Cell separation method

Abstract

A cell separation method, comprising the following steps: (1) centrifugally layering a cell sample in a centrifuge, wherein the layered cell sample comprises an inner cell layer and an outer cell layer which are sequentially distributed outwards from the center of the centrifuge; (2) acquiring cell sample layering images in the process of centrifugal layering, and processing the acquired cell sample layering images to obtain the volume Vouter of the outer cell layer obtained by layering; and (3) transferring the outer cell layer, and stopping transferring when the volume Vouter of the outer cell layer in the centrifuge is equal to Vouter set, wherein Vouter set is a target volume value, and in the process of transferring the outer cell layer, the cell sample layering images are acquired in real time, and the acquired cell sample layering images are processed to calculate the volume Vouter of the remaining outer cell layer in the centrifuge.

Description

A cell separation method Technical Field

[0001] The invention belongs to the field of cell separation, and particularly relates to a cell separation method. Background Art

[0002] With the rapid development of artificial intelligence, intelligence is becoming increasingly evident across industries. However, there are still deficiencies in the accuracy and intelligence of cell separation. In hospitals, biological or medical research institutes, and other places where large numbers of cells need to be separated, the cell separation process relies entirely on visual judgment and manual operation after the cell fluid is separated by centrifuge. This not only wastes a lot of manpower and financial resources, but also may lead to the loss of target cells or even separation failure due to operational errors.

[0003] Summary of the Invention

[0004] The purpose of the present invention is to provide a cell separation method, which improves the suction accuracy, increases the work efficiency, and reduces the errors and losses caused by manual operation errors.

[0005] In order to achieve the above object, a technical solution adopted by the present invention is:

[0006] A cell separation method comprises the following steps:

[0007] (1) The cell sample is centrifuged and stratified in a centrifuge. The stratified cell sample includes an inner cell layer and an outer cell layer that are sequentially distributed outward from the center of the centrifuge;

[0008] (2) Collecting cell sample stratification images during centrifugal stratification, processing the collected cell sample stratification images, and obtaining the volume V of the outer cell layer after stratification based on the processing results 外 , wherein the outer cell layer volume V 外 It is obtained by the following formula: V 外 =πR 2 h-πr 2 h,

[0009] R is the distance between the outermost side of the outer cell layer and the center of the centrifuge, r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and h is the height of the cell sample;

[0010] (3) Transfer the outer cell layer. When the volume of the outer cell layer in the centrifuge is V 外 Equal to V 外设 Stop transfer when V 外设 is the target volume value, wherein the cell sample layer images are collected in real time during the transfer of the outer cell layer, and the collected cell sample layer images are processed to calculate the volume V of the remaining outer cell layer in the centrifuge 外 .

[0011] By the above operation, a quantitative outer cell layer solution can be retained in the centrifuge.

[0012] According to some implementation aspects of the present invention, in step (2), the collected cell sample layered image is processed as follows:

[0013] S1, perform Gaussian filtering on the image;

[0014] S2, performing edge recognition on the filtered image;

[0015] S3. Perform expansion processing on the edge detection result.

[0016] The white straight line after the expansion process is the stratification boundary of the cell sample. By processing the stratified image of the cell sample in this step, the specific position of the outer cell layer can be obtained, which facilitates the calculation of the volume of the outer cell layer.

[0017] According to some embodiments of the present invention, in step (2), a cell sample stratification image is collected at an interval of n seconds; in step (3), a cell sample stratification image is collected at an interval of m seconds, where m is less than n. In step (2), stratification may not be completed, and the cell sample stratification image is collected after a longer interval to reduce the workload; when the stratification is completed and stabilized, the volume of the outer cell layer after stratification needs to be calculated as soon as possible, and the cell sample stratification image is collected after a shorter interval to facilitate the transfer of the outer cell layer in a shorter time.

[0018] According to some implementation aspects of the present invention, n is greater than or equal to 20 and less than or equal to 50, and preferably n is 30; m is greater than 0 and less than or equal to 5, and preferably m is 1.

[0019] According to some implementation aspects of the present invention, a cell sample layer image may be collected at intervals of n1 seconds, a cell sample layer image may be collected at intervals of n2 seconds, and then a cell sample layer image may be collected at intervals of n3 seconds...n seconds. i Collect a cell sample layer map in seconds, n1, n2, n3...n i It can be reduced in sequence, which makes the setting more flexible, reduces the workload and obtains the volume of the outer cell layer more quickly.

[0020] According to some embodiments of the present invention, in step (3), the centrifuge speed is reduced before transferring the outer cell layer. This operation can better and faster aspirate the outer cell layer while ensuring normal cell stratification, thereby ensuring aspiration accuracy.

[0021] According to some embodiments of the present invention, before step (3), if the volume of the outer cell layer remains unchanged during centrifugation, the outer cell layer is transferred; if the volume of the outer cell layer changes during centrifugation, the centrifugation is continued until the volume of the outer cell layer remains unchanged. If the volume of the outer cell layer remains unchanged during centrifugation, it indicates that stratification is complete, and step (3) is performed only after stratification is complete. This ensures the accuracy of aspirating the outer cell layer in step (3) and the accuracy of separation.

[0022] According to some embodiments of the present invention, after step (3), the method further includes: (4) transferring the inner cell layer, when the volume V of the inner cell layer in the centrifuge is 内 Equal to V 内设 Stop transfer when V 内设 is the target volume value, wherein the cell sample layered images are collected in real time during the transfer process, and the collected cell sample layered images are processed, and the volume V of the inner cell layer after layering is obtained based on the processing results 内 , the volume of the inner cell layer V 内 It is obtained by the following formula: V 内 =πr 2 h-πr1 2 h,

[0023] Where r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and r1 is the distance between the innermost side of the inner cell layer and the center of the centrifuge.

[0024] This step can ensure that a quantitative amount of inner cell layer solution is retained in the centrifuge.

[0025] According to some embodiments of the present invention, the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = R - [k + L × {(AY i-1 ) / (Y i -Y i-1 )}],

[0026] The pixels corresponding to multiple scales are Y1, Y2, Y3...Y i , Y i The scale indicated is Y i-1 The scale indicated is close to the center of the centrifugal chamber of the centrifuge, and the distance between two adjacent scales is L; the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is A; if the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is located between the pixels corresponding to the two adjacent scales (Y i 、Y i-1 ), k is Y i-1 The distance between the corresponding scale and the inner wall of the centrifugal chamber; or if the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, then k is Y i-1The distance between the scale corresponding to Y1 and the scale corresponding to Y2 is obtained by performing the above operation to calculate the volume of the outer cell layer.

[0027] According to some embodiments of the present invention, i is 2, 3, 4, 5, 6, 7 or 8.

[0028] According to some implementation aspects of the present invention, there are 7 scales, namely, the first scale, the second scale, the third scale, the fourth scale, the fifth scale, the sixth scale, and the seventh scale, which are distributed from the outermost side of the centrifugal chamber to the center thereof; the pixel corresponding to the first scale is Y1, the pixel corresponding to the second scale is Y2, the pixel corresponding to the third scale is Y3, the pixel corresponding to the fourth scale is Y4, the pixel corresponding to the fifth scale is Y5, the pixel corresponding to the sixth scale is Y6, and the pixel corresponding to the seventh scale is Y7, wherein , Y7 is greater than Y6, Y6 is greater than Y5, Y5 is greater than Y4, Y4 is greater than Y3, Y3 is greater than Y2, and Y2 is greater than Y1; if the stratification boundary between the outer cell layer and the inner cell layer is located between the horizontal line corresponding to the first scale and the horizontal line corresponding to the second scale, the spacing between the first scale and the second scale is L, the pixel corresponding to the first scale is Y1, the pixel corresponding to the second scale is Y2, the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is A, k is 0, then the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r=RL×{(A-Y1) / (Y2-Y1)};

[0029] If the delamination boundary between the outer cell layer and the inner cell layer corresponds to one of the scale lines, then the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = Rk,

[0030] Among them, the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, and k is the distance between the scale line and the scale corresponding to Y1.

[0031] According to some implementation aspects of the present invention, V 外设 20-60ml.

[0032] Another technical solution adopted in the present invention is:

[0033] A cell separation method comprises the following steps:

[0034] (1) The cell sample is centrifuged and stratified in a centrifuge. The stratified cell sample consists of an inner cell layer and an outer cell layer distributed outward from the center of the centrifuge;

[0035] (2) Collecting cell sample stratification images during centrifugal stratification, processing the collected cell sample stratification images, and obtaining the volume V of the outer cell layer after stratification 外 , wherein the outer cell layer volume V 外It is obtained by the following formula: V 外 =πR 2 h-πr 2 h,

[0036] R is the distance between the outermost side of the outer cell layer and the center of the centrifuge, r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and h is the height of the cell sample;

[0037] In step (2), the collected cell sample layered image is processed as follows:

[0038] S1, perform Gaussian filtering on the image;

[0039] S2, performing edge recognition on the filtered image;

[0040] S3. Perform expansion processing on the edge detection result.

[0041] (3) Transfer the outer cell layer. When the volume of the outer cell layer in the centrifuge is V 外 Equal to V 外设 Stop transfer when V 外设 is the target volume value, wherein during the process of transferring the outer cell layer, the cell sample layered images are collected in real time, and the collected cell sample layered images are processed;

[0042] (4) Transfer the inner cell layer. When the volume of the inner cell layer in the centrifuge is V 内 Equal to V 内设 Stop transfer when V 内设 is the target volume value, wherein the cell sample layered images are collected in real time during the transfer process, and the collected cell sample layered images are processed, and the volume V of the inner cell layer after layering is obtained based on the processing results 内 , the volume of the inner cell layer V 内 It is obtained by the following formula: V 内 =πr 2 h-πr1 2 h,

[0043] Where r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and r1 is the distance between the innermost side of the inner cell layer and the center of the centrifuge.

[0044] According to some implementation aspects of the present invention, the method is implemented by the following apparatus, comprising:

[0045] A centrifuge comprising a top cover, a base, and a first delivery channel, wherein the top cover is made of a transparent material, the top cover and the base enclosing a centrifugal chamber, the top cover being provided with radially extending graduations; the first delivery channel extending into the centrifugal chamber for transferring an outer cell layer, wherein a cell sample is centrifuged and stratified in the centrifuge, the stratified cell sample comprising an inner cell layer and an outer cell layer sequentially distributed outward from the center of the centrifuge;

[0046] An image acquisition module, configured to acquire layered images of cell samples;

[0047] An image processing module is used to process the collected cell sample layer image, wherein the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained based on the scale corresponding to the layer boundary between the inner cell layer and the outer cell layer and the pixels corresponding to the scale;

[0048] a liquid transfer module, the liquid transfer module being in communication with the first delivery channel and configured to provide power for transferring the outer cell layer;

[0049] a driving module, the driving module being connected to the centrifuge for driving the centrifuge to rotate;

[0050] A controller is connected to the image processing module, the liquid transfer module, and the driving module.

[0051] According to some embodiments of the present invention, a support portion is provided on the upper side of the top cover, and the support portion is arranged along the radial direction of the top cover. The support portion includes a support platform and a convex portion arranged on the support platform. The support platform has a horizontal plane and inclined planes located below both sides of the horizontal plane, wherein one of the inclined planes is inclined toward a direction close to the other inclined plane, and the scale is provided on the inclined plane, and the scale forms an acute angle with the horizontal plane.

[0052] According to some embodiments of the present invention, the device further comprises a second delivery channel, a first pinch valve, and a second pinch valve, wherein the second delivery channel extends into the centrifugal chamber for transferring the inner cell layer; the first pinch valve is in communication with the first delivery channel, and the second pinch valve is in communication with the second delivery channel;

[0053] In step (3), when transferring the outer cell layer, the first pinch valve is opened and the second pinch valve is closed.

[0054] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0055] The cell separation method provided by the present invention collects sample cell stratification images after the cell sample is stratified, processes the collected cell sample stratification images, obtains the volume of the outer cell layer after stratification based on the processing results, transfers the outer cell layer according to the volume, and retains a quantitative outer cell layer solution in the centrifuge, thereby improving separation accuracy, improving work efficiency, and reducing losses caused by human operational errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG1 is a schematic diagram of a stratified image of sample cells after stratification according to an embodiment;

[0057] FIG2 is a schematic diagram of a sample cell layer image after Gaussian filtering according to an embodiment;

[0058] FIG3 is a schematic diagram of a sample cell layer image after edge recognition according to an embodiment;

[0059] FIG4 is a schematic diagram of a cell layer image of a sample after expansion treatment according to an embodiment;

[0060] Figure 5 is an exploded view of a separation device according to an embodiment of the present invention;

[0061] 6 is a structural diagram of a centrifuge from a first perspective of a separation device according to an embodiment of the present invention;

[0062] 7 is a structural diagram of a centrifuge of a separation device according to an embodiment of the present invention from a second perspective;

[0063] Figure 8 is a front view of a centrifuge of a separation device according to an embodiment of the present invention;

[0064] FIG9 is a layered image of a cell sample according to an embodiment of the present invention;

[0065] FIG10 is a pixel diagram corresponding to seven scales according to an embodiment of the present invention.

[0066] In the above figures, the reference numerals are: 1-outer cell layer; 2-inner cell layer; 3-base; 4-top cover; 5-first interface, 6-second interface, 7-scale; 81-first pinch valve; 82-second pinch valve; 9-support platform, 91-inclined surface, 92-horizontal surface; 10-image acquisition module; 11-delivery pump; 12-convex portion. DETAILED DESCRIPTION

[0067] The present invention will be further described below with reference to the embodiments shown in the accompanying drawings.

[0068] The cell separation method in this example can obtain different types of high-purity immune cells or stem cells from blood (peripheral blood) and bone marrow to obtain the final desired cell products for downstream applications such as cell therapy.

[0069] A cell separation method comprises the following steps:

[0070] (1) The cell sample is centrifuged and stratified in a centrifuge. The stratified cell sample consists of an inner cell layer 2 and an outer cell layer 1, which are distributed outward from the center of the centrifuge;

[0071] (2) During the centrifugal stratification process, the image acquisition module is used to collect the cell sample stratification image, and the image processing module is used to process the collected cell sample stratification image. Based on the processing results, the volume V of the outer cell layer after stratification is obtained. 外 , where the outer cell layer volume V 外 It is obtained by the following formula: V 外 =πR 2 h-πr 2 h,

[0072] Where R is the distance from the outermost outer cell layer to the center of the centrifuge (i.e., the radius of the centrifuge chamber of the centrifuge), r is the distance from the outermost inner cell layer to the center of the centrifuge, and h is the height of the cell sample (i.e., the height of the centrifuge chamber of the centrifuge).

[0073] (3) Transfer the outer cell layer. When the volume of the outer cell layer in the centrifuge is V 外 Equal to V 外设 Stop transfer when V 外设 The target volume value can be a range. In this step, the collected cell sample layer image is processed in the transfer outer cell layer, and the volume V of the remaining outer cell layer after stratification is obtained based on the processing result. 外 .

[0074] In step (1), the stratified cell sample includes an inner cell layer and an outer cell layer, wherein the inner cell layer is close to the center of the centrifuge and the outer cell layer is arranged around the periphery of the inner cell layer.

[0075] In step (2), a layered image of the cell sample is collected and processed as follows:

[0076] S1, perform Gaussian filtering on the image to smooth and remove noise;

[0077] S2, performing edge recognition on the filtered image;

[0078] S3. Perform expansion processing on the edge detection result.

[0079] The white straight line after the expansion process is the stratification boundary of the cell sample. By processing the stratified image of the cell sample in this step, the specific position of the outer cell layer 1 can be obtained, and the volume of the outer cell layer can be calculated.

[0080] In step (2), an image acquisition module is used to collect layered images of the cell sample. The image acquisition module is located above the centrifuge and is a camera. A standard light source is set directly above the centrifuge to provide an auxiliary light source to improve the image capture quality.

[0081] The specific method of step S2 is as follows:

[0082] 1) Calculate edge gradient and direction (SobelX and SobelY kernels filter the smoothed image in the horizontal and vertical directions to find the edge gradient and direction of each pixel);

[0083] 2) Non-maximum suppression (after obtaining the gradient magnitude and direction, perform a full scan of the image to remove any unnecessary pixels that may not constitute an edge. Check whether the pixel is a local maximum in the neighborhood of its gradient direction. Otherwise, it will be suppressed (returned to zero). In short, the result is a binary image with "thin edges";

[0084] 3) Hysteresis thresholding determines which edges are true edges and which are not. This requires two thresholds, minVal and maxVal. Any edge with an intensity gradient greater than maxVal is considered an edge, while any edge less than minVal is considered a non-edge and is therefore discarded. Edges between these two thresholds are classified as edges or non-edges based on their connectivity. If they are connected to a "definite edge" pixel, they are considered part of an edge; otherwise, they are discarded.

[0085] In step (2), the volume of the outer cell layer after stratification is calculated based on the expansion processing result. In this step, if the volume of the inner and outer cell layers 1 does not change within a time threshold, it means that the stratification is completed (or if the volume of the inner and outer cell layers 1 does not change within several time thresholds, the stratification is completed), and step (3) is performed; if the volume of the inner and outer cell layers 1 changes within the time threshold, the stratification is not completed, and centrifugal stratification is continued until the volume of the outer cell layer 1 does not change; in this step, the time threshold can be 50-150s, for example 90s.

[0086] (3) Transferring the outer cell layer 1 (sucking the outer cell layer out of the centrifuge). If the volume of the outer cell layer 1 is less than a volume threshold, the outer cell layer 1 is not transferred. If the volume of the outer cell layer 1 is greater than a volume threshold, the outer cell layer 1 is transferred. During the transfer process, the volume V of the remaining outer cell layer 1 in the centrifuge is also calculated. 外 , when the volume V of the remaining outer cell layer 1 外 Reach V 外设 The transfer is completed when the volume of the cell layer inside and outside the centrifuge is V 外设 is the target volume of the outer cell layer that needs to be retained in the centrifuge.

[0087] In some embodiments, in step (3), a volume threshold may be 20-60 ml, V 外设 It can be 20-60ml.

[0088] During the process of collecting cell sample images in step (2), a cell sample stratification image is collected at intervals of n seconds; in step (3), a cell sample stratification image is collected at intervals of m seconds, where m is less than n, 20≤n≤50, and 0<m≤5. In step (2), stratification may not be completed yet. It is recommended to collect cell sample stratification images after a longer interval to reduce workload. When stratification is completed and stabilized, the volume of the outer cell layer 1 after stratification needs to be calculated as soon as possible. Cell sample stratification images are collected after a shorter interval to facilitate the transfer of the outer cell layer 1 in a shorter time.

[0089] In step (3), when transferring the outer cell layer, the centrifuge speed is first reduced to the speed threshold before transferring the outer cell layer 1. For example, when the outer cell layer 1 is not transferred, the centrifuge speed is 2000-3000 rpm. When the stratification is completed, when transferring the outer cell layer 1, the centrifuge speed is reduced to 800-1500 rpm. This operation can ensure that the outer cell layer 1 is sucked out better and faster while ensuring normal cell stratification, thereby ensuring the suction accuracy.

[0090] Step (3) also includes step (4): setting the volume value V to be retained in the centrifuge 内设 (can be a range) of the inner cell layer 2, aspirate the inner cell layer 2 (serum), when the volume V of the inner cell layer 2 in the centrifuge 内 Equal to V 内设 Stop transfer when V 内设 is the target volume value, wherein, during the transfer process, the cell sample layered images are collected in real time, and the collected cell sample layered images are processed, and the volume V of the inner cell layer 2 after layering is obtained based on the processing results 内 , the volume V of the inner cell layer 2 内 It is obtained by the following formula: V 内 =πr 2 h-πr1 2 h,

[0091] Where r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and r1 is the distance between the innermost side of the inner cell layer and the center of the centrifuge.

[0092] The cell separation method of this example can retain a certain amount of outer cell layer solution and a certain amount of inner cell layer solution in the centrifuge, and has high separation accuracy.

[0093] According to the storage capacity of the centrifuge and the volume of the target cell solution required, the following example is set:

[0094] A cell sample centrifugation method includes the following steps:

[0095] First, initialization

[0096] Initialize all hardware devices to the ideal state, prepare cell samples, pipelines and some preliminary work. The cell samples are peripheral blood.

[0097] Second, cell separation

[0098] (1) The cell sample enters the centrifuge, and the centrifuge rotates to the target speed (2500 rpm). The cell sample is stratified. The stratified cell sample includes an outer cell layer 1 (red blood cells) and an inner cell layer 2 (serum) distributed outward from the center of the centrifuge;

[0099] (2) The fill light is turned on, and the industrial camera starts taking pictures to collect layered images of the cell sample, as shown in Figure 1, except for 1 and 2, which represent blanks;

[0100] The collected cell sample layered images are processed as follows:

[0101] S1. Perform Gaussian filtering on the cell layer image taken by the layer camera to smooth and remove noise. The result is shown in Figure 2.

[0102] S2. Use the Canny algorithm to perform edge recognition on the filtered cell layer image, as shown in Figure 3;

[0103] S3. Dilation processing is performed on the edge detection result. The result is shown in FIG4 . In FIG4 , the white straight line after dilation processing is the stratification boundary of the cell sample.

[0104] Next, the volume of the outer cell layer 1 can be obtained based on the expansion treatment results. If the volume of the inner and outer cell layers 1 does not change for 90 consecutive seconds, the stratification is completed;

[0105] (3) After stratification, if the volume of outer cell layer 1 is less than 40 ml, outer cell layer 1 is not transferred; if it is greater than 40 ml, the centrifuge speed is first reduced to 1000 rpm. After the speed reduction is completed, outer cell layer 1 (red blood cells) is transferred.

[0106] The camera takes a picture every second and calculates the volume of the remaining outer cell layer (red blood cells). When the volume of the inner and outer cell layers reaches 40ml, the outer cell layer transfer is completed. Next, the inner cell layer 2 (serum) is aspirated. When the volume of the inner cell layer 2 (serum) in the centrifuge reaches 30ml, the transfer is stopped. The remaining cell fluid in the centrifuge is the target for testing. Because the portion between plasma and serum needs to be tested, some plasma is aspirated, leaving some plasma and serum in the centrifuge.

[0107] The above method is implemented by the following device, which includes a centrifuge, an image acquisition module 10, an image processing module, and a driving module.

[0108] Referring to Figures 5-8 , the centrifuge includes a top cover 4, a base 3, a first delivery channel, and a second delivery channel. The top cover 4 is positioned above the base 3. Together, the top cover 4 and the base 3 form a centrifuge chamber with a storage space. Cell samples enter the centrifuge chamber for centrifugal stratification. The base 3 and top cover 4 are made of a transparent material, such as transparent plastic.

[0109] The first delivery channel extends into the centrifugal chamber for transferring the outer cell layer, and the second delivery channel extends into the centrifugal chamber for transferring the inner cell layer. The outlet end of the first delivery channel is closer to the center of the centrifugal chamber than the outlet end of the second delivery channel. That is, during transfer, the outer cell layer first flows out of the centrifugal chamber from the first delivery channel, and the inner cell layer then flows out of the centrifugal chamber from the second delivery channel. The outer cell layer and the inner cell layer flow out from different channels.

[0110] The liquid transfer module is connected to the first delivery channel and the second delivery channel to provide power for transferring the outer cell layer and the inner cell layer. The liquid transfer module includes a delivery pump 11, which can be a peristaltic pump.

[0111] The first conveying channel is partially located inside the centrifugal chamber and partially extends outside the centrifugal chamber. The portion of the first conveying channel extending outside the centrifugal chamber has a first interface 5. The second conveying channel is partially located inside the centrifugal chamber and partially extends outside the centrifugal chamber. The portion of the second conveying channel extending outside the centrifugal chamber has a second interface 6. Both the first interface 5 and the second interface 6 are located outside the centrifugal chamber, and the conveying pump 11 is connected to both the first interface 5 and the second interface 6.

[0112] The device further includes a first pinch valve 81 and a second pinch valve 82 . The first pinch valve 81 is connected to the first delivery channel, and the second pinch valve 82 is connected to the second delivery channel.

[0113] In step (3), when transferring the outer cell layer, first open the first pinch valve 81, close the second pinch valve 82, and start the delivery pump 11 to transfer the outer cell layer 1. When aspirating the inner cell layer 2, open the second pinch valve 82, close the first pinch valve 81, and start the delivery pump 11 to transfer the inner cell layer 2.

[0114] In this example, a support portion is provided on the upper side of the top cover 4, and the top cover 4 is disc-shaped. The support portion is radially arranged along the top cover 4, the middle part of the support portion is concentric with the top cover 4, and a through hole is opened in the middle of the support portion. The support portion includes a support platform 9 and a convex portion 12. The convex portion 12 is arranged on the support platform 9, and the cross-section of the convex portion 12 is square; the support platform 9 has a horizontally arranged horizontal plane 92 and inclined planes 91 on both sides of the horizontal plane 92. The inclined plane 91 is located below the horizontal plane 92, one side of the inclined plane 91 is connected to the upper side of the horizontal plane 92, and the other side of the inclined plane 91 is connected to the upper side of the top cover 4, one of the inclined planes 91 is inclined toward the direction close to the other inclined plane 91, and the scale 7 is arranged on one inclined plane 91, and the scale 7 forms an acute angle with the horizontal plane 92, that is, the scale 7 is inclined, and the angle between the scale 7 and the horizontal plane 92 is equal to the angle between the inclined plane 91 and the horizontal plane 92, and the scale 7 faces the upper surface of the top cover 4. Multiple scale marks 7 are provided, arranged in parallel and distributed along the length of the support portion. The projections of the scale marks 7 on the horizontal plane of the top cover 4 are perpendicular to the extension of the support portion. The scale marks 7 are distributed sequentially from the outermost edge of the centrifugal chamber to the center of the centrifugal chamber. The spacing between adjacent scale marks can be consistent or inconsistent. In the collected cell sample layered image, the scale marks appear as horizontal lines on the image (see Figure 9).

[0115] The support platform 9 can be made of metal, and the scale is recessed on the inclined surface 91 of the support platform 9 .

[0116] 7 , the two inclined surfaces 91 of the support platform 9 are inclined toward each other, and both inclined surfaces 91 form acute angles with the horizontal plane 92 , and the angles are equal.

[0117] In this example, the driving module is connected to the centrifuge to drive the centrifuge to rotate, and the driving module can be a driving motor; the controller is connected to the image processing module, the liquid transfer module, and the driving module, and the controller is used to control the actions of the liquid transfer module and the driving module according to the processing results of the image processing module.

[0118] In steps (2) and (3) of the cell separation method of this example, the stratified image of the cell sample is processed by an image processing module, wherein during the processing, the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained based on the scale corresponding to the stratification boundary between the inner cell layer and the outer cell layer and the pixels corresponding to the scale.

[0119] The stratified image of the cell sample collected in this example is shown in Figure 9. A horizontal line (indicated by a in Figure 9) corresponds to a scale, and Y represents pixels. It can be seen that if the stratification boundary of the outer cell layer and the inner cell layer is located between two horizontal lines, the distance r between the outermost side of the inner cell layer and the center of the centrifuge can be obtained by the pixels corresponding to the stratification boundary of the outer cell layer and the inner cell layer and the distance between the scales corresponding to the two horizontal lines.

[0120] Specifically, the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = R - [k + L × {(AY i-1 ) / (Y i -Y i-1 )}],

[0121] The pixels corresponding to multiple scales are Y1, Y2, Y3...Y i , Y i The scale indicated is the same as Y i-1 The scales indicated are set adjacently, Y i The scale indicated is Y i-1 The scale is close to the center of the centrifugal chamber, and the distance between two adjacent scales is L. On the image, the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is A. If the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is located between the pixels corresponding to the two adjacent scales (Y i 、Y i-1 ), k is Y i-1 The distance between the corresponding scale and the inner wall of the centrifugal chamber. If the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, then k is Y i-1 The distance between the corresponding scale and the scale corresponding to Y1.

[0122] In one embodiment, there are 7 scales, and the first scale, the second scale, the third scale, the fourth scale, the fifth scale, the sixth scale, and the seventh scale are distributed in sequence from the outermost side of the centrifugal chamber to the middle side. The first scale corresponds to the inner wall of the centrifugal chamber, and the spacing between the first scale centrifugal chamber walls is 0. The seventh scale is close to the center of the centrifugal chamber. The spacing between the first scale and the second scale is 7.5 mm, the spacing between the second scale and the third scale is 5 mm, the spacing between the third scale and the fourth scale is 5 mm, the spacing between the fourth scale and the fifth scale is 5 mm, the spacing between the fifth scale and the sixth scale is 5 mm, and the spacing between the sixth scale and the seventh scale is 5 mm. In the collected cell sample layered image, the pixel corresponding to the first scale is Y1, the pixel corresponding to the second scale is Y2, the pixel corresponding to the third scale is Y3, the pixel corresponding to the fourth scale is Y4, the pixel corresponding to the fifth scale is Y5, the pixel corresponding to the sixth scale is Y6, and the pixel corresponding to the seventh scale is Y7, among which Y7 is greater than Y6, Y6 is greater than Y5, Y5 is greater than Y4, Y4 is greater than Y3, Y3 is greater than Y2, and Y2 is greater than Y1.

[0123] Referring to Figure 10, the stratification boundary between the outer cell layer and the inner cell layer is located between Y4 and Y5, Y1 is 65, Y2 is 116, Y3 is 156, Y4 is 195, Y5 is 234, Y6 is 271, and Y7 is 306. The pixel A corresponding to the stratification boundary between the outer cell layer and the inner cell layer is 210, the distance between Y1 and Y2 is 7.5 mm, the distance between Y2 and Y3 is 7.5 mm, the distance between Y3 and Y4 is 5 mm, the distance between Y4 and Y5 is 5 mm, the distance between Y5 and Y6 is 5 mm, and the distance between Y6 and Y7 is 5 mm.

[0124] The distance r between the outermost side of the inner cell layer and the center of the centrifuge was calculated by the following formula: r = R - [k + L x {(A - Y4) / (Y5 - Y4)}].

[0125] Among them, A is 210, Y4 is 195, Y5 is 234, L is 5mm, k is 7.5+5+5=17.5mm. After substituting into the above formula, we can obtain r=R-(17.5+1.92), L×{(A-Y4) / (Y5-Y4)}=5×{(210-195) / (234-195)}=1.92.

[0126] If the delamination boundary between the outer cell layer and the inner cell layer is located between the horizontal line corresponding to the first scale and the horizontal line corresponding to the second scale, the distance between the first scale and the second scale is L, the pixel corresponding to the first scale is Y1, the pixel corresponding to the second scale is Y2, the pixel corresponding to the delamination boundary between the outer cell layer and the inner cell layer is A, and k is 0, then the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = RL × {(A-Y1) / (Y2-Y1)},

[0127] For example, if Y1 is 65, Y2 is 116, A is 90, and L is 7.5 mm, then the result is: r = R - 3.68.

[0128] If the delamination boundary between the outer cell layer and the inner cell layer corresponds to one of the scale lines, then the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = Rk,

[0129] Among them, the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, and k is the distance between the scale line and the scale corresponding to Y1.

[0130] Accordingly, r1 is obtained by the above method. If the innermost limit of the inner cell layer is located between the two horizontal lines, the distance r1 between the innermost limit of the inner cell layer and the center of the centrifuge can be obtained by the distance between the pixels corresponding to the innermost limit of the inner cell layer and the scales corresponding to the two horizontal lines. r1 is obtained by the following formula: r1 = R-[k+L×{(BY i-1 ) / (Yi -Y i-1 )}],

[0131] The pixels corresponding to multiple scales are Y1, Y2, Y3...Y i , Y i The scale indicated is the same as Y i-1 The scales indicated are set adjacently, Y i The scale indicated is Y i-1 The scale is close to the center of the centrifugal chamber, and the distance between two adjacent scales is L. On the image, the pixel corresponding to the innermost boundary of the inner cell layer is B. If the pixel corresponding to the innermost boundary of the inner cell layer is located between the pixels corresponding to the two adjacent scales (Y i 、Y i-1 ), k is Y i-1 The distance between the corresponding scale and the inner wall of the centrifugal chamber. If the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, then k is Y i-1 The distance between the corresponding scale and the scale corresponding to Y1.

[0132] The image processing module specifically uses the OpenCV image processing algorithm to perform artificial intelligence recognition and position calibration on the layered images of cell samples to prepare for cell sorting.

[0133] By processing the layered images of the cell sample, the volumes of the inner and outer cell layers inside the centrifuge chamber can be obtained, and the outer and inner cell layers can be transferred according to the volumes of the inner and outer cell layers inside the centrifuge chamber, so as to retain a certain amount of outer and inner cell layer solutions in the centrifuge.

[0134] The cell separation method in this example collects cell sample stratification images after the cell sample is stratified, then filters, identifies edges, and dilates the cell sample stratification images. Based on the expansion processing results, the volume of the outer cell layer after stratification is calculated, and the outer cell layer is transferred according to the volume. This greatly simplifies manual operation, changing the original manual cell separation to automatic cell separation based on machine vision algorithms, achieving the retention of a quantitative outer cell layer solution in the centrifuge, improving separation accuracy, increasing work efficiency, reducing losses caused by manual operation errors, and providing strong guarantees for subsequent cell operations.

[0135] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A cell separation method, characterized in that: The steps include: (1) The cell sample is centrifuged and stratified in a centrifuge, and the stratified cell sample includes an inner cell layer and an outer cell layer distributed in sequence from the center of the centrifuge to the outside; (2) Collecting cell sample stratification images during the centrifugal stratification process, processing the collected cell sample stratification images, and obtaining the volume V of the outer cell layer after stratification 外 , wherein the outer cell layer volume V 外 Obtained by the following formula: V 外 =πR 2 h-πr 2 h, R is the distance between the outermost side of the outer cell layer and the center of the centrifuge, r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and h is the height of the cell sample; (3) Transfer the outer cell layer. When the volume of the outer cell layer in the centrifuge is V 外 Equal to V 外设 When the transfer stops, V 外设 is the target volume value, wherein the cell sample layer images are collected in real time during the transfer of the outer cell layer, and the collected cell sample layer images are processed to calculate the volume V of the remaining outer cell layer in the centrifuge 外 .

2. A cell separation method, characterized in that: The steps include: (1) The cell sample is centrifuged and stratified in a centrifuge. The stratified cell sample consists of an inner cell layer and an outer cell layer that are distributed outward from the center of the centrifuge; (2) Collecting cell sample stratification images during the centrifugal stratification process, processing the collected cell sample stratification images, and obtaining the volume V of the outer cell layer after stratification 外 , wherein the outer cell layer volume V 外 Obtained by the following formula: V 外 =πR 2 h-πr 2 h, R is the distance between the outermost side of the outer cell layer and the center of the centrifuge, r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and h is the height of the cell sample; In step (2), the collected cell sample layered image is processed as follows: S1, perform Gaussian filtering on the image; S2, performing edge recognition on the filtered image; S3, dilating the edge detection result; (3) Transfer the outer cell layer. When the volume of the outer cell layer in the centrifuge is V 外 Equal to V 外设 When the transfer stops, V 外设 is the target volume value, wherein the cell sample layered images are collected in real time during the outer cell layer transfer process, and the collected cell sample layered images are processed; (4) Transfer the inner cell layer. When the volume of the inner cell layer in the centrifuge is V 内 Equal to V 内设 When the transfer stops, V 内设 is the target volume value, wherein the cell sample stratification image is collected in real time during the transfer process, and the collected cell sample stratification image is processed, and the volume V of the inner cell layer after stratification is obtained based on the processing result 内 , the volume of the inner cell layer V 内 Obtained by the following formula: V 内 =πr 2 h-πr1 2 h, Among them, r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and r1 is the distance between the innermost side of the inner cell layer and the center of the centrifuge.

3. The cell separation method according to claim 1, characterized in that In step (2), the collected cell sample layered image is processed as follows: S1, perform Gaussian filtering on the image; S2, performing edge recognition on the filtered image; S3. Perform expansion processing on the edge detection result.

4. The cell separation method according to claim 1 or 2, characterized in that: In step (2), a cell sample stratification image is acquired at an interval of n seconds; in step (3), a cell sample stratification image is acquired at an interval of m seconds, where m is less than n.

5. The cell separation method according to claim 3, characterized in that: n is greater than or equal to 20 and less than or equal to 50, and m is greater than 0 and less than or equal to 5.

6. The cell separation method according to claim 1 or 2, characterized in that: In step (3), the centrifuge speed is reduced before transferring the outer cell layer.

7. The cell separation method according to claim 1 or 2, characterized in that: Before step (3), if the volume of the outer cell layer remains unchanged during the centrifugation process, the outer cell layer transfer operation is performed; if the volume of the outer cell layer changes during the centrifugation process, the centrifugation operation is continued until the volume of the outer cell layer remains unchanged.

8. The cell separation method according to claim 1 or 2, characterized in that: The distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = R - [k + L × {(AY i-1 ) / (Y i -Y i-1 )}], The pixels corresponding to multiple scales are Y1, Y2, Y3...Y i , Y i The scale indicated is Y i-1 The scale indicated is close to the center of the centrifugal chamber of the centrifuge, and the distance between two adjacent scales is L; the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is A; If the pixel corresponding to the stratification boundary between the outer cell layer and the inner cell layer is located between the pixels corresponding to the two adjacent scales (Y i , Y i-1 ), k is Y i-1 The distance between the corresponding scale and the inner wall of the centrifugal chamber; or if the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, then k is Y i-1 The distance between the corresponding scale and the scale corresponding to Y1.

9. The cell separation method according to claim 8, characterized in that: i is 2, 3, 4, 5, 6, 7 or 8.

10. The cell separation method according to claim 9, characterized in that: There are 7 scales, the first scale, the second scale, the third scale, the fourth scale, the fifth scale, the sixth scale, and the seventh scale are distributed in sequence from the outermost side to the center of the centrifugal chamber; The pixel corresponding to the first scale is Y1, the pixel corresponding to the second scale is Y2, the pixel corresponding to the third scale is Y3, the pixel corresponding to the fourth scale is Y4, the pixel corresponding to the fifth scale is Y5, the pixel corresponding to the sixth scale is Y6, and the pixel corresponding to the seventh scale is Y7, wherein Y7 is greater than Y6, Y6 is greater than Y5, and Y5 is greater than Y4. Y4 is greater than Y3, Y3 is greater than Y2, and Y2 is greater than Y1; If the delamination boundary between the outer cell layer and the inner cell layer is located between the horizontal line corresponding to the first scale and the horizontal line corresponding to the second scale, the spacing between the first scale and the second scale is L, the pixel corresponding to the first scale is Y1, the pixel corresponding to the second scale is Y2, the pixel corresponding to the delamination boundary between the outer cell layer and the inner cell layer is A, and k is 0, then the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r = RL × {(A-Y1) / (Y2-Y1)}; If the delamination boundary between the outer cell layer and the inner cell layer corresponds to one of the scale lines, then the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained by the following formula: r=Rk, Among them, the scale corresponding to Y1 corresponds to the inner wall of the centrifugal chamber, and k is the distance between the scale line and the scale corresponding to Y1.

11. The cell separation method according to claim 1 or 2, characterized in that: V 外设 20-60ml.

12. The cell separation method according to claim 1, characterized in that: After step (3), the method further includes: (4) Transfer the inner cell layer. When the volume of the inner cell layer in the centrifuge is V 内 Equal to V 内设 When the transfer stops, V 内设 is the target volume value, wherein the cell sample stratification image is collected in real time during the transfer process, and the collected cell sample stratification image is processed, and the volume V of the inner cell layer after stratification is obtained based on the processing result 内 , the volume of the inner cell layer V 内 Obtained by the following formula: V 内 =πr 2 h-πr1 2 h, Among them, r is the distance between the outermost side of the inner cell layer and the center of the centrifuge, and r1 is the distance between the innermost side of the inner cell layer and the center of the centrifuge.

13. The cell separation method according to any one of claims 1 to 12, characterized in that: The method is implemented by the following device, which includes: A centrifuge, the centrifuge comprising a top cover, a base, and a first delivery channel, the top cover being made of a transparent material, the top cover and the base forming a centrifugal chamber, the top cover being provided with scales extending along its radial direction, the first delivery channel extending into the centrifugal chamber for transferring the outer cell layer; An image acquisition module, which is used to collect layered images of cell samples; An image processing module, wherein the image processing module is used to process the collected cell sample layered image, wherein the distance r between the outermost side of the inner cell layer and the center of the centrifuge is obtained according to the scale corresponding to the layered boundary between the inner cell layer and the outer cell layer and the pixels corresponding to the scale; a liquid transfer module, the liquid transfer module being in communication with the first delivery channel and being used to provide power for transferring the outer cell layer; a drive module, the drive module being connected with the centrifuge and being used to drive the centrifuge to rotate; A controller is connected to the image processing module, the liquid transfer module and the driving module.

14. The cell separation method according to claim 13, characterized in that: A support portion is provided on the upper side of the top cover, and the support portion is provided along the radial direction of the top cover. The support portion includes a support platform and a convex portion provided on the support platform. The support platform has a horizontal plane and inclined planes located below both sides of the horizontal plane, wherein one of the inclined planes is inclined toward a direction close to the other inclined plane, and the scale is provided on one of the inclined planes, and a projection of the scale on the horizontal plane where the top cover is located is perpendicular to the extension direction of the support portion.

15. The cell separation method according to claim 14, characterized in that: The scale is recessed on the inclined surface of the support platform.

16. The cell separation method according to claim 13, characterized in that: The centrifuge also includes a second transfer channel extending into the centrifugal chamber for transferring the inner cell layer.

17. The cell separation method according to claim 13, characterized in that: The image acquisition module is a camera or a camera.

Images