KR20230057983A - Method of managing cell culture fluid using cell image processing technology and automatic cell incubator using the same - Google Patents

Abstract

The present invention relates to a cell culture medium management method through cell image image processing capable of maintaining the cell culture medium in an optimal state by identifying the density of cells, the number and size of clusters, etc. using image information obtained by photographing the cell culture medium, and an automatic method using the same It relates to a cell cultivator, and more particularly, image information acquisition step of acquiring image information by photographing the cell culture medium through an electron microscope; a histogram smoothing step for clearly distinguishing the background, single cells, and clusters included in the image information; In the image information through the histogram smoothing step, the highest value of the three RGB values for each pixel is converted to the maximum value, and the other two values are converted to the minimum value to more clearly distinguish the background, single cell, and cluster area separation step; a morphology calculation step of removing noise from image information through the region separation step and restoring the shape of the cluster; and a cluster detection step of detecting the outline of the cluster based on the binary image through the morphology calculation step and detecting the area of the cluster through the number of pixels inside the outline. It relates to a method and an automatic cell culture machine using the same.

Description

Cell culture medium management method through cell image image processing and automatic cell culture device using the same

The present invention relates to a cell culture solution management method through cell image image processing and an automatic cell culture device using the same. In particular, the cell culture solution by identifying the density of cells, the number and size of clusters, etc. using image information obtained by photographing the cell culture solution. It relates to a method for managing a cell culture medium through image processing of a cell image capable of maintaining a cell in an optimal state and an automatic cell culture device using the same.

In general, suspension cells such as NK cells do not grow by attaching to a certain surface, but float and grow. At this time, aggregation (cluster) occurs to facilitate interaction with each other while exchanging various factors necessary for growth. .

In particular, in the case of immune cells, it is known that clusters should form with each other at the beginning of culture so that interactions occur and grow well, and clusters tend to increase in size over time during in vitro cell culture.

Although this aggregation phenomenon plays an important role in the growth of cells, if this aggregation phenomenon occurs excessively and the size of the cluster increases, it is difficult to exchange nutrients and oxygen, which may limit growth. That is, controlling the size of clusters is very important in culturing floating cells.

On the other hand, it is known that the growth by interaction in the initial stage of culture is good, so it is common to culture while initially culturing in a small-capacity flask and then transferring to a large-capacity flask.

Therefore, until now, the size of the cluster has been periodically observed to check the culture state of the cells, and if the size of the cluster exceeds a certain standard value, the cluster is released through agitation. Clusters and cells are observed and the criteria are followed even during the work of moving the cells.

However, since the researcher determines the cell culture state by making a judgment based on the microscopic image during cell culture, there is a problem with the reproducibility of the experiment because the standard is different for each researcher. During the culturing period, continuous observation by the researcher is required.

Republic of Korea Patent No. 10-1679602 (November 21, 2016)

The technical problem to be solved by the present invention is to detect information such as the density of cells and the size and number of clusters by using image processing technology based on images taken with an electron microscope to solve the above-mentioned problems, An object of the present invention is to provide a method for managing a cell culture medium through image processing of a cell image capable of automatically managing a cell culture medium and an automatic cell culturing device using the same.

Another technical problem to be solved by the present invention is a cell culture solution through cell image image processing that expands the culture area of the culture vessel by using a roller that presses the top of the culture bag while moving the top of the culture bag located in a closed culture device. It is to provide a management method and an automatic cell cultivator using the same.

Another technical problem to be solved by the present invention is to install a culture bag on top of a closed incubator, install a roller module that can adjust the culture area of the culture bag according to the culture condition of cells on the top of the culture bag, and install a transparent material The culture at the bottom of the transparent panel using an inverted electron microscope mounted on a 3-dimensional stage that can move in three dimensions at the bottom of the culture bag located on the top of the panel of An object of the present invention is to provide a method for managing a cell culture medium through image processing of a cell image capable of inspecting the culture state of a bag and an automatic cell culture device using the same.

The technical problems to be achieved in the present invention are not limited to the above-mentioned technical problems, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below. You will be able to.

A cell culture medium management method through cell image image processing according to an embodiment of the present invention for achieving the above technical problem includes an image information acquisition step of acquiring image information by photographing a cell culture medium through an electron microscope; a histogram smoothing step for clearly distinguishing the background, single cells, and clusters included in the image information; In the image information through the histogram smoothing step, the highest value of the three RGB values for each pixel is converted to the maximum value, and the other two values are converted to the minimum value to more clearly distinguish the background, single cell, and cluster area separation step; a morphology calculation step of removing noise from image information through the region separation step and restoring the shape of the cluster; and a cluster detection step of detecting the outer line of the cluster based on the binary image through the morphology calculation step and detecting the area of the cluster through the number of pixels inside the outer line.

In this case, the method may further include a shaking step of shaking the cell culture medium when the area of one of the clusters detected through the cluster detecting step is greater than or equal to a reference value or the number of clusters is greater than or equal to a reference value.

In addition, in the image information acquisition step, image information is acquired by photographing a part of the cell culture medium through an electron microscope, and when the area of the cluster detected through the cluster detection step is less than a reference value, but the number of clusters is greater than or equal to the reference value. In this case, it is characterized in that the above-described steps are performed after acquiring image information by photographing another part of the cell culture medium.

In addition, a cell culture medium management method through cell image image processing according to another embodiment of the present invention includes an image information acquisition step of acquiring image information by photographing a cell culture medium through an electron microscope; a histogram smoothing step for clearly distinguishing the background, single cells, and clusters included in the image information; In the image information through the histogram smoothing step, the highest value of the three RGB values for each pixel is converted to the maximum value, and the other two values are converted to the minimum value to more clearly distinguish the background, single cell, and cluster area separation step; a cell density detection step of detecting a density of cells by obtaining a ratio of the number of pixels occupied by the background, single cells, and clusters to the total number of pixels of the video image from the image information obtained in the region separation step; and a cell culture medium adjusting step of increasing the amount of the cell culture medium when the cell density through the cell density detecting step is greater than or equal to a reference value.

At this time, in the cell density detection step, when counting the cells inside the cluster, the brightness value from 0 to 255 of one pixel is divided into a plurality of ranges, and the weight value corresponding to the divided range is multiplied to determine the number of cells inside the cluster. characterized by counting cells.

On the other hand, it is characterized by providing an automatic cell culture machine using the cell culture medium management method through the above-described cell image image processing.

In addition, the automatic cell culturing machine according to the present invention includes a culture medium storage container; a transparent culture bag made of a flexible material in which the set initial cell culture medium is accommodated and the culture medium is supplied from the culture medium reservoir; a tube pump for adjusting the supply amount of the culture solution supplied from the culture solution reservoir to the culture bag by adjusting the pressure of the culture solution reservoir and the tube connected to the culture bag; A transparent plate to which the culture bag is coupled to an upper portion; A roller module controlling the capacity of the culture bag while moving in close contact with the transparent plate with the culture bag interposed at the top of the transparent plate; a shaking module connected to the transparent plate to shake the transparent plate; an electron microscope movably disposed below the transparent plate and photographing the cell culture medium inside the culture bag; and a controller module controlling the tube pump, the roller module, the shaking module, and the electron microscope, wherein the controller module transfers image information obtained through the electron microscope to a cell culture medium management method through image processing of the cell image. and controlling operation states of the tube pump, the roller module, and the shaking module based on the processed result.

The above aspects of the present invention are only some of the preferred embodiments of the present invention, and various embodiments reflecting the technical features of the present invention are detailed descriptions of the present invention to be detailed below by those of ordinary skill in the art. It can be derived and understood based on.

The cell culture medium management method through cell image image processing and the automatic cell cultivator using the method according to the present invention described above have the following effects.

First, it is possible to improve the reproducibility and detection accuracy of the experiment, and by minimizing human intervention, it is possible to derive the same high-quality experimental results even without skilled technicians.

In addition, the production cost can be lowered in various aspects, such as the reduction of labor costs and contamination sources consumed when culturing natural killer (NK) cells, and in addition, it can be reborn as a treatment that is more accessible to cancer patients, thereby reducing the trend of cancer deaths, which are increasing every year. There are advantages to lowering it.

In addition, the culture area of the culture bag located at the bottom can be automatically adjusted by using the pressure and movement of the roller located at the top, and since it is installed in a sealed case, it has the advantage of minimizing the influence of external contaminants. .

In addition, since a roller module for controlling the culture area is installed at the top of the culture bag installed in the sealed interior and the cell culture state of the culture bag can be captured as an image at the bottom, even in a state where the culture bag is not leaked out of the closed incubator case. It has the advantage of being able to check the cell culture state.

The effects obtainable in the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the description below. will be.

1 is a diagram according to an embodiment of a cell culture medium management method through cell image image processing according to the present invention;
2 is a picture of an image processing image according to the present invention;
3 is a view according to an embodiment of a cell culture medium management method through cell image image processing according to the present invention;
4 is a diagram showing a pixel value distribution graph according to each channel of a video information image;
5 is a diagram showing a pixel value distribution graph after histogram smoothing for each channel;
6 is a diagram showing a method for estimating the number of cells in consideration of the Z-axis of a cluster;
7 is a circular cluster image detection and center region display image;
8 is an image showing cell counting;
9 is a view showing the appearance of an automatic cell culture device according to the present invention;
10 is a view showing the configuration of an automatic cell culture device according to the present invention;
11 is a view showing the inside of the solution case;
12 is a view according to an embodiment of a syringe pump module installed inside a solution case;
13 is a view according to an embodiment of a tube pump;
14 is a diagram explaining a Peltier module and the operating principle of the Peltier module;
15 is a diagram according to an embodiment of an internal module;
16 is a view showing a roller module;
17 is a diagram according to an embodiment of a 3D stage;
18 is a view explaining the operating principle of the internal air circulation fan;
19 is a view according to an embodiment of a cell culture method according to the present invention;
20 is a view according to an embodiment of an apparatus for automatically adjusting a culture area of a culture bag according to the present invention;
21 is a view explaining a situation in which the culture area of the culture bag is varied using a roller module;
Figure 22 is a view of the roller installed in the culture case from one point on the top;
Figure 23 is a view of the roller installed in the culture case from another point on the top;
24 is an enlarged view of the edge of the roller frame;
25 is a view according to one embodiment of a roller bracket;
26 is an external structural diagram of an automatic cell culturing apparatus using an inverted electron microscope according to the present invention;
27 is a view according to an embodiment of the configuration of an automatic cell culture system using an inverted electron microscope according to the present invention;
28 is a view of the internal configuration of an automatic cell culturing machine using an inverted electron microscope according to the present invention from one side of the upper side;
29 is a view of the internal configuration of an automatic cell culturing machine using an inverted electron microscope according to the present invention from another upper side;
30 is a view of the arrangement of an inverted electron microscope and a culture bag as seen from one side of the upper part;
31 is a view of the arrangement of an inverted electron microscope and a culture bag as seen from another side of the upper part;
Fig. 32 is a view for explaining an example of a cultured state of cells taken with an inverted electron microscope;

Since the present invention can make various changes and have various embodiments, specific embodiments are illustrated and described in the drawings. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention.

Terms including ordinal numbers such as second and first may be used to describe various components, but the components are not limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a second element may be termed a first element, and similarly, a first element may be termed a second element, without departing from the scope of the present invention. The terms and/or include any combination of a plurality of related recited items or any of a plurality of related recited items.

It is understood that when an element is referred to as being "connected" or "connected" to another element, it may be directly connected or connected to the other element, but other elements may exist in the middle. It should be. On the other hand, when an element is referred to as “directly connected” or “directly connected” to another element, it should be understood that no other element exists in the middle.

In the description of the embodiments, each layer (film), region, pattern or structure is "on" or "under" the substrate, each layer (film), region, pad or pattern. The substrate formed on includes all those formed directly or through another layer. The criteria for upper/upper or lower/lower of each layer will be described based on drawings. In addition, since the thickness or size of each layer (film), region, pattern, or structure in the drawing may be modified for clarity and convenience of description, it does not entirely reflect the actual size.

Terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

Hereinafter, the present invention will be described in detail by describing preferred embodiments of the present invention with reference to the accompanying drawings. Like reference numerals in each figure indicate like elements.

1 is a diagram according to an embodiment of a cell culture medium management method through cell image image processing according to the present invention, FIG. 2 is a picture of an image processing image according to the present invention, and FIG. 3 is a cell image image processing according to the present invention. 1 can be seen as a cell culture management method through cell image image processing for detecting the size and number of clusters inside the cell culture medium, and FIG. It can be seen as a cell culture medium management method through cell image image processing for detecting the density of cells inside the culture medium.

In addition, the cell culture medium management method through cell image image processing according to the present invention can be regarded as being performed by a controller module in an automatic cell cultivator, which will be described later.

First, referring to FIGS. 1 and 2, the cell culture medium management method through cell image image processing according to the present invention includes an image information acquisition step (S10), a histogram smoothing step (S20), a region separation step (S30), and a morphology calculation step. (S40) and a cluster detection step (S50) may be included, and a shaking step (S60) may be further included.

The image information acquisition step (S) is a step of acquiring image information by photographing the cell culture medium through an electron microscope. First, it is possible to obtain a microscopy image, that is, image information, which is stored in real time by taking a picture with a digital (electronic) microscope installed in an internal module during the cell culture process.

In this way, image information is composed of a total of three channels of RGB (Red, Green, Blue), and can be expressed as a three-dimensional matrix when expressed as a matrix.

In addition, image information obtained through an electron microscope is not easy to classify because the color of the background, single cells (cells that do not form clusters), and clusters are similar. Here, the background can be viewed as a culture medium, and the culture medium may consist of a small amount of reagents, blood plasma, and a medium solution constituting most of the culture medium.

The histogram equalization step (S20) is a step for clearly distinguishing the background, single cells, and clusters included in the image information, and clearly distinguishes the background, single cells, and clusters in the image information (original image). In order to do this, each channel (R, G, B) is separated and histogram smoothing is performed.

4 is a diagram showing a pixel value distribution graph according to each channel of a video information image, and each curve represents the distribution of B, G, and R channels according to color.

5 is a diagram showing a pixel value distribution graph after histogram smoothing for each channel.

[Equation 1] and [Equation 2] are applied to each channel to perform histogram equalization and maximize the contrast ratio, and the distribution of pixel values spreads over the range of 0 to 255 as shown in FIG. 5 .

In [Equation 1] and [Equation 2], i = position value of a pixel, hist[j] = frequency of contrast values, and N = total number of pixels of an image.

Then, when each smoothed channel (three two-dimensional matrices) is merged and the image is displayed, the image is expressed as the Histogram Equalization in FIG. 2, and the background (B) and cluster (G) , single cells (R) appear distinctly for each color.

In the zone separation step (S30), the highest value among the three RGB values for each pixel in the image information through the histogram equalization step (S20) is converted to the maximum value, and the remaining two values are converted to the minimum value, and the background , It is a step to more clearly distinguish between single cells and clusters.

In the region separation step (S30), the channel value having the largest value for each pixel may be converted to '255' and the remaining channel values to '0' to increase convenience in image processing. For example, since an image consists of three channels (BGR), when one pixel value is checked, it has a total of three values (B, G, R) such as (183, 23, 54). Therefore, if the first factor (B) channel with the highest value is converted to '255' and the remaining two factor values are converted to '0' and expressed as (255, 0, 0), a more distinct blue color (in this case, the background area ) image can be obtained. Through this operation, the Zone Separation image of FIG. 2 can be obtained, and it can be seen that the division of each region becomes more clear when compared with the previous Histogram Equalization image.

Afterwards, if the G (Green) channel corresponding to the cluster area is separately separated to extract the cluster area, it appears as a two-dimensional matrix consisting of '0' and '255'. Pixels excluding the cluster area (pixel value 255) are displayed in black (pixel value 0).

The morphology calculation step (S40) is a step of removing noise from image information through a region separation step and restoring the shape of the cluster, and morphology calculation (erosion and dilation) may be performed to remove noise included in the image. That is, noise is removed in the erosion operation, and then an image such as noise removal and redetection in FIG. 2 can be obtained by restoring the shape of the existing cluster eroded through the expansion operation.

The cluster contour detection step (S50) is a step of detecting the outline of the cluster based on the binary image through the morphology calculation step (S40) and detecting the area of the cluster through the number of pixels inside the outline. .

The shaking step (S60) is a step of shaking the cell culture medium when the area of one of the clusters detected through the cluster detection step is greater than or equal to a reference value or the number of clusters is greater than or equal to a reference value.

That is, the outline can be detected based on the binary image on which the morphology operation has been performed as described above. When the outline is detected, the number of outlines (closed curves) included in the image and the internal area (number of pixels) of each outline can be detected. At this time, if the area of the largest cluster is greater than or equal to the shaking operation reference value α, the shaking operation may be performed.

On the other hand, in the image information acquisition step (S10), image information is obtained by photographing a part of the cell culture medium through an electron microscope, and the area of the cluster detected through the cluster detection step is less than the reference value, but the number of the clusters is greater than the reference value. In this case, the above-described steps may be performed after acquiring image information by photographing another part of the cell culture medium.

That is, if the area of the largest cluster is less than the reference value, but the number of clusters is greater than the criterion, it is considered that the growth of the cells has progressed considerably, and since the growth of the cluster is expected, move the microscope to another point and retake , the above operation can be repeated.

Referring to FIG. 3, the cell culture medium management method through cell image image processing according to the present invention includes image information acquisition step (S10), histogram smoothing step (S20), region separation step (S30), cell density detection step (S70), and cell It may be configured including a culture medium control step (S80).

Descriptions of the image information acquisition step (S10), the histogram smoothing step (S20), and the region separation step (S30) may refer to the above description.

6 is a diagram showing a method for estimating the number of cells in consideration of the Z-axis of a cluster, FIG. 7 is an image of detecting a circular cluster image and displaying a central region, and FIG. 8 is an image showing cell counting.

The cell density detection step (S70) is a step of detecting the cell density by obtaining the ratio of the number of pixels occupied by the background, single cells, and clusters to the total number of pixels of the video image from the image information obtained in the above-described region separation step (S30). .

The cell culture medium adjusting step (S80) is a step of increasing the amount of the cell culture medium according to a predetermined value when the cell density through the cell density detecting step (S70) is greater than or equal to the reference value.

That is, in the region separation step (S30), the density of clusters and the density of single cells can be detected through the ratio of the number of pixels corresponding to each region to the total number of image pixels (N) as follows.

1) Density of cluster = number of cluster area pixels (number of pixels with 255 pixel value in G channel)/total number of pixels (N)

2) Single cell density = number of pixels in single cell area (number of pixels with 255 pixel value in R channel)/number of total pixels (N)

3) Proportion occupied by cells = cluster density + single cell density

Accordingly, based on the ratio occupied by the cells, if the total occupancy ratio is greater than or equal to the reference value, the roller module to be described later may be operated to expand the culture area and maintain the cell density below the reference value.

At this time, in the cell density detection step (S70), when counting the cells inside the cluster, the brightness value of one pixel from 0 to 255 is divided into a plurality of ranges, and the weight value corresponding to the divided range is multiplied to obtain the inside of the cluster. of cells can be counted.

That is, from the image information obtained through the electron microscope, only a plane image (2D image) based on the x-axis and the y-axis can be obtained. Therefore, since information on the z-axis is unknown, a mapping table for this can be created.

Although the shape of the cluster is diverse, it can be assumed that the shape of the cluster is closer to a circle when observing a 2D image, and the cell pixel group with the darkest pixel is in the center. Therefore, it can be determined that the length of the z-axis is equal to the diameter of the cluster observed in the 2D image.

Therefore, as shown in FIG. 7, based on the circular cluster in the 2D image, the darkest pixel value (region indicated in red in FIG. 7) located in the center of the cluster and the diameter of the cluster (outline length / π) are mapped. Tables can be configured like '7-1' and '7-2' in 6.

'7-2' of '7-1' in FIG. 6 is a simplified example of mapping table information. If data is collected based on a spherical cluster, the If 3 cells are included in the z-axis (diameter of the cluster) when a pixel value in the range of step 1 to step 7 is detected, the weight of each range can be set to 3. In this case, the weight may represent the number of cells included in the corresponding diameter.

The mapping table may include the length of the z-axis (the diameter of the sphere), the brightness (pixel value) of the cluster according to the light transmittance when the diameter has the corresponding diameter, and information on the number of cells that can be included in the corresponding diameter. The number of cells that can be included in the diameter is based on the size (A) of the cells detected in the cell counting inside the cluster in FIG. /diameter of one cell).

In addition, the mapping table-based cell counting method inside the atypical cluster is as follows.

The number of cells inside an atypical cluster can be detected by estimating the z-axis length using the brightness (pixel value) inside the cluster based on the data collected from the spherical cluster.

That is, a pixel group having an area of the representative cell size (A) is formed, and the z-axis length corresponding to the pixel value and the number of cells included in the z-axis length are calculated using the average pixel value of each group and the mapping table. It is possible to detect the number of cells as much as the area of each pixel group.

In 7-1 of FIG. 6, 400x400 is a simplified representation of one cluster, and the cluster is composed of cells having a cell size (A) of 100x100. Accordingly, the cell cluster region is divided into 100×100 areas, the average pixel value is calculated for each region, and the cell cluster region can be classified into a total of 7 stages as shown in 7-2 of FIG. 6 according to the range of pixel values.

7 levels, for example, 0~35, 36~71, 72~107, 108~143, 144~179, 180~215, 216~255, each pixel area classified into 7 levels is an area of cell size (A). Since it is divided, assuming that it is one cell on the 2D image, and counting the cells by considering the 3D z-axis, the number of cells can be detected by multiplying the weight (the number of cells included in the diameter) included in the mapping table.

7-3 of FIG. 6 shows the number of pixel regions for each step, each step has a corresponding weight, and the total number of cells can be detected by multiplying the weight by the number of pixel regions for each step.

Meanwhile, FIG. 8 shows an image according to a cell counting algorithm. When counting single cells, clusters are detected and cells inside the clusters are counted to estimate the number of single cells.

That is, the detection of clusters may proceed with cell counting based on cluster area information detected in the cluster detection step described above.

In addition, for the cell count inside the cluster, histogram smoothing may be performed using only pixels corresponding to the cluster region by utilizing the pixel index corresponding to the cluster region detected in the cluster estimation and extraction of FIG. 2 .

In addition, when the contrast ratio is maximized for the inner region of the cluster, it is possible to detect the inner cells because cells clustered inside the cluster are clearly distinguished.

In addition, individual cells inside the cluster are detected in the cluster area where histogram smoothing has been performed, outlines for individual cells are extracted, and the size of each cell constituting the cluster can be listed by detecting the size inside each outline.

For example, if the number of outlines (closed curves) detected inside cells is 11, a list such as [5, 9, 16, 13, 8, 10, 12, 9, 14, 18, 6] is constructed, and the size list The median value of is extracted, and the extracted median value can be determined as the representative size (A) of an individual cell (one cell).

After that, when each size is reordered in the above list, the median value in [5, 6, 8, 9, 9, 10, 12, 13, 14, 16, 18] corresponds to 10 in the middle of the list, and the cell size 10 (10 pixels inside the outline) can be judged as a representative size of one cell.

In addition, when estimating the number of single cells, the number of cells can be derived by dividing the area (number of pixels marked in red) of single-region cells (indicated by R) by 'A' in the above-described region separation step (S30).

According to the present invention, an automatic cell culture device can be provided using the cell culture medium management method through cell image image processing described above.

The automatic cell culturing machine according to the present invention includes a culture medium storage container; a transparent culture bag made of a flexible material in which the set initial cell culture medium is accommodated and the culture medium is supplied from the culture medium reservoir; a tube pump for adjusting the supply amount of the culture solution supplied from the culture solution reservoir to the culture bag by adjusting the pressure of the culture solution reservoir and the tube connected to the culture bag; A transparent plate to which the culture bag is coupled to an upper portion; A roller module controlling the capacity of the culture bag while moving in close contact with the transparent plate with the culture bag interposed at the top of the transparent plate; a shaking module connected to the transparent plate to shake the transparent plate; an electron microscope movably disposed below the transparent plate and photographing the cell culture medium inside the culture bag; and a controller module controlling the tube pump, the roller module, the shaking module, and the electron microscope.

The controller module processes the image information obtained through the electron microscope based on the cell culture medium management method through the cell image image processing, and based on the processed result, the operation of the tube pump, the roller module, and the shaking module state can be controlled.

Hereinafter, an automatic cell culture device according to the present invention will be described with reference to the drawings.

9 shows an external appearance of an automatic cell culturing device using the image processing technology according to the present invention.

Referring to FIG. 9 , the automatic cell culture device 100 utilizing the image processing technology according to the present invention is sealed by a solution case 10 and an external case 20, and an external case 20 in contact with the solution case 10 ) The controller module 140 is installed in the upper part of the inside.

The solution case 10 and the outer case 20 are made of stainless steel, and it is preferable to additionally use a heat insulating material inside the solution case 10 .

10 is an example of the configuration of an automatic cell culturing device using the image processing technology according to the present invention.

Referring to FIG. 10 , an automatic cell cultivator (100, hereinafter referred to as an automatic cell cultivator) utilizing image processing technology includes a syringe pump module 110, a tube pump 120, a peltier module 130, and a solution case 10. It can be seen that the culture medium reservoir 135 is built in, and the controller module 140, the inner module 150, and the inner air circulation fan 160 are built in the outer case 20.

The syringe pump module 110 includes a syringe 112 . The tube pump 120 adjusts the pressure of the middle portion of the tube 121 connecting the culture medium reservoir 135 and the culture bag 153, thereby transferring the cell culture medium from the culture medium reservoir 135 to the inside of the culture bag 153. and adjusting the amount of the drug. The Peltier module 130 controls the internal temperature of the solution case 10 .

The controller module 140 includes a syringe pump module 110, a tube pump 120, a peltier module 130, and a shaking module 153 constituting the internal module 150, a roller module 155, an electron microscope 157 ) and the operation of the 3D stage 156, the cell culture state is determined using the image information of the cells being cultured in the culture bag 153 obtained through the electron microscope 157, and the external case ( 20), a control device 141 for controlling carbon dioxide (not shown) and a heat source (not shown) supplied to the inside, mounted on the outer surface of the outer case and inputting user instructions to the control device 141 or automatic cell cultivator ( 100) and a touch screen 142 displaying the status and a timer 143. The timer 143 is used when performing an automatic cell culturing method to be described later.

The culture bag 153 positioned on top of the transparent plate 152 uses the cell culture medium and chemicals supplied through the tube 121 to culture the cells already introduced therein. The shaking module 154 shakes the transparent plate 152, and the roller module 155 reciprocates while applying pressure to the top of the culture bag 153 to adjust the cell culture area in the culture bag 153. The 3-dimensional stage 156 moves the mounted electron microscope 157 in 3 dimensions, and the internal air circulation fan 160 filters contaminants contained in the air inside the external case 20 while filtering the external case 20. circulate the air inside.

The solution case 10 and the outer case 20 each have a hole (not shown) through which the tube 121 passes.

Hereinafter, components and functions of the automatic cell culturing device 100 will be described.

11 shows the inside of the solution case.

Referring to FIG. 11 , it can be seen that a syringe pump module 110, a tube pump 120, and a Peltier module 130 are built into the solution case 10.

12 is an example of a syringe pump module installed inside a solution case.

Referring to FIG. 12 , the syringe pump module 110 includes a syringe frame 111 and a syringe 112 . When the pump 114 fixed to the syringe frame 111 is activated, the amount of the culture solution flowing into or discharged from the syringe 112 can be controlled by the push rod 113 moving. The culture solution of the syringe 112 flows out through the tube 121.

13 is an embodiment of a tube pump.

Referring to FIG. 13, the tube pump 120 applies pressure to the outer surface of the tube 121 passing through the inside, thereby increasing the amount of culture solution supplied from the syringe module 110 to the culture bag 153 using the tube 121. Adjust the Since both the amount of the culture medium and the injection of the culture medium can be automatically performed, it is possible to fundamentally block the inflow of contaminants, which occurred in conventional devices that were manually performed. The supply amount can be adjusted by finely adjusting a motor (not shown) that operates the tube pump 120 .

14 explains the Peltier module and the operating principle of the Peltier module.

Referring to FIG. 14, the Peltier module 130 is installed in the solution case 10 to cool the internal air of the solution case 10, thereby maintaining the preservation temperature of the culture medium located inside the solution case 10. let it be

15 is an embodiment of an internal module.

Referring to FIG. 15, the internal module 150 includes a transparent plate 152 (not shown), a culture bag 153, a shaking module 154, a roller module 155, a 3D stage 156, and an electron microscope 157 ), which are all fixed to the frame 151.

The culture bag 153 is located on top of the transparent plate 152, and the shaking module 154 shakes the transparent plate 152 so that the cells are evenly cultured inside the culture bag 153. The shaking module 154 uses a barbell gear (not shown) operated by a motor (not shown) to shake the transparent plate 152 using various rotational forces of the motor.

16 shows the roller module in detail.

Referring to FIG. 16, the roller module 155 is a guide rail 155-1 using guide rails 155-1 installed on both sides of the culture bag 153 and roller brackets 155-3 installed on both ends. It includes a roller 155-4 fixed to both ends of the roller frame 155-2 and the roller bracket 155-3 moving along the inside. It is preferable that the surface of the roller 155-4 moves from one side of the culture bag 153 to the other side, and the cell culture area increases as the movement is made.

17 is an example of a 3D stage.

Referring to FIG. 17 , since the 3D stage 156 can move in the x-y direction and the x-z direction, it can eventually be moved to a desired location in the 3-dimensional space inside the internal module 150.

Although not shown in detail for simplification of the drawing, by installing the electron microscope 157 on the upper part of the 3D stage 156, as shown in FIG. It is preferable to take an image in an upward direction from the .

18 explains the operating principle of the internal air circulation fan.

Referring to FIG. 18 , an internal air circulation fan 160 preferably fixed to the frame 151 circulates the internal air of the external case 20 . The internal air circulation fan 160 does not discharge the internal air to the outside of the outer case 20 but continuously circulates the internal air, and at this time, it is possible to filter impurities contained in the internal air.

19 is an embodiment of a cell culture method according to the present invention.

Referring to FIG. 19, the cell culture method 1100 of FIG. 11 according to the present invention includes a check timer check step 1110, a shaker use step 1120, and a culture area change step 1130. .

In the test timer confirmation step 1110, the cell detection algorithm, the shaker utilization step 1120 and the culture area change step 1130 are performed at predetermined times using the timer 143, assuming that the predetermined time is 12 hours If so, it is preferable to perform the cell detection algorithm every 12 hours.

In the step of utilizing the shaker (1120), the clustering of the cells being cultured in the culture bag 153 is detected using the image of the culture bag 153, and the shaking module 154 (or shaker) is activated as needed to form the culture bag 153. ) allows cells to be cultured in a uniform distribution, and image generation step 1121, image processing step 1122, clustering detection step 1123, shaker drive determination step 1124 and shaker drive step 1125 include

In the image generation step 1121, the electron microscope 157 captures the cells cultured in the culture bag 153 from the lower part of the culture bag 153.

In the image processing step 1122, the image generated in the image generating step 1121 is processed. This is because measuring the clustering or cell density described later using the image obtained from the electron microscope 157 is less precise. am.

In the clustering detection step 1123, cell clustering is detected using the image processed in the image processing step 1122. Since it is desirable to ensure that the size of the clustering of cells is uniform, in order to confirm this, the clustering of cells must first be detected.

In the shaker driving determination step 1124, the detected clustering is compared with a preset standard size, and when the size of the detected clustering is less than the standard size (1124, No), the culture area change step 1130 described later is performed. .

In the shaker driving step 1125, the shaking module 154 (or shaker) is driven to reduce the clustering size when the size of the detected clustering is greater than or equal to the reference size (1124, Yes). When the shaker driving step 1125 ends, the image generating step 1121 is re-performed.

In the shaker driving step 1125, based on the driving time and RPM setting values of the shaking module 154, a plurality of vibration modes having different driving times and different RPM values are set in pairs, and one of the plurality of vibration modes Examples using or a combination of them will be possible.

In the culture area change step 1130, the culture bag area is increased by activating the roller module 155 from the density of the cells cultured in the culture bag 153 using the image of the culture bag 153, or the syringe pump module ( 110) and tube pump 120 are activated to perform a function of adding culture medium to the culture bag 153, image generation step 1131, image processing step 1132, cell density detection step 1133, culture area It includes an expansion determination step (1134) and a culture area expansion & culture solution addition step (1135).

In the image generation step 1131, the electron microscope 157 captures the cells cultured in the culture bag 153 from the lower part of the culture bag 153.

In the image processing step 1132, the image generated in the image generating step 1131 is processed.

In the cell density detection step 1133, the cell density is detected from the image processed in the image processing step 1132. The density of the cells cultured in the culture bag 153 is preferably maintained at a preset standard density.

In the culture area expansion determination step 1134, the detected cell density is compared with a preset reference density, and when the detected cell density is less than the reference density (1134, No), the test timer check step 1110 is performed. .

In the culture area expansion & culture solution addition step (1135), when the detected cell density is equal to or greater than the standard density (1134, Yes), the culture area is expanded or culture solution is added.

At this time, it is possible to increase the increase rate of the culture bag area to the same area, but an embodiment in which the area to be added is increased every time the number of increases is also possible. In addition, it is possible to add the same amount of culture medium added to the culture bag 153, but an embodiment in which the amount added at each addition is also possible.

The plurality of steps 1122, 1123, 1124, 1132, 1133, and 1134 shown in FIG. 19 are an image processing device (not shown) installed inside the automatic cell culture machine 100 or an image installed outside the automatic cell culture machine 100. It is performed using a processing device (not shown). If an externally installed image processing device (not shown) is used, a communication means (not shown) should be provided inside the automatic cell culturing device 100 .

20 is an example of an apparatus for automatically adjusting the culture area of a culture bag according to the present invention.

Referring to FIG. 20, the apparatus 20 for automatically adjusting the culture area of a culture bag according to the present invention includes a roller module 201, a culture bag 202, a panel 203, a microscope 204, and a control device 205. And, it is sealed by the culture case 200.

In the culture bag 202, there are culture medium and cells to be cultured. When the cells included in the culture bag 202 are cultured using a culture medium, a small area will be sufficient at the beginning of the culture, but it will be easily understood that the culture area of the culture bag 202 must be widened as the cells proliferate.

In the present invention, it is proposed to adjust the culture area of the culture bag 202 by using a roller module 201 that applies a constant pressure to the culture bag 202 from the top of the culture bag 202.

The culture bag 202 is placed on top of a transparent panel 203 that is not bent by the pressure of the roller module 201, and on the other hand, the culture bag 202 is cultured by the pressure of the roller module 201 It is easy to adjust the area, and on the other hand, it is preferable to allow the microscope 204 located at the bottom of the panel 203 to monitor the culture situation in the culture bag 202.

Supplying the culture solution to the inside of the culture bag 202 is the pump 101, the syringe 102, the tube pump 103 and the culture solution storage container constituting the culture solution supply device 10 installed in the sealed culture storage case 100. It is performed using (104). Pressure is applied to the tube pump 103 to deliver the culture medium stored in the culture medium container 104 to the culture bag 202. At this time, the culture medium preservation case 100 and the culture case 200 have a certain hole This allows the tube to pass through, and also seals the periphery of the hole through which the tube passes, thereby preventing contamination of the culture medium and the culture case 200 when the culture medium is supplied.

In order to make the amount of the culture solution supplied to the culture bag 202 more precise, by applying pressure to the outside of the tube installed in the middle of the tube passing through, It would be desirable to adjust the delivery timing and delivery amount of the culture medium.

The controller 205 controls the operation of the roller module 201 and the microscope 204.

21 describes a situation in which the culture area of a culture bag is varied using a roller module.

Referring to FIG. 21, a roller module 201 located on the top of the culture bag 202 seated on the top of the panel 203 moves one side of the culture bag 202 to the other with a constant force from the top of the culture bag 202. separate from the side. 21 is a side cross-sectional view of a state in which the panel 203, the culture bag 202, and the roller module 201 are moved, and D1, D2, and D3 are moved by the roller module 201 from one side of the culture bag 202. It means the length of the culture area to be determined. Here, one side of the culture bag 202 is preferably a surface through which the culture solution is introduced into the culture bag 202 through the tube. Therefore, it can be seen that the culture area in FIG. 21a is the narrowest, and the culture area increases toward FIGS. 21b and 21c.

As described above, the roller module 201, the culture container 202, the panel 203, and the microscope 204 constituting the automatic culture area control device 20 are installed in the closed culture case 200, and the roller If the movement of the module 201 is performed automatically, external contaminants will not be able to intervene when changing the culture area.

For convenience of description, the roller module 201 has been treated like a roller in the above, but the configuration of the roller module 201 will be described below.

22 is a view of a roller installed in the culture case from an upper point.

23 is a view of the roller installed in the culture case from another point on the top.

22 and 23, the roller module 201 includes a guide rail 201-1, a roller frame 201-2, a roller bracket 201-3, a roller 201-4, a roller shaft ( 201-5), a belt 201-6, a thumb screw 201-7, and an inner wall 201-8.

The roller frame 201-2 equipped with rollers 201-4 at the bottom moves along the guide rail 201-1 using roller brackets 201-3 installed on both edges. The guide rails 201-1 are installed on both sides of the inner wall 201-8 along the moving direction.

The roller bracket 201-3 may move the roller frame 201-2 in a manner of moving along the belt 201-6 surrounding the two roller shafts 201-5. In addition, the roller bracket 201-3 is fixed to the belt 201-6, and the belt 201-6 moves between the roller shafts 201-5 to move the roller frame 201-2. example is also possible. In the first embodiment, a rotating member (not shown) having threads corresponding to the threads formed on the belt 201-6 should be installed inside the roller bracket 201-3, and the rotating member should have a motor installed therein (not shown). city) will work. In the second embodiment, one of the two roller shafts 201-5 is rotated by a motor (not shown), and the other roller shaft 201-5 is rotated by a belt 201-5. It is preferable to rotate freely according to the movement of 6).

24 is an enlarged view of the edge of the roller frame.

25 is an embodiment of a roller bracket.

24 and 25, the roller shaft 201-4a formed at the end of the roller 201-4 is inserted into the roller shaft fixing groove 201-3a of the roller bracket 201-3, and the roller frame It can be seen that the roller bracket 201-3 to which the roller shaft 201-4a is mounted is fixed to the edge of 201-2 by thumb screws 201-7. At this time, the thumb screw 201-7 applies pressure to the roller shaft 201-4a while moving downward in the roller shaft fixing groove 201-3a, thereby fixing the roller shaft 201-4a to the roller bracket 201-3a. 3) while fixing the roller bracket 201-3 to the roller frame 201-2.

26 is an external structure of an automated cell culturing apparatus using an inverted electron microscope according to the present invention.

27 is an example of the configuration of an automated cell culture system using an inverted electron microscope according to the present invention.

26 and 27, the automatic cell cultivator using an inverted electron microscope according to the present invention is sealed by a culture medium storage case 100 and a culture case 200, and is stored inside the culture medium storage case 100. The existing culture medium is delivered to the culture case 200 through a tube.

The culture case 200 includes a roller module 201, a culture bag 202, a panel 203, an inverted electron microscope 204, a three-dimensional stage 205, a communication module 206, a control device 207 and frame 208.

Figure 28 is an example of the internal configuration of an automatic cell culturing machine using an inverted electron microscope according to the present invention on one side of the top.

Fig. 29 is an example of the internal configuration of an automatic cell culturing machine using an inverted electron microscope according to the present invention viewed from another upper side.

Hereinafter, the internal configuration of the automatic cell culturing machine will be described with reference to FIGS. 28 and 29 .

The roller module 201 is located on the upper part of the culture bag 202 and moves along the guide rail 201-2 installed on the inner wall 201-1 so that the roller 201-3 is constantly attached to the culture bag 202. By applying pressure, the function of increasing the culture area of the culture bag 202 is performed.

The culture bag 202 is located on top of the transparent panel 203, and cultures cells from the inside using the culture medium supplied through a tube (not shown) from the outside, that is, the culture medium preservation case 100. An inverted electron microscope (204, not shown) is installed on the 3D stage 205 to photograph the cells cultured in the culture bag 202 under the transparent panel 203. The 3D stage 205 may move in a 3D direction in a space formed under the culture bag 202 .

The communication module 206 transmits an image captured by the inverted electron microscope 204 to the outside. The controller 207 controls the operation of the roller module 201, the inverted electron microscope 204 and the three-dimensional stage 205. The roller module 201, the transparent panel 203, and the three-dimensional stage 205 are each fixed to the frame 208 installed inside the culture case 200.

Fig. 30 is an example of an arrangement situation of an inverted electron microscope and a culture bag as seen from one side of the top.

31 is an example of an arrangement situation of an inverted electron microscope and a culture bag as seen from another side of the upper part.

30 and 31, the lens of the inverted electron microscope 204 is installed in the form of taking an image in the upper direction, and the lens of the inverted electron microscope 204 captures the lower part of the culture bag 202. You can see that it has been installed.

As described above, the culture area of the culture bag 202 can be varied according to the movement of the roller module 201 located above the culture bag 202, and it is determined that it is necessary to expand the predetermined culture area. When the roller module 201 will move.

The expansion of the predetermined culture area can be grasped through the cell culture state of the culture bag photographed by the inverted electron microscope 204 .

Fig. 32 illustrates an example of a cultured state of cells photographed under an inverted electron microscope.

Referring to FIG. 32 , a right image is generated using a color difference between the left image, the number of cell pixels and background pixels is derived from the right image, and the cultivation area is expanded when it is determined that the expansion criterion is satisfied.

In the above, the technical idea of the present invention has been described with the accompanying drawings, but this is an exemplary description of preferred embodiments of the present invention, but does not limit the present invention. In addition, it is obvious that anyone skilled in the art can make various modifications and imitations without departing from the scope of the technical idea of the present invention.

10: solution case
110: syringe pump module
120: tube pump
130: Peltier module
135: culture medium container
20: outer case
140: controller module
150: internal module
151: frame 152: transparent plate
153: culture bag 154: shaking module
155: roller module 156: three-dimensional stage
157 electron microscope
160: internal air circulation fan
100: culture medium storage case
101: pump 102: syringe
103: tube pump 104: culture medium container
200: culture case
201: roller module 202: culture bag
203: panel 204: microscope
205: control device

Claims (7)

Image information acquisition step of acquiring image information by photographing the cell culture medium through an electron microscope;
a histogram smoothing step for clearly distinguishing the background, single cells, and clusters included in the image information;
In the image information through the histogram smoothing step, the highest value of the three RGB values for each pixel is converted to the maximum value, and the other two values are converted to the minimum value to more clearly distinguish the background, single cell, and cluster area separation step;
a morphology calculation step of removing noise from image information through the region separation step and restoring the shape of the cluster; and
A cell culture solution management method through cell image image processing comprising a cluster detection step of detecting the outline of the cluster based on the binary image through the morphology calculation step and detecting the area of the cluster through the number of pixels inside the outline. . According to claim 1,
Cell culture medium management method through cell image image processing further comprising a shaking step of shaking the cell culture medium when the area of any one of the clusters detected through the cluster detection step is greater than or equal to a reference value or the number of clusters is greater than or equal to a reference value. According to claim 1,
In the image information acquisition step,
Obtaining image information by photographing a part of the cell culture medium through an electron microscope;
When the area of the clusters detected through the cluster detection step is less than the reference value but the number of clusters is greater than the reference value, image information is obtained by photographing other parts of the cell culture medium, and then cell image image processing is performed to perform the above-described steps. Cell culture management method through Image information acquisition step of acquiring image information by photographing the cell culture medium through an electron microscope;
a histogram smoothing step for clearly distinguishing the background, single cells, and clusters included in the image information;
In the image information through the histogram smoothing step, the highest value of the three RGB values for each pixel is converted to the maximum value, and the other two values are converted to the minimum value to more clearly distinguish the background, single cell, and cluster area separation step;
a cell density detection step of detecting a density of cells by obtaining a ratio of the number of pixels occupied by the background, single cells, and clusters to the total number of pixels of the video image from the image information obtained in the region separation step; and
A cell culture management method through cell image image processing comprising a cell culture medium adjusting step of increasing the amount of the cell culture medium when the cell density through the cell density detection step is greater than or equal to a reference value. According to claim 4,
In the cell density detection step,
When counting the cells inside the cluster,
A cell culture solution management method through cell image image processing that divides the brightness values of one pixel from 0 to 255 into multiple ranges and counts the cells inside the cluster by multiplying the weight values corresponding to the divided ranges. An automatic cell cultivator using the cell culture medium management method according to any one of claims 1 to 5 through image processing of cell images. According to claim 6,
Culture medium storage container;
a transparent culture bag made of a flexible material in which the set initial cell culture medium is accommodated and the culture medium is supplied from the culture medium reservoir;
a tube pump for adjusting the supply amount of the culture solution supplied from the culture solution reservoir to the culture bag by adjusting the pressure of the culture solution reservoir and the tube connected to the culture bag;
A transparent plate to which the culture bag is coupled to an upper portion;
A roller module controlling the capacity of the culture bag while moving in close contact with the transparent plate with the culture bag interposed at the top of the transparent plate;
a shaking module connected to the transparent plate to shake the transparent plate;
an electron microscope movably disposed below the transparent plate and photographing the cell culture medium inside the culture bag; and
A controller module for controlling the tube pump, the roller module, the shaking module, and the electron microscope,
The controller module processes the image information obtained through the electron microscope based on the cell culture medium management method through the cell image image processing, and based on the processed result, the operation of the tube pump, the roller module, and the shaking module Automated cell incubator with condition control.

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