Single-cell suspension and preparation method thereof

Abstract

The invention provides a single-cell suspension and a preparation method thereof. Specifically, the preparation method comprises the following steps: 1) mixing a condensed-state cell sample to be dispersed with a pretreatment solution, and carrying out ultrasonic treatment; 2) mixing the sample obtained in the step 1) with the enzymatic hydrolysate, and carrying out an enzymatic hydrolysis reaction; 3) adding a nanometer material with the surface modified with a target cell specific marker, controlling the distribution of the nanometer material through an external magnetic field, and enriching target single cells; 4) separating the sample obtained in the step 3) to obtain a target single-cell suspension; wherein the conditions of the ultrasonic treatment and the enzymolysis reaction are controlled by an intelligent control system. According to the technical scheme, ultrasonic treatment and enzymolysis reaction are combined, an intelligent control system is used for controlling ultrasonic conditions and enzymolysis conditions respectively, and a nano material labeling technology is combined, so that the finally obtained cell suspension is high in cell viability and high in purity.

Description

Technical Field

[0001] The present disclosure relates to the field of cell biotechnology, and in particular to a single cell suspension and a preparation method thereof. Background Art

[0002] In biotechnology research, the preparation of highly active single-cell suspensions is the basis of many experiments and applications. Traditional cell dispersion methods usually include mechanical dispersion, enzymatic hydrolysis, ultrasonic treatment and other means. Although these methods can separate cells to a certain extent, they still have some significant disadvantages. For example, mechanical dispersion may cause cell membrane damage, and the unevenness during enzymatic hydrolysis can easily cause cell breakage or loss of activity, while ultrasonic treatment may cause irreversible damage to cells due to improper operation. With the rapid development of single-cell technology, scientists have put forward higher requirements for the purity and activity of cell suspensions. These traditional methods seem to be unable to cope with emerging challenges. Summary of the invention

[0003] In view of this, the purpose of the present disclosure is to provide a single cell suspension and a preparation method.

[0004] Based on the above objectives, the present disclosure provides a method for preparing a single cell suspension, comprising:

[0005] 1) mixing the condensed cell sample to be dispersed with the pretreatment solution and performing ultrasonic treatment;

[0006] 2) mixing the sample obtained in step 1) with the enzymatic hydrolysis solution to perform an enzymatic hydrolysis reaction;

[0007] 3) adding nanomaterials with surface-modified target cell-specific markers, controlling the distribution of the nanomaterials by applying an external magnetic field, and enriching the target single cells;

[0008] 4) Separating the sample obtained in step 3) to obtain a target single cell suspension; wherein the conditions of the ultrasonic treatment and the enzymatic hydrolysis reaction are controlled by an intelligent control system.

[0009] In some embodiments, the controlling by an intelligent control system specifically includes:

[0010] Obtain and determine the corresponding ultrasound model and enzymatic hydrolysis model according to the type of target single cells;

[0011] The ultrasonic treatment conditions are controlled by using an ultrasonic model, and the enzymatic hydrolysis reaction conditions are controlled by using an enzymatic hydrolysis model;

[0012] Obtaining and adjusting the conditions of the ultrasonic treatment and the conditions of the enzymatic hydrolysis reaction according to the particle size, cell activity data, temperature, pH and enzymatic hydrolysis solution concentration of the sample using a feedback model; wherein,

[0013] The ultrasound model, the enzymatic hydrolysis model and the feedback model are respectively trained machine learning algorithm models.

[0014] In some embodiments, the ultrasonic treatment conditions include at least one of ultrasonic frequency, ultrasonic intensity and ultrasonic time; and / or

[0015] The conditions of the enzymatic hydrolysis reaction include at least one of temperature, shaking speed, enzyme type, enzymatic hydrolysis solution concentration and enzymatic hydrolysis time.

[0016] In some embodiments, the enzymatic hydrolysis solution comprises dithiothreitol, a chelating agent and at least one enzyme; wherein the at least one enzyme is selected from at least one of trypsin, collagenase, hyaluronidase and lysozyme.

[0017] In some embodiments, in the enzymatic hydrolysis reaction system of step 2), the content of dithiothreitol is 0.8-1.2 mM; the content of the chelating agent is 1.5-3.5 mM; the content of trypsin is 0.15-0.3%; the content of collagenase is 0.08-0.12%; the content of hyaluronidase is 0.04-0.06%; and the content of lysozyme is 0.1-0.2%.

[0018] In some embodiments, the nanomaterial comprises iron oxide nanoparticles, wherein the diameter of the iron oxide nanoparticles is 10 to 50 nm; and / or

[0019] The specific marker is selected from an antibody or a peptide segment; wherein the antibody or the peptide segment specifically recognizes the target single cell.

[0020] In some embodiments, the strength of the external magnetic field in step 3) is 3000-3500 Gs; the direction of the external magnetic field can be dynamically adjusted.

[0021] In some embodiments, the condensed cell sample is selected from animal or plant tissue, tumor tissue, microbial colony or sludge sample.

[0022] In some embodiments, the ultrasonic treatment includes a first stage and a second stage; wherein the frequency of the first stage is 20kHz to 30kHz; and the frequency of the second stage is 40kHz to 60kHz.

[0023] Based on the same inventive concept, the disclosed embodiments also provide a target single cell suspension obtained by any of the aforementioned preparation methods; wherein the cell activity of the target single cell suspension is ≥90%, and the cell purity is ≥95%.

[0024] As can be seen from the above, the single cell suspension and preparation method provided by the present disclosure first combine ultrasonic treatment and enzymatic hydrolysis reaction, and use an intelligent control system to control the ultrasonic conditions and enzymatic hydrolysis conditions respectively, so as to achieve refined control and full-process monitoring feedback, so that the dispersion process is efficient, stable and the repeatability is significantly improved; combined with nanomaterial labeling technology, the target cells are preliminarily screened and enriched during the dispersion process, which greatly improves the purity of the single cell suspension, and the cell activity and purity of the cell suspension finally obtained are high. In addition, the preparation method of the single cell suspension disclosed in the present disclosure has a wide range of applicability and has great application prospects in the fields of in vitro culture, drug screening, genetic engineering and environmental governance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 An imaging diagram showing a sludge sample provided by an embodiment of the present disclosure after ultrasonic dispersion is shown;

[0027] Figure 2 An image of the sludge sample provided by the embodiment of the present disclosure after enzymatic hydrolysis is shown;

[0028] Figure 3 An image of the sludge sample after filtration provided by an embodiment of the present disclosure is shown;

[0029] Figure 4 An image of the sludge sample provided by an embodiment of the present disclosure after centrifugation is shown. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0031] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should be understood by people with ordinary skills in the field to which the present disclosure belongs. The "first", "second" and similar words used in the embodiments of the present disclosure do not indicate any order, quantity or importance, but are only used to distinguish different components. "Including" or "comprising" and similar words mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0032] The cell dispersion technology of related technologies often relies on the experience of operators, resulting in poor experimental repeatability and difficulty in achieving refined control. In order to overcome this problem, the embodiment of the present disclosure introduces an intelligent control system to achieve full monitoring and dynamic adjustment of the dispersion process.

[0033] In order to facilitate understanding of the technical solution of the present disclosure, the intelligent control system involved in the present disclosure is introduced below.

[0034] The intelligent control system includes a sensor unit, an execution unit and a control module. Here, the control module is connected to the sensor unit and the execution unit respectively.

[0035] The sensor unit consists of multiple high-precision sensors such as pH sensor, temperature sensor, ultrasonic sensor, concentration sensor, cell activity detector, etc., which can monitor the temperature, pH, enzyme solution concentration, ultrasonic intensity, etc. in the reactor in real time. It should be noted that the various data collected by the sensor unit are transmitted to the control module through a wired or wireless network for analysis and optimization by the algorithm.

[0036] The execution unit includes an automatic enzymatic solution adding device, an ultrasonic generator, a temperature control device, and a stirring device. The control module can not only control these devices to place the sample with the best operating parameters according to the preset model, but also adjust the operating parameters of these devices according to the data provided by the sensor unit to ensure the smooth progress of the dispersion process. It should be noted that the sensitivity and response speed of the execution unit have been strictly adjusted, and parameter adjustments can be completed within milliseconds.

[0037] The control module includes an ultrasonic model and an enzymatic hydrolysis model. Here, the ultrasonic model can be used to control the conditions of the ultrasonic treatment, and the enzymatic hydrolysis model can be used to control the conditions of the enzymatic hydrolysis reaction; the ultrasonic model and the enzymatic hydrolysis model are respectively trained machine learning algorithm models. Optionally, the machine learning algorithm can be a gradient descent method, a simulated annealing algorithm, a genetic algorithm, a differential evolution algorithm, or an ant colony algorithm, which is not limited in the present disclosure.

[0038] The training process of the ultrasonic model and the enzymatic hydrolysis model is illustrated. First, prepare the cell dispersion experimental data of various condensed cell samples. Here, the condensed cell samples include but are not limited to animal and plant tissues, tumor tissues, microbial colonies, sludge samples, etc. The cell dispersion experimental data include ultrasonic conditions, enzymatic hydrolysis conditions and corresponding cell activities. Here, the ultrasonic conditions include but are not limited to ultrasonic frequency, ultrasonic intensity, ultrasonic time, etc.; the enzymatic hydrolysis conditions include but are not limited to the parameters of the enzymatic hydrolysis solution, the enzymatic hydrolysis time, the enzymatic hydrolysis temperature, etc. Next, using the cell dispersion experimental data of each cell sample, the preset machine learning algorithm model is trained respectively to obtain the predicted correspondence between cell activity and ultrasonic conditions and enzymatic hydrolysis conditions, such as ultrasonic conditions and enzymatic hydrolysis conditions corresponding to cell activity ≥90%; ultrasonic conditions and enzymatic hydrolysis conditions corresponding to cell activity ≥95%, etc. Then, a single cell suspension is prepared based on the predicted correspondence, the cell activity is monitored, and the monitoring results are used as new cell dispersion experimental data to update the predicted correspondence until the training standard is reached, that is, the ultrasonic model and enzymatic hydrolysis model corresponding to each cell sample are obtained.

[0039] It should be understood that, due to the differences in suitable ultrasonic conditions and enzymatic hydrolysis conditions for different cell samples, different cell samples correspond to different ultrasonic models and enzymatic hydrolysis models. It can be understood by those skilled in the art that for similar cell samples, such as sludge collected from different rivers, the same ultrasonic model and enzymatic hydrolysis model can be applied. The user can select a matching pair model in the intelligent control system according to the different samples, and the present disclosure does not describe this in detail.

[0040] In some embodiments, the control module further includes a feedback model. Here, the feedback model can be used to provide feedback on the parameters of the ultrasonic treatment and the enzymatic treatment. For example, if the cell activity decreases, the intelligent control system will automatically reduce the ultrasonic intensity and compensate for the dispersion effect by increasing the ultrasonic time or extending the enzymatic treatment time. It should be noted that the feedback model can be a trained neural network model.

[0041] In order to make the technical solution of the present disclosure clearer and easier to understand, the preparation method of the single cell suspension provided by the present disclosure is described in detail below in conjunction with specific embodiments.

[0042] Firstly, the agglomerated cell sample to be dispersed is mixed with a pretreatment solution and then subjected to ultrasonic treatment.

[0043] Here, the condensed cell sample can be animal or plant tissue, tumor tissue, microbial colony, sludge sample, etc.

[0044] In some embodiments, the condensed cell sample can be pretreated. Exemplarily, the condensed cell sample is a tumor tissue, and the tumor tissue can be cut into small pieces of about 1 to 2 mm using sterile scissors and a scalpel to disperse the sample, so as to facilitate the technical effects of ultrasonic treatment and enzymatic treatment. Exemplarily, the condensed cell sample is a sludge sample, and the sample can be stirred to disperse.

[0045] Furthermore, the pretreatment solution may be a phosphate buffered saline buffer (PBS buffer for short), or a PBS buffer containing a degrading enzyme.

[0046] It should be noted that the frequency and intensity of ultrasound are key influencing factors during the dispersion process. Too strong ultrasound can cause damage to the cell membrane, while too weak ultrasound cannot effectively disperse the cells. Based on this, the embodiment of the present disclosure dynamically adjusts the ultrasound intensity through an intelligent control system. Exemplarily, the first stage (corresponding to the initial stage) uses low-frequency ultrasound to treat the sample, and gradually transitions to high-frequency ultrasound to enhance the dispersion effect. Here, low-frequency ultrasound can be 20kHz to 30kHz, such as 20kHz, 25kHz, and 30kHz; high-frequency ultrasound can be 40kHz to 60kHz, such as 40kHz, 45kHz, 50kHz, 55kHz, and 60kHz.

[0047] It should be understood that, based on the foregoing description, different samples have different corresponding ultrasonic models, such as the specific selection of low-frequency ultrasound, duration, etc.; while the intelligent control system performs ultrasonic control based on the ultrasonic model, the intelligent control system also obtains cell activity data and particle size in real time, and uses a feedback model to dynamically adjust the ultrasonic treatment conditions. For example, if the particle size is larger, the ultrasonic treatment time can be extended; for another example, if the particle size is smaller, the ultrasonic treatment time can be shortened.

[0048] Next, the ultrasonically treated sample and the enzymatic hydrolysis solution are mixed to carry out an enzymatic hydrolysis reaction.

[0049] Different cell types have different requirements for enzymatic solutions. In related methods, the concentration and addition time of the enzymatic solution are usually fixed, which is difficult to adapt to the needs of complex samples.

[0050] The disclosed embodiments provide a multi-component composite enzymatic hydrolysate, which achieves gentler and more effective cell dispersion by combining a variety of enzymes and adjuvants.

[0051] In some embodiments, the composite enzymatic hydrolysis solution includes basic enzymes and auxiliary components, wherein the auxiliary components help to further improve the enzymatic hydrolysis effect.

[0052] The basic enzymes and auxiliary components are described in detail below.

[0053] Basic enzymes include at least one of trypsin, collagenase, lysozyme and hyaluronidase. Among them, trypsin is used to cut protein molecular chains, mainly acting on the protein structure in the cell membrane and extracellular matrix; collagenase specifically degrades collagen and is suitable for collagen-rich tissues such as connective tissue and tumor matrix; hyaluronidase targets hyaluronic acid, a polysaccharide commonly found in the extracellular matrix, and promotes the degradation of intercellular matrix and cell release; lysozyme is an alkaline enzyme that can hydrolyze mucopolysaccharides in bacteria.

[0054] The auxiliary ingredients include at least one of dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), antioxidants, and cell membrane protectants. Among them, DTT is a common reducing agent that can break disulfide bonds and promote the degradation of the extracellular matrix; EDTA is a chelating agent that interferes with the stability of the intercellular matrix by chelating metal ions, further promoting the dispersion of cells; antioxidants and cell membrane protectants can effectively reduce oxidative stress and membrane structure damage, increase cell activity, and prevent cell damage during enzymatic hydrolysis. Optionally, the antioxidant can be vitamin C; the cell membrane protectant can be bovine serum albumin (BSA).

[0055] The configuration and use of the composite enzymatic solution are illustrated as follows:

[0056] In some embodiments, under sterile conditions, trypsin, collagenase and hyaluronidase are first dissolved in PBS buffer respectively, and then the three are mixed in an optimized ratio. Then, a certain amount of DTT and EDTA are added and stored at low temperature (4°C) for standby use. It should be noted that the enzymatic solution should be freshly prepared before use. In the dispersion experiment for tumor tissue, after 20 minutes of treatment with the above-mentioned new composite enzymatic solution, the tissue structure was obviously loose and the cell separation was significantly improved. Compared with the single enzymatic solution treatment in the related art, the cell activity was increased by about 15%, the purity of the single cell suspension was increased by more than 20%, and the cell damage rate during the enzymatic process was greatly reduced. Exemplarily, the content of each component of the configured composite enzymatic solution in the enzymatic system can be 0.25% trypsin, 0.1% collagenase, 0.05% hyaluronidase, DTT 1mM, EDTA 2mM. It should be understood that since the solution is added before ultrasonic treatment, the content of each component in the prepared enzymatic solution is greater than the content in the enzymatic hydrolysis system. Those skilled in the art can configure it according to the proportional relationship, and the present disclosure does not make specific limitations on this.

[0057] In some alternative embodiments, trypsin, collagenase, hyaluronidase, DTT, and EDTA are dissolved in PBS buffer respectively and stored at low temperature (4° C.) for standby use. In this way, each component can be placed in a different container during use, and the intelligent control system can not only control the addition speed of the enzymolysis solution, but also control the addition amount and addition time of each component in the enzymolysis solution, such as trypsin first, then collagenase, etc. It should be understood that the intelligent control system can control the addition of each component of the enzymolysis solution so that it meets the requirements of the enzymolysis model for the enzymolysis solution.

[0058] In some embodiments, in the enzymatic reaction system, the content of dithiothreitol is 0.8-1.2 mM, for example, 1.0 mM, 1.1 mM; the content of chelating agent is 1.5-3.5 mM, for example, 1.5 mM, 2.0 mM, 2.3 mM, 3.0 mM, etc.; the content of trypsin is 0.15-0.3%, for example, 0.15%, 0.2%, 0.25%, etc.; the content of collagenase is 0.08-0.12%, for example, 0.10%, 0.11%, etc.; the content of hyaluronidase is 0.04-0.06%, for example, 0.04%, 0.05%, 0.06%, etc.; the content of lysozyme is 0.1-0.2%, for example, 0.1%, 0.15%, 0.2%.

[0059] During the dispersion process, the intelligent control system can determine the corresponding enzymatic hydrolysis model based on the sample type, and control the use of the enzymatic hydrolysis solution based on the enzymatic hydrolysis model, such as temperature, shaking speed, enzymatic hydrolysis solution concentration (basic enzyme type and concentration in the enzymatic hydrolysis solution), enzymatic hydrolysis time, and addition rate.

[0060] For example, for samples with dense tissue (such as tumor tissue, hardened connective tissue), a low concentration of enzymatic solution is first used for pretreatment to soften the tissue structure; then the concentration of the enzymatic solution is gradually increased, and the enzymatic solution time is appropriately extended to ensure complete dissociation. For microbial aggregates or sludge samples, the concentration of the enzymatic solution and the treatment time can be appropriately reduced to avoid cell breakage caused by excessive enzymatic solution.

[0061] For example, when treating tumor tissue, the system will first pre-treat with a lower concentration of trypsin, and then gradually increase the proportion of collagenase according to the tissue degradation to ensure complete degradation of the extracellular matrix and gentle release of cells.

[0062] It should be understood that the intelligent control system can also obtain and adjust the enzymatic hydrolysis conditions using a feedback model based on cell activity data, temperature, pH and enzymatic hydrolysis solution concentration.

[0063] In the process of cell dispersion, a single physical or chemical method is often difficult to achieve the desired effect, especially when specific cell types need to be dispersed and enriched. To this end, the disclosed embodiment combines nanomaterial labeling technology on the basis of ultrasonic treatment and enzymatic treatment, and achieves efficient dispersion and preliminary screening of target cells by specific labeling of the cell surface.

[0064] In some embodiments, iron oxide nanoparticles are used as labeling materials, and iron oxide nanoparticles have good biocompatibility, magnetic response characteristics, and are easy to surface modify. Optionally, the diameter of the iron oxide nanoparticles is controlled between 10 and 50 nm, such as 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, and 50 nm, which helps them to fully penetrate into the sample and bind to the surface of the target cells.

[0065] In order to achieve specific labeling, the surface of the nanoparticles is modified with antibodies or peptides targeting the target cells. For example, for a specific type of cancer cells, antigens specifically expressed on the surface of cancer cells, such as HER2, CD44, etc., can be selected to be labeled. It should be noted that the surface modification is achieved through covalent bonding or adsorption, and the surface modification density is optimized to ensure that the nanoparticles have sufficient targeting and stability.

[0066] After the enzymatic hydrolysis reaction, nanomaterials with surface modified target cell-specific markers are added to the enzymatic hydrolysis solution, and the distribution of the nanomaterials is controlled by an external magnetic field, so that target single cells can be enriched. Specifically, this step can include two stages: adding nanoparticles and magnetic field-assisted dispersion.

[0067] In some embodiments, adding nanoparticles can be: adding nanomaterials to the enzymatic solution and incubating it with the sample. Here, the incubation time and temperature need to be adjusted according to the sample type and the characteristics of the nanomaterials, generally incubating at 37° C. for 30 minutes to ensure the effectiveness of the labeling.

[0068] In some embodiments, magnetic field-assisted dispersion can be: in the process of nanomaterial labeling, the distribution of nanoparticles in the sample is controlled by an external magnetic field. By adjusting the intensity and direction of the magnetic field, the dispersion effect of the target cells can be enhanced and preliminary enrichment can be achieved. It should be noted that the process is carried out in a fully enclosed sterile environment to avoid the influence of external contamination.

[0069] In some embodiments, the strength of the external magnetic field is 3000-3500 Gs, for example, 3100 Gs, 3200 Gs, 3300 Gs, 3350 Gs, 3400 Gs, 3500 Gs, etc.

[0070] Finally, the enriched sample is further separated to obtain a target single cell suspension. Here, the method for further separation can be gradient centrifugation, such as Percoll density gradient centrifugation, and the purity of the target cells can be further improved. Exemplarily, the cancer cell sample obtained by the preparation method disclosed in the present invention has a cell purity of 98% after dispersion, the number of specifically labeled target cells is increased by more than 30% compared with related methods, and the cell activity is maintained at more than 90%.

[0071] It can be seen that the method for preparing a single cell suspension provided in the embodiments of the present disclosure combines an intelligent control system, a new composite enzymatic hydrolysate and nanomaterial labeling technology, and has achieved significant progress in improving cell dispersion efficiency, maintaining cell activity and enhancing cell purity. Among them, the intelligent control system's refined control of the ultrasonic and enzymatic processes makes the cell dispersion process more efficient, especially when dealing with complex samples, such as multicellular tissues and high-density microbial samples, the dispersion efficiency is at least 50% higher than that of related methods; the application of the new composite enzymatic solution, through mild enzymatic hydrolysis and antioxidant protection, minimizes cell damage during the dispersion process, and increases cell activity by 15 to 20 percentage points compared with related methods; the introduction of nanomaterial labeling technology enables the target cells to be preliminarily screened and enriched during the dispersion process, and the purity of the single cell suspension obtained after dispersion is more than 20% higher than that of related methods, which is especially suitable for enriching rare or difficult-to-separate cell types; the intelligent control system can realize full-process monitoring and feedback, which significantly improves the stability and repeatability of the dispersion process; the experimental results between different batches are highly consistent, suitable for large-scale industrial applications, and widely applicable to animal and plant tissues, tumor tissues, microbial colonies, granular sludge, etc., with broad application prospects, and can be applied to in vitro culture, drug screening, genetic engineering and environmental governance.

[0072] The preparation method provided in the embodiments of the present disclosure is exemplified below by taking tumor tissue samples and sludge samples as examples.

[0073] Example 1 Single cell dispersion of tumor tissue

[0074] (1) Sample preparation and pretreatment

[0075] About 2 grams of tumor tissue from the patient was taken and placed in ice-cold PBS buffer. Sterile scissors and a scalpel were used to cut the tumor tissue into small pieces of about 1 to 2 mm.

[0076] The sheared tissue sample is transferred to a 50 ml centrifuge tube, 5 ml of pre-cooled PBS buffer is added, and 1 ml of an enzymatic pretreatment solution (such as a low-concentration compound enzymatic solution) containing 1% bovine serum albumin (BSA) is added, and after the addition, the overall system contains 0.1% trypsin, 0.05% collagenase and 0.025% hyaluronidase. Here, the addition of PBS buffer, BSA, etc. can be performed by an intelligent control system.

[0077] (2) Ultrasonic treatment

[0078] Place the centrifuge tube in the ultrasonic processor of the intelligent control system and start the control module. The control module matches the ultrasonic model of the tumor tissue and controls the ultrasonic treatment conditions, such as setting the initial ultrasonic frequency to 25kHz and the intensity to 50W / cm 2 The treatment time was 3 minutes; the ultrasonic frequency was gradually increased to 40kHz and the intensity was adjusted to 70W / cm 2 , the processing time is extended to 5 minutes. During this process, the control module uses the feedback module to adjust the ultrasonic conditions based on the cell activity data, temperature, pH, enzyme solution concentration and other parameters monitored in real time by the sensor unit to ensure the smooth progress of the dispersion process.

[0079] (3) Enzymatic hydrolysis

[0080] According to the characteristics of tumor tissue, a composite enzymatic hydrolysis solution was prepared, and the target concentration after adding the enzymatic hydrolysis system was 0.25% trypsin, 0.1% collagenase, 0.05% hyaluronidase, 1mM DTT and 2mM EDTA to promote the degradation of the extracellular matrix.

[0081] After ultrasonic treatment, the sample was removed from the ultrasonic processor, and the intelligent control system controlled the addition of 5 ml of the prepared compound enzymatic solution, and incubated in a 37°C constant temperature shaker for 30 minutes. During the incubation period, the control module monitored the progress of the enzymatic reaction in real time, adjusted the temperature and shaker speed to ensure uniform dispersion and efficient release of the cells.

[0082] (4) Nanomaterial labeling and magnetic field-assisted dispersion:

[0083] The anti-HER2 antibody modified with the surface of iron oxide nanoparticles was added to the composite enzymatic solution at a final concentration of 50 μg / mL. The labeled enzymatic solution was incubated with the tumor tissue for 30 minutes at 37°C with slight shaking.

[0084] Subsequently, an external magnetic field was used to control the distribution of iron oxide nanoparticles in the sample, the magnetic field strength was adjusted to 3000 Gs, and the direction of the magnetic field was dynamically changed during the incubation process to improve the uniformity of labeling.

[0085] After labeling, the labeled cells are initially separated by a magnetic separator to obtain enriched HER2-positive cancer cells.

[0086] (5) Cell separation and suspension preparation:

[0087] The dispersed sample was centrifuged at low speed (300 g, 5 minutes), the supernatant was discarded, and 5 ml of PBS was added to resuspend the cells, followed by Percoll density gradient centrifugation (40% and 80% Percoll gradient) to separate cell populations of different densities.

[0088] The single cell population at the interface was collected, and the single cell suspension finally obtained was subjected to activity detection (using trypan blue staining method), and the cell activity was about 95% and the cell purity reached 98%.

[0089] Example 2 Microbial single cell dispersion of sludge samples

[0090] (1) Sample preparation and pretreatment:

[0091] Take about 5 g of sludge sample, place it in a sterile 50 ml centrifuge tube, add 10 ml of pre-cooled PBS buffer, and stir gently to mix well.

[0092] (2) Ultrasonic treatment

[0093] The sludge sample was placed in an ultrasonic processor for treatment. The control module set the initial ultrasonic frequency to 30 kHz and the intensity to 60 W / cm 2 The treatment time is 3 minutes to destroy large sludge flocs. The control module automatically adjusts the ultrasonic frequency to 45kHz and increases the intensity to 80W / cm 2 , the treatment time is extended to 7 minutes to ensure that the sludge flocs are fully dispersed while avoiding excessive treatment that causes microbial cell rupture. At the same time, the control module adjusts the ultrasonic conditions based on parameters such as cell activity monitored in real time by the sensor unit. Figure 1 The image of the sludge sample provided by the embodiment of the present disclosure after ultrasonic dispersion is shown; specifically, the image of the ultrasonically dispersed suspension is diluted 500 times. Figure 1 It can be seen that the particles of the sludge sample are small and the dispersion is high.

[0094] (3) Enzymatic hydrolysis

[0095] According to the characteristics of various microorganisms in the sludge samples, a composite enzymatic hydrolysate was prepared, and its target concentration after adding the enzymatic hydrolysis system was 0.2% trypsin, 0.15% lysozyme, 0.1% collagenase, 1mM DTT and 3mM EDTA to promote the degradation of extracellular aggregates.

[0096] The control module controls the addition of the composite enzymatic hydrolysate to the sludge sample after ultrasonic treatment, and the enzymatic hydrolysis conditions, such as incubation at 37°C for 45 minutes. The control module adjusts the incubation temperature and time according to the real-time monitored cell dispersion, cell activity, etc., to ensure the uniformity and efficiency of the enzymatic hydrolysis process.

[0097] It should be noted that the control module can control the addition speed and concentration of the enzymatic solution, which will not be described in detail here. Figure 2 Shown are images of a 500-fold dilution after enzymatic digestion and termination of digestion with serum protein.

[0098] (4)Nanomaterial labeling and dispersion:

[0099] In order to specifically separate a certain type of target microorganisms (such as nitrifying bacteria) in the sludge sample, anti-nitrifying bacteria antibodies modified on the surface of iron oxide nanoparticles were added to the composite enzymatic hydrolysate at a concentration of 100 μg / mL.

[0100] Under an external magnetic field, the magnetic field gradient method was used to enrich the labeled microbial cells, the magnetic field strength was adjusted to 3500 Gs, and the magnetic field direction was dynamically adjusted to enhance the enrichment effect. Figure 3 Shown is an image of an enriched sample diluted 500-fold after filtration through an 8 μm filter.

[0101] (5) Cell separation and suspension preparation:

[0102] After enrichment by magnetic separator, the enriched cells are centrifuged at low speed, for example, 400 g for 10 minutes, the supernatant is discarded, 10 ml of PBS is added for resuspending, and the image is diluted 500 times as shown in the figure. Figure 4 Multiple gradient centrifugation steps were then performed to separate microbial populations of different densities.

[0103] The final microbial single cell suspension was tested by trypan blue staining, and the cell activity reached 90%, and the purity of the target microorganism was increased to more than 95%.

[0104] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0105] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A method for preparing a single cell suspension, characterized in that: include: 1) mixing the condensed cell sample to be dispersed with the pretreatment solution and performing ultrasonic treatment; 2) mixing the sample obtained in step 1) with the enzymatic hydrolysis solution to perform an enzymatic hydrolysis reaction; 3) adding nanomaterials with surface-modified target cell-specific markers, controlling the distribution of the nanomaterials by applying an external magnetic field, and enriching the target single cells; 4) Separating the sample obtained in step 3) to obtain a target single cell suspension; wherein the conditions of the ultrasonic treatment and the enzymatic hydrolysis reaction are controlled by an intelligent control system.

2. The preparation method according to claim 1, characterized in that: The control by the intelligent control system specifically includes: Obtain and determine the corresponding ultrasound model and enzymatic hydrolysis model according to the type of target single cells; The ultrasonic treatment conditions are controlled by using an ultrasonic model, and the enzymatic hydrolysis reaction conditions are controlled by using an enzymatic hydrolysis model; Obtaining and adjusting the conditions of the ultrasonic treatment and the conditions of the enzymatic hydrolysis reaction according to the particle size, cell activity data, temperature, pH and enzymatic hydrolysis solution concentration of the sample using a feedback model; wherein, The ultrasound model, the enzymatic hydrolysis model and the feedback model are respectively trained machine learning algorithm models.

3. The preparation method according to claim 2, characterized in that: The ultrasonic treatment conditions include at least one of ultrasonic frequency, ultrasonic intensity and ultrasonic time; and / or The conditions of the enzymatic hydrolysis reaction include at least one of temperature, shaking speed, enzyme type, enzymatic hydrolysis solution concentration and enzymatic hydrolysis time.

4. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis solution comprises dithiothreitol, a chelating agent and at least one enzyme; wherein the at least one enzyme is selected from at least one of trypsin, collagenase, hyaluronidase and lysozyme.

5. The preparation method according to claim 4, characterized in that: In the enzymatic hydrolysis reaction system of step 2), the content of dithiothreitol is 0.8-1.2 mM; the content of the chelating agent is 1.5-3.5 mM; the content of trypsin is 0.15-0.3%; the content of collagenase is 0.08-0.12%; the content of hyaluronidase is 0.04-0.06%; and the content of lysozyme is 0.1-0.2%.

6. The preparation method according to claim 1, characterized in that: The nanomaterial comprises iron oxide nanoparticles, wherein the diameter of the iron oxide nanoparticles is 10 to 50 nm; and / or The specific marker is selected from an antibody or a peptide segment; wherein the antibody or the peptide segment specifically recognizes the target single cell.

7. The preparation method according to claim 1, characterized in that: The intensity of the external magnetic field in step 3) is 3000-3500 Gs; the direction of the external magnetic field can be dynamically adjusted.

8. The preparation method according to claim 1, characterized in that: The condensed cell sample is selected from animal or plant tissues, tumor tissues, microbial colonies or sludge samples.

9. The preparation method according to claim 1, characterized in that: The ultrasonic treatment includes a first stage and a second stage; wherein the frequency of the first stage is 20kHz to 30kHz; and the frequency of the second stage is 40kHz to 60kHz.

10. The target single cell suspension obtained by the preparation method according to any one of claims 1 to 9; wherein The cell activity of the target single cell suspension is ≥90%, and the cell purity is ≥95%.

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