Mesenchymal stem cell separation and culture method based on real-time monitoring

Abstract

The invention relates to the technical field of stem cells, in particular to a mesenchymal stem cell separation and culture method based on real-time monitoring. The method comprises the following steps: S1, collecting a bone marrow sample; step S2, primary separation; s3, carrying out enzyme digestion; step S4, carrying out secondary separation; and S5, changing the liquid. According to the method, due to the fact that the first-time liquid changing time has a certain influence on reproduction and growth of the mesenchymal stem cells, the separation quality is guaranteed and the separation efficiency is improved by monitoring the key step, namely liquid changing, of the adherent method in real time, refining the liquid changing time and accurately determining the first-time liquid changing time, and meanwhile the separation efficiency is improved. A camera device and an image processing technology are adopted, the color change and bubble state of a culture solution are monitored in real time, culture conditions are adjusted in time, it is ensured that the growth environment of cells is appropriate, through an automatic monitoring and feedback mechanism, the requirement for manual intervention is reduced, and operation simplicity and reliability are improved; the separation and culture efficiency of the mesenchymal stem cells is improved.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, and in particular to a method for isolating and culturing mesenchymal stem cells based on real-time monitoring. Background Art

[0002] Mesenchymal stem cells (MSCs), a branch of stem cell research, are cells capable of self-replication and multidirectional differentiation. They can continuously self-renew and, under specific conditions, transform into one or more of the cells that make up human tissues or organs. MSCs are found not only in bone marrow but also in skeletal muscle, periosteum, and trabecular bone. Existing MSC isolation and culture methods suffer from low efficiency, long production times, and poor cell viability. Therefore, a real-time monitoring isolation and culture method is urgently needed to optimize culture conditions by analyzing real-time monitoring data.

[0003] Chinese patent publication number: CN110885785B discloses a method for isolating and culturing mesenchymal stem cells. The technical point of the method is to shorten the isolation time of mesenchymal stem cells, increase the yield of mesenchymal stem cells, and enhance their proliferation ability and biological efficacy through a high-temperature induction step. It can be seen that existing stem cell isolation and culture methods lack a method that can achieve purification of mesenchymal stem cells by optimizing culture conditions and refining the fluid exchange time, and can ensure separation quality and improve separation efficiency. Summary of the Invention

[0004] To this end, the present invention provides a method for isolating and culturing mesenchymal stem cells based on real-time monitoring, which is used to overcome the problem in the prior art that it is difficult to ensure the accurate time of medium replacement, thereby hindering cell proliferation and causing cells to overgrow when the culture medium is insufficiently nutritious, making it difficult to ensure separation quality.

[0005] To achieve the above objectives, the present invention provides a method for isolating and culturing mesenchymal stem cells based on real-time monitoring, comprising:

[0006] Step S1, collecting and processing a bone marrow sample under sterile conditions to obtain primary tissue;

[0007] Step S2, performing initial separation on the original tissue to obtain a suspension to be digested;

[0008] Step S3, dissolving the suspension to be digested with collagenase to release the mesenchymal stem cells, and then filtering and washing to form a cell suspension to be separated;

[0009] Step S4, placing the cell suspension to be separated on a culture dish coated with adhesion protein for adhesion, so that the target cells adhere to the culture dish;

[0010] Step S5, monitoring the adhesion process, and determining whether to replace the culture medium or modify the culture parameters based on the monitoring results;

[0011] The culture parameters include a preset stirring rate and a surface area of the culture medium;

[0012] The culture fluid images are acquired at preset acquisition intervals, the average grayscale value corresponding to each culture fluid image is calculated, and a curve of the average grayscale value changing over time is plotted to obtain an average grayscale curve;

[0013] The average grayscale curve is judged, and when an inflection point appears in the average grayscale curve and the real-time liquid replacement chromaticity is less than or equal to the standard liquid replacement chromaticity, the liquid is replaced;

[0014] When it is determined that no inflection point appears in the average grayscale curve and the single attachment time is greater than or equal to the standard medium change time, whether to change the medium or modify the culture parameters is determined based on the real-time oxygen dissolution rate.

[0015] Furthermore, in the step S5, it includes:

[0016] The culture dish is subjected to several fluid exchange operations. During the first attachment or attachment after any fluid exchange operation, images of the upper surface of the culture fluid are periodically captured at a preset time interval by a camera device to obtain a cumulative number of culture fluid images, calculate the average grayscale value corresponding to each culture fluid image, plot a curve of the average grayscale value changing with time, obtain an average grayscale curve, and update the average grayscale curve when the culture fluid image is captured in the next acquisition period.

[0017] Furthermore, in the step S5, it also includes:

[0018] Get the average grayscale curve drawn in the current acquisition cycle and make a judgment.

[0019] If an inflection point appears in the average grayscale curve, the real-time fluid change chromaticity is compared with the standard fluid change chromaticity to determine whether to perform fluid change in the current adhesion cycle;

[0020] If no inflection point appears in the average grayscale curve, the single adhesion time is compared with the standard medium change time to determine whether to perform medium change in the current adhesion cycle.

[0021] Furthermore, in the step S5, it also includes, when it is determined that an inflection point appears in the average grayscale curve, obtaining the real-time liquid change chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle, comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, and if the real-time liquid change chromaticity is less than or equal to the standard liquid change chromaticity, performing liquid change.

[0022] Furthermore, in the step S5, it also includes, when it is determined that an inflection point appears in the average grayscale curve, obtaining the real-time liquid change chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle, comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, and if the real-time liquid change chromaticity is greater than the standard liquid change chromaticity, comparing the single adhesion time with the standard liquid change time to determine whether to change the liquid in the current adhesion cycle.

[0023] Furthermore, in the step S5, it also includes, when it is determined that an inflection point appears in the average grayscale curve, obtaining the real-time liquid change chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle, comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, and if the real-time liquid change chromaticity is greater than the standard liquid change chromaticity, comparing the single adhesion time with the standard liquid change time to determine whether to change the liquid in the current adhesion cycle.

[0024] Furthermore, in the step S5, when it is determined that the real-time liquid exchange chromaticity is greater than or equal to the standard liquid exchange chromaticity, or when no inflection point appears in the average grayscale curve, a single adherence time is obtained, and the single adherence time is compared with the standard liquid exchange time.

[0025] If the single adhesion time is less than the standard medium change time, continue the adhesion operation and obtain the monitoring results of the next adhesion cycle for judgment;

[0026] If the single attachment time is longer than or equal to the standard medium change time, determine whether to change the medium or modify the culture parameters based on the real-time oxygen dissolution rate.

[0027] Furthermore, the culture parameters are modified based on the real-time oxygen dissolution rate, including:

[0028] Obtain the real-time oxygen dissolution rate in the culture medium and compare it with the standard oxygen dissolution rate.

[0029] When the real-time oxygen dissolution rate is greater than or equal to the standard oxygen dissolution rate, the preset liquid replacement dose is adjusted to the corrected liquid replacement dose, and the liquid is replaced. The culture medium with the corrected liquid replacement dose is added to the cell suspension to be separated, and the monitoring results of the next attachment cycle are continued to be determined;

[0030] When the real-time oxygen dissolution rate is lower than the standard oxygen dissolution rate, the morphology of bubbles formed in the culture medium is analyzed, and the preset stirring rate or the surface area of the culture medium is adjusted based on the analysis result.

[0031] Further, analyzing the morphology of bubbles formed in the culture medium, and adjusting the preset stirring rate or the surface area of the culture medium based on the analysis results include,

[0032] Get the maximum bubble area difference among all bubbles, and compare the standard bubble area difference with the maximum bubble area difference.

[0033] If the maximum bubble area difference is less than or equal to the standard bubble area difference, determining whether to increase the preset stirring rate based on the bubble burst frequency;

[0034] If the maximum bubble area difference is greater than the standard bubble area difference, the sign of the maximum bubble area difference is analyzed, and the preset stirring rate is increased or the surface area of the culture medium is increased based on the analysis result.

[0035] Furthermore, collecting bone marrow samples under sterile conditions and processing them include,

[0036] Under sterile conditions, a bone marrow sample was obtained from the donor;

[0037] Washing the bone marrow sample with a buffer and centrifuging the sample;

[0038] The cleaned bone marrow sample is cut to obtain several tissue samples.

[0039] Compared with the existing technology, the beneficial effect of the present invention is that, since fluid exchange is a key step in the adhesion method, the time of the first fluid exchange has a certain impact on the reproductive growth of mesenchymal stem cells. Therefore, by refining the fluid exchange time and accurately determining the time of the first fluid exchange, the separation quality is guaranteed and the separation efficiency is improved. At the same time, a camera device and image processing technology are used to monitor the color changes and bubble status of the culture medium in real time, and the culture conditions are adjusted in time to ensure that the cell growth environment is suitable. Through the automated monitoring and feedback mechanism, the need for manual intervention is reduced, the simplicity and reliability of the operation are improved, and the separation and culture efficiency of mesenchymal stem cells is improved.

[0040] Furthermore, when the bubbles are large, the stirring rate is increased to effectively control the size of the bubbles and ensure that the bubbles are within an appropriate range. When the bubbles are small, it is preferred to increase the surface area of the culture container to promote the entry of oxygen and the discharge of carbon dioxide, ensuring a high efficiency of gas exchange, thereby improving the dissolution efficiency of oxygen and further improving the growth and differentiation efficiency of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the process of isolating and culturing mesenchymal stem cells based on real-time monitoring according to an embodiment of the present invention;

[0042] Figure 2 A schematic diagram of the process of collecting bone marrow samples and processing the same according to an embodiment of the present invention;

[0043] Figure 3Schematic diagram of a process for adjusting a preset stirring rate or a surface area of a culture medium based on analysis results according to an embodiment of the present invention;

[0044] Figure 4 This is a flow chart of determining whether to increase a preset stirring rate based on the bubble burst frequency according to an embodiment of the present invention. DETAILED DESCRIPTION

[0045] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0046] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0047] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside", and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0048] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0049] See also Figure 1 and Figure 2 As shown, Figure 1 Schematic diagram of the process of isolating and culturing mesenchymal stem cells based on real-time monitoring according to an embodiment of the present invention. Figure 2 The present invention provides a method for isolating and culturing mesenchymal stem cells based on real-time monitoring, which includes:

[0050] Step S1, collecting bone marrow samples under sterile conditions and processing them to obtain original tissue, including:

[0051] Step S101, extracting a bone marrow sample from a donor under sterile conditions;

[0052] Step S102, washing and centrifuging the bone marrow sample using a buffer solution;

[0053] Step S103, cutting the cleaned bone marrow sample to obtain several tissue samples;

[0054] Step S2, performing initial separation on the original tissue to obtain a suspension to be digested;

[0055] Step S3, enzymatic digestion, dissolving the suspension to be digested with collagenase to release the mesenchymal stem cells, and then filtering and washing to form a cell suspension to be separated;

[0056] Step S4, secondary separation, placing the cell suspension to be separated on a culture dish coated with adhesion protein for adhesion, so that the target cells adhere to the culture dish;

[0057] Step S5: changing the medium, monitoring the adhesion process, and determining whether to change the medium or modify the culture parameters based on the monitoring results;

[0058] The culture parameters include a preset stirring rate and a surface area of the culture medium;

[0059] Among them, the culture fluid image is obtained by a camera device at a preset acquisition interval, the average grayscale value corresponding to each culture fluid image is calculated, and a curve of the change of the average grayscale value over time is drawn to obtain an average grayscale curve, and the average grayscale curve is judged. When it is judged that an inflection point appears in the average grayscale curve and the real-time fluid change chromaticity is less than or equal to the standard fluid change chromaticity, fluid change is performed; when it is judged that no inflection point appears in the average grayscale curve and the single wall attachment time is greater than or equal to the standard fluid change time, it is determined whether to change the fluid or correct the culture parameters based on the real-time oxygen dissolution rate.

[0060] In this embodiment, the preset collection interval is set to 5 hours; bone marrow is selected as the tissue source of mesenchymal stem cells, and adipose tissue or umbilical cord blood can also be selected as the tissue source of mesenchymal stem cells; a bone marrow sample is extracted from a donor (such as a mouse or a human) under sterile conditions, and the collected bone marrow sample is placed in a sterile centrifuge tube, and the bone marrow sample is washed with physiological saline or a suitable buffer (such as PBS), gently shaken to remove blood and impurities on the surface, and the supernatant is removed by centrifugation (300-400g, 5-10 minutes) to ensure that the bone marrow sample is clean, and the washed bone marrow sample is cut into small pieces, cut into 1-2 mm cubes to increase the surface area for enzymatic digestion, and cut with a sterile scalpel or scissors to ensure the sterility of the cutting tool. Place the cut tissue pieces in an appropriate amount of digestive enzyme solution (collagenase). The digestion conditions include a temperature of 37°C and a time of 1 hour. During the digestion process, shake the mixture gently periodically to promote the uniform action of the enzyme. After digestion, filter the digestion solution using a sterile cell strainer to remove undigested tissue pieces. Resuspend the filtered cell suspension in physiological saline or culture medium and adjust the cell concentration to 1×10 6 to 1×10 7 cells / mL, filter the resuspended cell suspension using a sterile cell strainer to remove cell clumps and impurities to obtain a bone marrow cell suspension; trypsin is used as the adhesion protein.

[0061] Since medium change is a key step in the adherence method, the time of the first medium change has a certain impact on the reproductive growth of mesenchymal stem cells. Therefore, by refining the medium change time and accurately determining the first medium change time, the separation quality is guaranteed and the separation efficiency is improved. At the same time, the camera device and image processing technology are used to monitor the color changes and bubble status of the culture medium in real time, and the culture conditions are adjusted in time to ensure that the cell growth environment is suitable. Through the automated monitoring and feedback mechanism, the need for manual intervention is reduced, the simplicity and reliability of the operation are improved, and the separation and culture efficiency of mesenchymal stem cells is improved.

[0062] Specifically, in step S5, a standard chromaticity change rate is set, and the culture dish is subjected to several liquid replacement operations. When the dish adheres to the wall for the first time or after any liquid replacement operation, the camera device periodically captures images of the upper surface of the culture liquid at a preset time acquisition interval to obtain a cumulative number of culture liquid images, calculate the average grayscale value corresponding to each culture liquid image, and draw a curve of the change of the average grayscale value over time to obtain an average grayscale curve. When the culture liquid image is captured in the next acquisition cycle, the average grayscale curve is updated.

[0063] Through associative image recognition technology, the color changes of the culture medium are regularly monitored to characterize the changes in the pH value of the culture medium, indirectly indicating the metabolic state of the cells and the nutritional status of the culture medium. As the monitoring time progresses, the pH value of the culture medium decreases due to the production of acidic substances such as lactic acid by cell metabolism, and the color of the culture medium changes from red to yellow. By plotting the change curve of the image grayscale value versus the acquisition time, the degree of culture medium color change is intuitively displayed, so as to intelligently indicate the time for medium change, avoid the first medium change being too late, so that impurities adhere tightly to the wall and reduce the purity of the target separated cells.

[0064] Specifically, in step S5, the average grayscale curve drawn during the current acquisition period is obtained and judged.

[0065] If an inflection point appears in the average grayscale curve, the real-time medium change chromaticity is compared with the standard medium change chromaticity to determine whether to change the medium in the current attachment cycle;

[0066] If no inflection point appears in the average grayscale curve, the single attachment time is compared with the standard medium change time to determine whether to change the medium in the current attachment cycle.

[0067] In this embodiment, the preset time acquisition interval can be set to 10 minutes to 15 minutes, which can be selected according to the cell density in the actual adhesion operation and the specifications of the culture medium used; the standard slope represents the degree of grayscale change of the two collected images, representing the degree of color change of the culture medium. The more obvious the color change, the greater the rate of color change. The standard slope approaches zero and can be set between 0.1 and 0.3; an acquisition cycle is the length of time between the two end points of the preset time acquisition interval; for example, if the slope suddenly changes from -0.05 to -0.25, it means that the average grayscale curve has an inflection point. By comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, the real-time liquid change chromaticity = 175 (<180). Under this condition, the liquid change condition is triggered. The separation effect is characterized by monitoring the degree of color change of the culture medium. If it is determined that there is no inflection point in the average grayscale curve, it means that the color change of the culture medium is not obvious. This may be due to a short attachment time and the cells have not started metabolic activity. It may also be due to the mismatch between cell density and culture medium dosage, which hinders cell metabolism and makes the separation effect poor. The attachment time is then determined to analyze the reasons for the lack of obvious color change of the culture medium. If it is determined that there is an inflection point in the average grayscale curve, it means that the color change of the culture medium is more obvious. In this case, it is necessary to determine whether the culture medium needs to be replaced in time based on the color change.

[0068] Specifically, in step S5, a standard liquid exchange chromaticity is set. When it is determined that an inflection point appears in the average grayscale curve, the real-time liquid exchange chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle is obtained, and the real-time liquid exchange chromaticity is compared with the standard liquid exchange chromaticity.

[0069] If the real-time liquid change chromaticity is less than or equal to the standard liquid change chromaticity, perform liquid change;

[0070] If the real-time medium change chromaticity is greater than the standard medium change chromaticity, the single attachment time is compared with the standard medium change time to determine whether to change the medium in the current attachment cycle.

[0071] The standard medium exchange chromaticity set in this embodiment is expressed as the chromaticity value of lighter yellow in the grayscale image. The lighter yellow has a higher grayscale value, and the darker yellow has a lower grayscale value. The standard medium exchange chromaticity is set between 150 and 200, preferably set to 180, and is selected according to the actual culture dish specifications and cell density.

[0072] When the color change of the culture medium is determined to be more obvious, the color of the culture medium is specifically analyzed. If the real-time culture medium chromaticity is determined to be less than the standard culture medium chromaticity, it means that the color of the actual culture medium has reached the set standard chromaticity. In order to avoid excessive growth of cells without sufficient nutrients, the culture medium is replaced when the color turns to a lighter yellow to remove metabolic products in time and provide new nutrients. If the real-time culture medium chromaticity is determined to be greater than the standard culture medium chromaticity, it means that the color of the actual culture medium has not reached the set standard chromaticity. The attachment time will be determined to analyze the reason why the culture medium color has not reached the set standard chromaticity.

[0073] Specifically, in step S5, a standard fluid exchange time is set. When it is determined that the real-time fluid exchange chromaticity is greater than or equal to the standard fluid exchange chromaticity, or when no inflection point appears in the average grayscale curve, the single adhesion time is obtained and compared with the standard fluid exchange time.

[0074] If the single adhesion time is less than the standard medium change time, continue the adhesion operation and obtain the monitoring results of the next adhesion cycle for judgment;

[0075] If the single attachment time is longer than or equal to the standard medium change time, determine whether to change the medium or modify the culture parameters based on the real-time oxygen dissolution rate.

[0076] The single attachment time is the time from the moment the culture medium is added to the culture dish containing the cell suspension to be separated during the secondary separation of the cell suspension to be separated, until the real-time medium change chromaticity is determined to be greater than or equal to the standard medium change chromaticity during the current cycle. The attachment cycle is the time from the moment the culture medium is added to the culture dish containing the cell suspension to be separated, until the medium change is determined to be complete during the secondary separation of the cell suspension to be separated, for the current attachment operation.

[0077] In this embodiment, the standard fluid change time is set to 20 hours, and the number of fluid changes is 4 times; the preset fluid change dose is related to the cell density and the culture dish size, and can be set to 1 micromole per liter; if no inflection point is detected in the average grayscale curve, or by comparing the real-time fluid change chromaticity with the standard fluid change chromaticity, when the real-time fluid change chromaticity = 210 (> 180), continue to monitor the culture, and when the single attachment time is greater than or equal to the standard fluid change time, trigger the monitoring of the oxygen dissolution rate. If the oxygen dissolution rate = 6 mg / L / h (< 7 mg / L / h), it is necessary to adjust the parameters through bubble morphology analysis. If the average bubble area = 0.2 mm 2 , maximum bubble area difference = +0.15mm 2 (>Standard deviation 0.1mm 2 ), in this case, the difference is positive, so the stirring rate is increased: the original rate is 50 rpm, and the corrected rate = 50 × (1 + 0.15 / 0.15) = 100 rpm. When the color change of the culture medium is not obvious or the color of the culture medium does not reach the standard color of the culture medium change, the actual attachment time is determined to characterize the cell growth status. If the single attachment time is determined to be less than the standard culture medium change time, it means that due to the short attachment time, the cell growth metabolic activity is not obvious, the amount of metabolites is small, and the culture medium color change is not obvious or does not reach the standard color of the culture medium change. If the single attachment time is determined to be greater than or equal to the standard culture medium change time, the bubble morphology in the culture medium is analyzed to determine whether the cell growth environment is optimized.

[0078] Specifically, culture parameters were modified based on real-time oxygen dissolution rates, including:

[0079] Obtain the real-time oxygen dissolution rate in the culture medium and compare it with the standard oxygen dissolution rate.

[0080] When the real-time oxygen dissolution rate is greater than or equal to the standard oxygen dissolution rate, the preset liquid replacement dose is adjusted to the corrected liquid replacement dose, and the liquid is replaced. The culture medium with the corrected liquid replacement dose is added to the cell suspension to be separated, and the monitoring results of the next attachment cycle are continued to be determined;

[0081] The corrected liquid replacement dose is the product of the preset liquid replacement dose and the correction step length, the correction step length is the sum of 1 and the correction degree, and the correction degree is the ratio of the portion of the real-time oxygen dissolution rate that exceeds the standard oxygen dissolution rate to the standard oxygen dissolution rate.

[0082] When the real-time oxygen dissolution rate is lower than the standard oxygen dissolution rate, the morphology of bubbles formed in the culture medium is analyzed, and the preset stirring rate or the surface area of the culture medium is adjusted based on the analysis result.

[0083] The standard oxygen dissolution rate in this embodiment represents the ideal dissolution rate of oxygen in the culture medium, reflecting the oxygen supply required by the cells during growth to ensure that the cells can carry out normal metabolic activities. Generally, for the culture of mesenchymal stem cells, the standard oxygen dissolution rate can be set between 5 mg / L / h and 10 mg / L / h. Rapidly proliferating cells require a higher oxygen dissolution rate.

[0084] By installing an adjustable-speed agitator, the culture medium is ensured to be evenly mixed in the culture vessel. By introducing a bubble generator into the culture system, the upward movement of bubbles is used to promote the circulation of the culture medium. The size and frequency of the bubbles are adjusted to ensure that the bubbles do not cause excessive impact on the cells. The introduction of bubbles through the bubble generator promotes the circulation of the culture medium and increases the solubility of oxygen. Therefore, the oxygen dissolution rate is used to characterize the environment in which cells grow, and the culture conditions can be optimized based on the monitoring results to improve the efficiency of cell growth and differentiation. When the real-time oxygen dissolution rate is determined to be greater than or equal to the standard oxygen dissolution rate, it indicates that the oxygen dissolution efficiency is normal. However, as the attachment time increases, the cell density is higher, while the culture medium dosage is lower, which is not conducive to the dispersed growth of cells, thereby hindering cell growth and metabolic activity. In this case, the medium needs to be changed, and the replacement medium dosage needs to be increased to better diffuse and disperse the cells, avoid cell overcrowding, and promote cell separation and growth. When the real-time oxygen dissolution rate is determined to be less than the standard oxygen dissolution rate, it indicates that the oxygen dissolution efficiency is affected, and the cause needs to be analyzed and the stirring and bubble technology needs to be optimized.

[0085] See Figure 3 As shown, it is a schematic diagram of a process for adjusting a preset stirring rate or a surface area of a culture medium based on analysis results according to an embodiment of the present invention;

[0086] Specifically, analyzing the morphology of bubbles formed in the culture medium, and adjusting the preset stirring rate or the surface area of the culture medium based on the analysis results include:

[0087] Step S5001, obtaining the average bubble area of each bubble in the culture medium;

[0088] Step S5002: subtract the average bubble area from the real-time bubble area of each bubble to obtain the corresponding real-time bubble area difference;

[0089] Step S5003, selecting the maximum value of the real-time bubble area differences to obtain the maximum bubble area difference;

[0090] Step S5004: compare the standard bubble area difference with the maximum bubble area difference.

[0091] If the maximum bubble area difference is less than or equal to the standard bubble area difference, determining whether to increase the preset stirring rate based on the bubble burst frequency;

[0092] If the maximum bubble area difference is greater than the standard bubble area difference, the sign of the maximum bubble area difference is analyzed, and the preset stirring rate is increased or the surface area of the culture medium is increased based on the analysis result.

[0093] In this embodiment, the maximum bubble area difference represents the maximum value of the difference between the bubble area of each bubble in the culture medium and the average bubble area. Since the size and frequency of the bubbles affect the dissolution efficiency of oxygen, the culture conditions can be adjusted in a targeted manner by real-time monitoring of the bubble morphology to improve the accuracy and efficiency of cell culture. That is, based on the real-time monitoring results, the stirring rate and the surface area of the culture medium are dynamically adjusted to ensure high gas exchange efficiency, promote oxygen dissolution, and meet the growth requirements of cells.

[0094] See Figure 4 As shown, it is a flow chart of determining whether to increase the preset stirring rate based on the bubble burst frequency according to an embodiment of the present invention;

[0095] Specifically, determining whether to increase the preset stirring rate based on the bubble collapse frequency includes:

[0096] Step S5014, shooting a video of the cultivation process to obtain a video stream;

[0097] Step S5024, read the video stream and process each frame of the image, convert the image into a grayscale image and perform Gaussian blur to reduce noise;

[0098] Step S5034, calculating the difference between the current frame and the previous frame to obtain the bubble burst frequency;

[0099] Among them, the bubble is identified by using the contour detection method, and the frequency of bubble bursting is calculated at regular intervals to obtain the bubble burst frequency;

[0100] Step S5044, comparing the bubble burst frequency with the minimum burst frequency, and determining whether to increase the preset stirring rate based on the comparison;

[0101] If the bubble burst frequency is lower than the minimum burst frequency, the preset stirring rate is increased to the first stirring rate;

[0102] If the bubble burst frequency is greater than or equal to the minimum burst frequency, the preset stirring rate will not be increased;

[0103] The first stirring rate is the product of the preset stirring rate and the first adjustment step, the first adjustment step is the sum of 1 and the first adjustment degree, and the first adjustment degree is the ratio of the part of the minimum burst frequency exceeding the bubble burst frequency to the minimum burst frequency.

[0104] The minimum rupture frequency in this embodiment represents the minimum frequency of bubble rupture in the liquid under the culture conditions of this embodiment. This frequency is used to measure whether the bubbles can effectively contact the liquid, thereby forming a sufficient gas-liquid interface to promote oxygen dissolution. If the frequency of bubble rupture is lower than the minimum rupture frequency, it means that the bubble rupture speed is insufficient, which will lead to insufficient formation of the gas-liquid interface, thereby affecting the oxygen dissolution efficiency and ultimately affecting cell growth and metabolism. The minimum rupture frequency can be set between 0.5 Hz and 2 Hz (hertz). The higher the minimum rupture frequency setting value, the higher the demand for gas exchange efficiency. The calculation method of the bubble rupture frequency is the existing technology and will not be repeated here.

[0105] Specifically, the analysis of the positive and negative values of the maximum bubble area difference includes:

[0106] If the maximum bubble area difference is a positive value, the preset stirring rate is increased to the second stirring rate;

[0107] If the maximum bubble area difference is negative, another medium is selected to perform the medium exchange operation based on the increased surface area of the medium;

[0108] The second stirring rate is the product of the preset stirring rate and the second adjustment step size, which is the sum of 1 and the second adjustment level. The second adjustment level is the ratio of the maximum bubble area difference exceeding the standard bubble area difference to the maximum bubble area difference. The medium change operation is to replace 50% of the medium (1 mL) to maintain cell density and nutrient supply.

[0109] In the case of large bubbles, increase the stirring rate to effectively control the size of the bubbles and ensure that the bubbles are within an appropriate range. In the case of small bubbles, increase the surface area of the culture container to promote the entry of oxygen and the discharge of carbon dioxide, ensure high efficiency of gas exchange, thereby improving the dissolution efficiency of oxygen and thus improving the growth and differentiation efficiency of cells.

[0110] Specifically, an example was set up: mouse bone marrow was obtained, digested with collagenase at 37°C for 1 hour, and subcultured using DMEM / F12 medium containing 10% fetal bovine serum. The subculture process was recorded, the standard medium change time was 20 hours, and the standard oxygen dissolution rate was 8 mg / L / h.

[0111] A comparative example was set up: mouse bone marrow was harvested and digested with collagenase at 37°C for 1 hour. The cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum. The subculture process was recorded, and the medium was changed at a fixed time (once every 48 hours). No real-time monitoring or dynamic adjustment was performed.

[0112] The experimental results are shown in Tables 1, 2, and 3, wherein Table 1 is a comparison of the data and results of the first-generation culture embodiment and the comparative example culture process, Table 2 is a comparison of the data and results of the second-generation culture embodiment and the comparative example culture process, and Table 3 is a comparison of the data and results of the third-generation culture embodiment and the comparative example culture process.

[0113] Table 1

[0114] Example Comparative Example Initial cell density <![CDATA[1×10 6 cells / mL]]> <![CDATA[1×10 6 cells / mL]]> First attachment time 6 hours 8 hours First fluid change time After 12 hours (according to the average grayscale curve) After 48 hours (fixed time) Total culture time 4 days 5 days Cell morphology observation Normal, fibrous and densely arranged but not crowded Some cells are aging and have abnormal morphology Survival rate 95％ 85％ Induction differentiation test Fat 85%, Cartilage 80%, Bone 78% Fat 70%, cartilage 65%, bone 60%

[0115] The first fluid change time is 12 hours later (according to the inflection point of the average grayscale curve, the real-time fluid change chromaticity is 190, which is less than the standard fluid change chromaticity of 200);

[0116] As can be seen from Table 1, the method of this embodiment optimizes the medium replacement time and culture parameters through real-time monitoring, shortens the total culture time, and increases cell survival rate and differentiation ability.

[0117] Table 2

[0118]

[0119]

[0120] The first medium change was after 10 hours (according to the average grayscale curve, there was no obvious inflection point, and the single attachment time was up to 15 hours);

[0121] As can be seen from Table 2, the example further showed advantages in the second generation culture, the total culture time was shorter, and the cell survival rate and differentiation ability of the example were significantly better than those of the comparative example.

[0122] Table 3

[0123] Example Comparative Example Initial cell density <![CDATA[0.8×10 6 cells / mL]]> <![CDATA[0.8×10 6 cells / mL]]> First attachment time 7 hours 9 hours First fluid change time After 14 hours (according to the average grayscale curve) After 48 hours (fixed time) Total culture time 4 days 5 days Cell morphology observation Normal, good growth Obvious aging and abnormal morphology Survival rate 93％ 78％ Induction differentiation test Fat 86%, Cartilage 81%, Bone 77% Fat 65%, Cartilage 60%, Bone 55%

[0124] The first fluid change was after 14 hours (according to the inflection point of the average grayscale curve, the real-time fluid change chromaticity was 185, which was less than the standard fluid change chromaticity of 200);

[0125] As shown in Table 3, the examples still maintained a relatively high survival rate and differentiation ability in the third generation of culture; the comparative examples showed serious cell aging in the third generation of culture, and their differentiation ability decreased significantly.

[0126] In general, the examples significantly shortened the subculture time (an average of 3.8 days per generation, compared to 4.5 days per generation in the control group); the cell survival rate of the examples was maintained between 93% and 95%, while that of the control group was only 78% to 85%; the cells of the examples exhibited superior multidirectional differentiation ability, with adipose, cartilage, and bone differentiation efficiencies all exceeding 80%, while the differentiation efficiency of the control group was lower.

[0127] The method of this embodiment is used to isolate and culture mesenchymal stem cells, which can effectively ensure cell survival rate, shorten culture time, normal cell morphology, and excellent differentiation ability. This method combines real-time monitoring technology to accurately control the fluid exchange time and culture parameters, significantly improving culture efficiency and cell quality, and is suitable for large-scale clinical applications and scientific research.

[0128] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

[0129] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for isolating and culturing mesenchymal stem cells based on real-time monitoring, characterized in that: include, Step S1, collecting and processing a bone marrow sample under sterile conditions to obtain primary tissue; Step S2, performing initial separation on the original tissue to obtain a suspension to be digested; Step S3, dissolving the suspension to be digested with collagenase to release the mesenchymal stem cells, and then filtering and washing to form a cell suspension to be separated; Step S4, placing the cell suspension to be separated on a culture dish coated with adhesion protein for adhesion, so that the target cells adhere to the culture dish; Step S5, monitoring the attachment process, and determining whether to replace the culture medium or modify the culture parameters based on the monitoring results, wherein the culture parameters include a preset stirring rate and the surface area of the culture medium; The culture fluid images are acquired at preset acquisition intervals by a camera device, the average grayscale value corresponding to each culture fluid image is calculated, and a curve of the average grayscale value changing over time is plotted to obtain an average grayscale curve; The average grayscale curve is judged, and when an inflection point appears in the average grayscale curve and the real-time liquid replacement chromaticity is less than or equal to the standard liquid replacement chromaticity, the liquid is replaced; When it is determined that no inflection point appears in the average grayscale curve and the single attachment time is greater than or equal to the standard medium change time, whether to change the medium or modify the culture parameters is determined based on the real-time oxygen dissolution rate.

2. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 1, characterized in that: In said step S5, it includes: The culture dish is subjected to several fluid exchange operations. When the culture dish adheres to the wall for the first time or after any fluid exchange operation, images of the upper surface of the culture liquid are periodically captured at a preset time acquisition interval to obtain a cumulative number of culture liquid images, calculate the average grayscale value corresponding to each culture liquid image, plot a curve of the average grayscale value changing with time, and obtain an average grayscale curve. The average grayscale curve is updated when the culture liquid image is captured in the next acquisition period.

3. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 2, characterized in that: In the step S5, it further includes: Get the average grayscale curve drawn in the current acquisition cycle and make a judgment. If an inflection point appears in the average grayscale curve, the real-time fluid change chromaticity is compared with the standard fluid change chromaticity to determine whether to perform fluid change in the current adhesion cycle; If no inflection point appears in the average grayscale curve, the single adhesion time is compared with the standard medium change time to determine whether to perform medium change in the current adhesion cycle.

4. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 3, characterized in that: In the step S5, it also includes obtaining the real-time liquid change chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle when it is determined that an inflection point appears in the average grayscale curve, comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, and if the real-time liquid change chromaticity is less than or equal to the standard liquid change chromaticity, performing liquid change.

5. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 4, characterized in that: In the step S5, it also includes obtaining the real-time liquid change chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle when it is determined that an inflection point appears in the average grayscale curve, comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, and if the real-time liquid change chromaticity is greater than the standard liquid change chromaticity, comparing the single adhesion time with the standard liquid change time to determine whether to change the liquid in the current adhesion cycle.

6. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 5, characterized in that: In the step S5, it also includes obtaining the real-time liquid change chromaticity of the culture medium collected at the end of the preset time collection interval in the current collection cycle when it is determined that an inflection point appears in the average grayscale curve, comparing the real-time liquid change chromaticity with the standard liquid change chromaticity, and if the real-time liquid change chromaticity is greater than the standard liquid change chromaticity, comparing the single adhesion time with the standard liquid change time to determine whether to change the liquid in the current adhesion cycle.

7. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 6, characterized in that: In the step S5, it further includes obtaining a single adherence time when it is determined that the real-time fluid exchange chromaticity is greater than or equal to the standard fluid exchange chromaticity, or when no inflection point appears in the average grayscale curve, and comparing the single adherence time with the standard fluid exchange time. If the single adhesion time is less than the standard medium change time, continue the adhesion operation and obtain the monitoring results of the next adhesion cycle for judgment; If the single attachment time is longer than or equal to the standard medium change time, determine whether to change the medium or modify the culture parameters based on the real-time oxygen dissolution rate.

8. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 7, characterized in that: Correction of culture parameters based on real-time oxygen dissolution rate includes: Obtain the real-time oxygen dissolution rate in the culture medium and compare it with the standard oxygen dissolution rate. When the real-time oxygen dissolution rate is greater than or equal to the standard oxygen dissolution rate, the preset liquid replacement dose is adjusted to the corrected liquid replacement dose, and the liquid is replaced. The culture medium with the corrected liquid replacement dose is added to the cell suspension to be separated, and the monitoring results of the next attachment cycle are continued to be determined; When the real-time oxygen dissolution rate is lower than the standard oxygen dissolution rate, the morphology of bubbles formed in the culture medium is analyzed, and the preset stirring rate or the surface area of the culture medium is adjusted based on the analysis result.

9. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 8, characterized in that: Analyze the morphology of bubbles formed in the culture medium and adjust the preset stirring rate or the surface area of the culture medium based on the analysis results, including: Get the maximum bubble area difference among all bubbles, and compare the standard bubble area difference with the maximum bubble area difference. If the maximum bubble area difference is less than or equal to the standard bubble area difference, determining whether to increase the preset stirring rate based on the bubble burst frequency; If the maximum bubble area difference is greater than the standard bubble area difference, the sign of the maximum bubble area difference is analyzed, and the preset stirring rate is increased or the surface area of the culture medium is increased based on the analysis result.

10. The method for isolating and culturing mesenchymal stem cells based on real-time monitoring according to claim 1, characterized in that: Collection and processing of bone marrow samples under sterile conditions includes: Under sterile conditions, a bone marrow sample was obtained from the donor; Washing the bone marrow sample with a buffer and centrifuging the sample; The cleaned bone marrow sample is cut to obtain several tissue samples.

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