electrical stimulation unit further provides an electrical stimulation to the micromass stained with the fluorescent substance.

6. The fluorescent image acquisition unit After the electrical stimulus is provided, 6. The apparatus according to claim 5, wherein an image of calcium oscillation occurring in the micromass stained with the fluorescent substance is acquired within 40 hours after 6 hours.

7. 1. A method of forming chondroprogenitor cell aggregates carried out by an apparatus, comprising: The method comprises: providing an electrical stimulus to micromasses disposed in a plurality of wells; acquiring images by photographing the micromass at predetermined time intervals; determining from the image whether to provide the electrical stimulus; Including, In the step of determining whether to provide the electrical stimulus, the method determines whether to provide the electrical stimulus based on the degree of aggregation of the micromass to which the electrical stimulus is provided.

8. the micromass is one of mesenchymal stem cells and fibroblasts; The method according to claim 7, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

9. The electrical stimulation a frequency greater than 0 and less than or equal to 20 Hz; An amplitude of -20V or more and 20V or less; 8. The method of claim 7, wherein the duty ratio is greater than 0 and less than or equal to 80%.

10. In the step of providing the electrical stimulation, 8. The method of claim 7, wherein the electrical stimulation is provided to the micromass continuously or intermittently for up to six days.

11. The method comprises: providing light to a well in which the fluorescently stained micromass is located so that the stained micromass emits fluorescence; acquiring a fluorescent image by photographing the micromass stained with the fluorescent substance; Further comprising:

8. The method of claim 7, wherein the fluorescently stained micromass is provided with an electrical stimulus.

12. In the step of acquiring the fluorescent image, After the electrical stimulus is provided, The method of claim 11, wherein an image of calcium oscillation occurring in the micromass stained with the fluorescent substance is acquired within 6 hours to 40 hours.

13. 8. The method of claim 7, wherein the step of acquiring the images occurs in parallel with the step of providing the electrical stimulus.

Images