Process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells

Abstract

The invention relates to the technical field of stem cell preparation and culture, and discloses a hospital-grade olfactory mucosa mesenchymal stem cell preparation and sterile culture process which comprises the following steps: collecting olfactory mucosa tissues from a donor olfactory mucosa area through a minimally invasive surgery, putting the collected tissues into a cold-chain transportation solution, and transporting at the temperature of 4 DEG C or below; a device for hospital-level olfactory mucosa mesenchymal stem cell preparation and sterile culture comprises a closed sterile bioreactor system used for controlling temperature, CO2 concentration, O2 concentration and pH value. Through electrochemical signal regulation and control, a real-time monitoring system and A I intelligent feedback, accurate control over cell proliferation, differentiation and anti-inflammatory functions is achieved, electric field parameters are optimized at different stages, cell proliferation, neural differentiation and anti-inflammatory capacity is improved, and through real-time monitoring and automatic feedback, it is ensured that the culture process is efficient, accurate and automatic, and the culture efficiency is improved. The cell culture success rate is obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell preparation and culture, and in particular to a process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells. Background Art

[0002] Olfactory mucosa mesenchymal stem cells are a type of stem cell with strong regenerative and immunomodulatory functions. In recent years, they have been widely used in nerve repair, anti-inflammatory therapy and other regenerative medicine fields. Olfactory mucosa mesenchymal stem cells not only have the characteristics of traditional mesenchymal stem cells, such as multidirectional differentiation potential and immunomodulatory ability, but also have unique neural differentiation potential. Therefore, they show good application prospects in the treatment of central nervous system diseases and nerve damage.

[0003] However, there are still many challenges in the current preparation process of olfactory mucosal mesenchymal stem cells. First, how to efficiently separate high-quality olfactory mucosal mesenchymal stem cells in a sterile environment is a key issue in the preparation process. Conventional tissue collection and processing methods may cause tissue degradation or decreased cell activity, thereby affecting the quality of cells. Secondly, the regulation of environmental parameters in traditional culture systems (such as temperature, oxygen, CO 2 Concentration, etc.) are not precise enough, which can easily lead to unstable cell growth conditions. In addition, functional regulation of olfactory mucosal mesenchymal stem cells (such as proliferation and differentiation) is also difficult, and traditional culture methods often cannot effectively enhance their specific functions (such as neural differentiation or anti-inflammatory function).

[0004] In order to solve these problems, there is an urgent need for a process that can efficiently prepare OM-MSCs in a sterile environment to ensure the high activity, functionality and consistency of the cells. To this end, the present invention proposes a process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells. Summary of the invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a process for the preparation and aseptic culture of hospital-grade olfactory mucosal mesenchymal stem cells, which solves the problem of how to efficiently prepare, culture and optimize the proliferation, differentiation and function of olfactory mucosal mesenchymal stem cells in a sterile environment, while ensuring that the activity and functionality of the cells remain stable after freezing.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: The process of preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells comprises the following steps:

[0007] S1. Collect olfactory mucosal tissue from the donor's olfactory mucosa area through minimally invasive surgery, and place the collected tissue in cold chain transport fluid and maintain it below 4°C during transportation;

[0008] S2. Under sterile conditions, the olfactory mucosa tissue was minced and enzymatically digested, the obtained tissue fragments were digested with an enzyme digestion solution, and then a single cell suspension was collected by centrifugation and filtration;

[0009] S3, culturing the isolated olfactory mucosa mesenchymal stem cells in a serum-free culture medium, and controlling the culture environment by an automated bioreactor system, including the regulation of temperature, CO2 concentration, and O2 concentration;

[0010] S4, applying electrochemical signals to the olfactory mucosal mesenchymal stem cells to stimulate cell proliferation, differentiation or function optimization by setting the electric field strength and frequency;

[0011] S5. Use a real-time monitoring system to monitor cell proliferation status, metabolic levels, and membrane potential changes, and dynamically adjust electrochemical signal parameters based on the monitoring results through an intelligent feedback mechanism;

[0012] S6. Perform phenotypic identification and multidirectional differentiation capacity testing on the expanded olfactory mucosal mesenchymal stem cells, and freeze or use qualified cells for clinical applications.

[0013] Preferably, the step S2 specifically includes the following steps:

[0014] S2.1. Aseptic processing: Cut the olfactory mucosa tissue into pieces in a biological safety cabinet, with the size of the tissue fragments being 1mm3-2mm3;

[0015] S2.2, enzyme digestion treatment: prepare a solution with a mass concentration of 0.2% collagenase I and 0.05% hyaluronidase, the digestion temperature is 35°C-37°C, and the digestion time is 20min-40min;

[0016] S2.3, stop enzyme digestion: when stopping the enzyme reaction, add an equal volume of 10% fetal bovine serum culture medium, the termination time is 1min-3min;

[0017] S2.4, Filtration and centrifugation: The mesh size used for filtration is 100μm-150μm, the centrifugal speed is 100g-300g, and the centrifugal time is 3min-7min;

[0018] S2.5. Cell resuspension: The cell resuspension density is controlled at 1×10 6 cells / mL-5×10 6 cells / mL.

[0019] Preferably, the step S3 specifically includes the following steps:

[0020] S3.1. Initial cell culture: The cell density was 1×10 6 cells / mL-5×10 6cells / mL;

[0021] S3.2. Bioreactor Setup:

[0022] The temperature is controlled at 35℃-38℃;

[0023] CO2 concentration is set at 3%-7%;

[0024] O2 concentration is set at 3%-10%;

[0025] S3.3. Automated culture medium replacement: The frequency of culture medium replacement is once every 12h-24h, depending on the cell density.

[0026] Preferably, the step S4 specifically includes the following steps:

[0027] S4.1. Electrode installation and connection: The spacing between platinum-coated electrodes is 3mm-7mm, and the electrode thickness is 0.05mm-0.2mm;

[0028] S4.2. Electrochemical signal stimulation during the proliferation stage: the electric field strength ranges from 0.3V / cm to 0.7V / cm, the frequency is 30Hz to 70Hz, each electrical stimulation lasts for 15min to 30min, and the electrical stimulation is performed once to three times a day for 3d to 4d.

[0029] S4.3. Electrochemical signal stimulation during the neural differentiation stage: the electric field strength ranges from 0.2V / cm to 0.5V / cm, the frequency ranges from 10Hz to 30Hz, the duration of each electrical stimulation is 10min to 20min, and the electrical stimulation is performed once or twice a day for 3d to 4d.

[0030] S4.4. Electrochemical signal stimulation in the anti-inflammatory function optimization stage: the electric field strength range is 0.1V / cm-0.4V / cm, the frequency is 20Hz-40Hz, each electrical stimulation lasts for 5min-15min, and the electrical stimulation is once a day for 2d-4d.

[0031] Preferably, the step S5 specifically includes the following steps:

[0032] S5.1. Microscopic imaging monitoring: The frequency of cell image acquisition is once every 6-12 hours, and the microscopic imaging analysis system monitors cell morphology and density in real time;

[0033] S5.2 Metabolic monitoring: monitor glucose, lactate and amino acid concentrations, and record metabolic levels in real time, with each test interval of 2h-6h;

[0034] S5.3. Electrophysiological monitoring: real-time monitoring of changes in cell membrane potential and dynamic adjustment of electric field strength and frequency;

[0035] S5.4. Intelligent feedback system adjustment: The system performs real-time data processing based on AI algorithm and adjusts electrochemical parameters according to the following formula.

[0036] Preferably, the calculation formula for adjusting the electrochemical parameters in step S5.4 is as follows: P(t)=α·E(t)+β·f(t)+γ·ΔV m (t), where P(t) represents the cell proliferation rate, E(t) is the electric field strength, f(t) is the frequency, and ΔV m (t) is the change of cell membrane potential, α, β and γ are the preset feedback parameters, the electric field strength range is 0.1V / cm-1V / cm, and the frequency range is 1Hz-100Hz.

[0037] Preferably, the step S6 specifically includes the following steps:

[0038] S6.1. Phenotypic identification: Each batch was tested at least 3 times, using 1×10 5 cells-5×10 5 cells, and the positive expression of surface markers CD73, CD90, and CD105, and the negative expression of CD34 and CD45 were detected by flow cytometry;

[0039] S6.2. Multidirectional differentiation test: Olfactory mucosal mesenchymal stem cells were cultured in neural differentiation induction medium and the concentrations of IL-10 and TGF-β in the culture supernatant were detected by ELISA;

[0040] S6.3. Cell cryopreservation: The cell concentration is controlled at 0.5×10 6 cells / mL-5×10 6 cells / mL, the freezing solution contained 5%-15% DMSO, the cooling rate was -0.5℃ / min--2℃ / min, and the cells were finally stored in a liquid nitrogen tank.

[0041] Preferably, the multidirectional differentiation test in step S6.2 specifically comprises the following steps:

[0042] Neural differentiation test: Olfactory mucosal mesenchymal stem cells were cultured in neural differentiation induction medium. After 10-20 days of differentiation, the expression of Nestin and MAP2 was detected by immunofluorescence.

[0043] Anti-inflammatory ability test: The concentrations of IL-10 and TGF-β in the culture supernatant were detected by ELISA method, with a sample volume of 50μL-200μL.

[0044] A device for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells, used in conjunction with the above-mentioned process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells, comprising:

[0045] Closed sterile bioreactor system for controlling temperature, CO 2 Concentration, O 2 concentration and pH value;

[0046] An electrochemical signal generating device equipped with platinum-coated electrodes for applying electric signals with an electric field strength ranging from 0.1V / cm to 1V / cm and a frequency ranging from 1Hz to 100Hz to regulate cell proliferation, differentiation and function;

[0047] Real-time monitoring system for integrated cell morphology monitoring, metabolic detection, and electrophysiological signal detection;

[0048] Intelligent feedback control module, used to dynamically adjust electric field parameters through AI algorithm.

[0049] The present invention provides a process for preparing and aseptically culturing hospital-grade olfactory mucosal mesenchymal stem cells, which has the following beneficial effects:

[0050] 1. The present invention can precisely regulate cell proliferation, neural differentiation and anti-inflammatory function through electrochemical signal regulation. The electric field strength and frequency settings at different stages enable cells to proliferate faster in the proliferation stage, effectively differentiate in the direction of nerve cells in the neural differentiation stage, and enhance their immunoregulatory function in the anti-inflammatory stage. The cells not only maintain a high proliferation rate during the culture process, but also optimize their functionality through specific electrical stimulation, significantly improving their effect in clinical treatment.

[0051] 2. The present invention can continuously monitor the proliferation state, metabolic level and membrane potential changes of cells through a real-time monitoring system to ensure that the cells are always in the best growth state during the culture process. The system collects cell images at regular intervals and analyzes cell morphology and density changes, and detects metabolites in the culture medium (such as glucose, lactic acid, etc.) to ensure the supply of nutrients and timely discharge of waste. Through precise electrophysiological monitoring, the system can provide real-time feedback on the cell's response to electrochemical signals to ensure that the electric field strength matches the cell state, thereby improving the accuracy of cell function regulation.

[0052] 3. The present invention uses an intelligent feedback system based on an AI algorithm to adjust electrochemical signal parameters in real time to adapt to the needs of cells at different stages. The system will automatically optimize stimulation conditions based on monitored data to ensure that cells are always in the best state during proliferation, differentiation and function optimization. Compared with traditional static parameter settings, the intelligent feedback mechanism of the present invention greatly improves the automation and personalized regulation level of the culture process, reduces manual intervention, and improves the success rate of cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1is a flow chart of the process of the present invention;

[0054] Figure 2 It is a framework diagram of the device of the present invention. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] Please see attached Figure 1 The embodiment of the present invention provides a process for preparing and aseptically culturing hospital-grade olfactory mucosal mesenchymal stem cells, comprising the following steps:

[0057] S1. Collect olfactory mucosal tissue from the donor's olfactory mucosa area through minimally invasive surgery, and place the collected tissue in cold chain transport fluid and maintain it below 4°C during transportation;

[0058] Specifically, in the process of preparing hospital-grade olfactory mucosal mesenchymal stem cells, tissue collection and transportation is the first step of the entire process and a key step to ensure cell activity and sterility. This step obtains tissue from the donor's olfactory mucosa area through minimally invasive surgery, and transports the collected olfactory mucosa tissue to the laboratory through strictly controlled cold chain transportation to maximize tissue activity and avoid contamination. During cold chain transportation, it is necessary to ensure that the transportation temperature and time are controlled within the appropriate range, thereby laying the foundation for subsequent cell isolation and culture.

[0059] In this embodiment, olfactory mucosal tissue is collected from the olfactory mucosa region of the donor through minimally invasive surgery. The surgery uses local anesthesia to ensure the comfort of the donor and the accuracy of the surgical operation. The collection tool is a sterile soft forceps or a special collection tool for nasal endoscope. The entire collection process needs to be carried out in a sterile environment to avoid the introduction of exogenous contamination.

[0060] The olfactory mucosa tissue collected should be 1mm 3 -5mm 3 The size of the tissue block is to ensure that there is enough cell source for subsequent processing without causing too much trauma to the donor.

[0061] After the tissue collection is completed, in this embodiment, the collected olfactory mucosa tissue is immediately placed in a sterile centrifuge tube containing a low-temperature transport fluid. The low-temperature transport fluid is composed of DMEM / F12 basic culture medium containing 1% penicillin-streptomycin to prevent bacterial contamination and maintain the physiological activity of the tissue.

[0062] The transportation temperature is controlled between 2℃-4℃, which can effectively slow down the metabolic process, prolong the survival time of tissues, and inhibit the growth of microorganisms.

[0063] The transportation time should be strictly controlled within 15min-45min to ensure that the tissue can reach the laboratory quickly and avoid the decrease of cell activity due to too long transportation.

[0064] In this embodiment, the entire process of cold chain transportation uses a closed transportation device to ensure that the sample is not exposed to the external environment during transportation and maintains sterility. At the same time, the cold chain transportation equipment should be equipped with a temperature monitoring device to record the temperature changes during transportation in real time. The temperature monitoring data needs to be checked after the tissue arrives at the laboratory to ensure that the temperature does not exceed 4°C during the entire transportation process, thereby providing the best tissue quality for the subsequent olfactory mucosal mesenchymal stem cell isolation.

[0065] In order to ensure that the olfactory mucosa tissue maintains stable biological characteristics during transportation, the change in cell metabolic rate during transportation can be estimated according to the formula during cold chain transportation. Assuming that the cell metabolic rate R is exponentially related to the temperature T, the following formula can be used to approximate the change in metabolic rate:

[0066]

[0067] Among them, R 0 is the baseline metabolic rate, T 0 is the reference temperature (usually 37°C), β is a constant related to the metabolic rate, and T is the actual transport temperature.

[0068] This formula can be used to analyze the impact of temperature control on metabolic rate, and thus deduce that during cold chain transportation, lower temperatures can effectively reduce the metabolic rate of cells and maximize the retention of active cells in tissues.

[0069] During transportation, temperature monitoring and time control should comply with standard specifications to ensure that the olfactory mucosal tissue in the transport fluid is maintained in an ideal state to facilitate subsequent cell isolation and culture.

[0070] S2. Under sterile conditions, the olfactory mucosa tissue was minced and enzymatically digested, the obtained tissue fragments were digested with an enzyme digestion solution, and then a single cell suspension was collected by centrifugation and filtration;

[0071] The S2 step specifically includes the following steps:

[0072] S2.1. Aseptic processing: Cut the olfactory mucosa tissue into pieces in a biological safety cabinet. The size of the tissue fragments should be 1 mm. 3 -2mm 3 ;

[0073] S2.2, enzyme digestion treatment: prepare a solution with a mass concentration of 0.2% collagenase I and 0.05% hyaluronidase, the digestion temperature is 35°C-37°C, and the digestion time is 20min-40min;

[0074] S2.3, stop enzyme digestion: when stopping the enzyme reaction, add an equal volume of 10% fetal bovine serum culture medium, the termination time is 1min-3min;

[0075] S2.4, Filtration and centrifugation: The mesh size used for filtration is 100μm-150μm, the centrifugal speed is 100g-300g, and the centrifugal time is 3min-7min;

[0076] S2.5. Cell resuspension: The cell resuspension density is controlled at 1×10 6 cells / mL-5×10 6 cells / mL.

[0077] Specifically, for step S2, the processing of the olfactory mucosal tissue is crucial, which directly determines the separation efficiency and cell activity of the olfactory mucosal mesenchymal stem cells. This step includes operations such as tissue shearing, enzymatic digestion, filtration and centrifugation. Under sterile conditions, the olfactory mucosal tissue is sheared to make its particle size suitable for enzymatic digestion. The tissue is then digested with an appropriate enzyme solution to release the cells from the matrix, and then a single cell suspension is collected by centrifugation and filtration to ensure that highly active, high-quality olfactory mucosal mesenchymal stem cells are obtained. Precise control of enzyme digestion time, temperature and other conditions has an important influence on the final cell output and functionality.

[0078] In this embodiment, the first step in processing the olfactory mucosal tissue is to cut the collected tissue into pieces. This operation is performed in a biological safety cabinet to ensure full control of the aseptic operation. Use sterile scissors to cut the olfactory mucosal tissue into pieces of 1 mm in size. 3 Up to 2mm 3 The size of the tissue particles in this step is very important. Too large particles may lead to incomplete enzymatic digestion, while too small particles may cause cell damage.

[0079] In this embodiment, the shredded tissue blocks are placed in an enzyme digestion solution for enzymolysis. The enzyme digestion solution is composed of 0.2% (w / v) collagenase I and 0.05% hyaluronidase. These two enzymes act on the extracellular matrix respectively, and can effectively decompose extracellular collagen and hyaluronic acid, so that olfactory mucosal mesenchymal stem cells are released from the tissue. The temperature of the digestion solution is controlled between 35°C and 37°C, and the digestion time is 20min-40min. The choice of digestion time is closely related to factors such as tissue amount and particle size, and needs to be optimized according to actual conditions.

[0080] The cell release and degradation rates during enzymatic digestion can be described by the following equations:

[0081] C(t)=C 0 ·(1-e -κt )

[0082] Where C(t) is the release rate of cells at time t, C 0 is the total amount of cells in the initial tissue, and κ is the enzymatic rate constant, which is related to the concentration of the enzyme and temperature. By adjusting the temperature and enzyme concentration, the rate and degree of cell release can be effectively controlled to avoid cell damage caused by excessive digestion.

[0083] In this embodiment, when digestion is completed, an equal volume of 10% fetal bovine serum medium is immediately added to terminate the enzyme reaction. Fetal bovine serum contains rich protein components, which can quickly neutralize the remaining enzyme activity, and the termination time is 1min-3min. By terminating the enzyme reaction in time, further damage to the cell surface structure by the enzyme is avoided, ensuring that the cells maintain good morphology and function.

[0084] In this embodiment, the digestion solution after the enzymatic hydrolysis is terminated is filtered through a sterile mesh with a pore size of 100 μm-150 μm to remove incompletely digested tissue fragments and retain a single cell suspension. The filtered suspension is centrifuged at 100g-300g for 3min-7min to separate the cell precipitate. By precisely controlling the centrifugal force and time, it is possible to ensure that a cell precipitate with high activity is obtained without causing excessive mechanical damage to the cells.

[0085] In this example, the cell pellet was resuspended in serum-free medium after centrifugation, and the resuspended density was controlled to 1×10 6 cells / mL-5×10 6 cells / mL to provide a stable initial cell number for the subsequent culture process.

[0086] S3. The isolated olfactory mucosal mesenchymal stem cells are cultured in serum-free medium, and the culture environment, including temperature, CO, is controlled by an automated bioreactor system. 2 Concentration and O 2 Concentration adjustment;

[0087] The S3 step specifically includes the following steps:

[0088] S3.1. Initial cell culture: The cell density was 1×10 6 cells / mL-5×10 6 cells / mL;

[0089] S3.2. Bioreactor Setup:

[0090] The temperature is controlled at 35℃-38℃;

[0091] CO 2 The concentration is set at 3%-7%;

[0092] O 2 The concentration is set at 3%-10%;

[0093] S3.3. Automated culture medium replacement: The frequency of culture medium replacement is once every 12h-24h, depending on the cell density.

[0094] Specifically, for step S3, precise control of culture conditions, such as temperature, CO 2 Concentration and O 2 The concentration will directly affect the proliferation efficiency and quality of cells. The use of an automated bioreactor system can ensure the precise control of environmental parameters and reduce the possibility of human error and contamination. The system can also maintain a stable nutrient supply to cells through automated culture medium replacement, thereby further improving the growth rate and consistency of cells.

[0095] In this example, the isolated olfactory mucosa mesenchymal stem cells were placed in a serum-free medium for preliminary culture. The initial cell density was controlled at 1×10 6 cells / mL-5×10 6 cells / mL to ensure that cells can be evenly distributed in the culture environment in the early stages and reduce the difference in growth rate caused by overcrowding or sparse cells.

[0096] In this embodiment, the culture process of olfactory mucosal mesenchymal stem cells is controlled by an automated bioreactor system. The system sets precise environmental parameters:

[0097] Temperature control: The culture temperature is set at 35℃-38℃. This temperature range helps maintain the optimal metabolic state of cells while avoiding the impact of temperature fluctuations on cell proliferation.

[0098] CO 2 Concentration control: CO 2 The concentration is set at 3%-7% to maintain appropriate CO 2 The pH level helps stabilize the culture medium, ensuring that the carbonate buffer system required for cell metabolism remains functional.

[0099] Oxygen concentration control: The oxygen concentration is controlled at 3%-10% (hypoxic conditions), which simulates the hypoxic environment in the body and has been shown to increase the proliferation rate of mesenchymal stem cells and enhance their ability to maintain stemness and differentiation potential.

[0100] Gas exchange and nutrient metabolism in the culture medium can be used to estimate the metabolic requirements of the cells using the following formula:

[0101]

[0102] Among them, Q o is the oxygen requirement of the cell, k L a is the oxygen transfer coefficient, is the oxygen saturation concentration in the culture medium, C o is the current oxygen concentration.

[0103] By adjusting the oxygen concentration and the gas exchange rate in the culture medium, it is possible to ensure that the cells have an adequate oxygen supply during the culture process.

[0104] In this embodiment, the automated bioreactor also has an automatic culture medium replacement function to ensure that the cells can continue to obtain sufficient nutrients and remove metabolic waste during the expansion process. According to the changes in cell density, the system automatically calculates and adjusts the culture medium replacement frequency, which is generally once every 24 to 72 hours, and the specific time is dynamically adjusted according to the growth rate and metabolic needs of the cells.

[0105] The metabolic demand and medium replenishment volume during the medium change process can be estimated by the following formula:

[0106]

[0107] Among them, V replace is the volume of each medium replacement, V total is the total culture medium volume, C metabolite is the current concentration of the metabolite, C threshold The upper limit of the tolerance concentration of metabolites. By monitoring the concentration changes of metabolites (such as lactate and amino acids), the volume and frequency of medium replacement are automatically adjusted to ensure that cells continue to proliferate in a good environment.

[0108] In this embodiment, the automated bioreactor monitors and adjusts environmental parameters in real time through sensors and control systems to ensure a stable growth environment for olfactory mucosal mesenchymal stem cells. By reducing manual operations, the bioreactor can reduce the risk of contamination and ensure the sterility and consistency of the culture process. This highly automated and precise control method provides a solid foundation for the large-scale clinical application of olfactory mucosal mesenchymal stem cells.

[0109] S4, applying electrochemical signals to the olfactory mucosal mesenchymal stem cells to stimulate cell proliferation, differentiation or function optimization by setting the electric field strength and frequency;

[0110] The S4 step specifically includes the following steps:

[0111] S4.1. Electrode installation and connection: The spacing between platinum-coated electrodes is 3mm-7mm, and the electrode thickness is 0.05mm-0.2mm;

[0112] S4.2. Electrochemical signal stimulation during the proliferation stage: the electric field strength ranges from 0.3V / cm to 0.7V / cm, the frequency is 30Hz to 70Hz, each electrical stimulation lasts for 15min to 30min, and the electrical stimulation is performed once to three times a day for 3d to 4d.

[0113] S4.3. Electrochemical signal stimulation during the neural differentiation stage: the electric field strength ranges from 0.2V / cm to 0.5V / cm, the frequency ranges from 10Hz to 30Hz, the duration of each electrical stimulation is 10min to 20min, and the electrical stimulation is performed once or twice a day for 3d to 4d.

[0114] S4.4. Electrochemical signal stimulation in the anti-inflammatory function optimization stage: the electric field strength range is 0.1V / cm-0.4V / cm, the frequency is 20Hz-40Hz, each electrical stimulation lasts for 5min-15min, and the electrical stimulation is once a day for 2d-4d.

[0115] Specifically, for step S4, the electrochemical signal acts on the cells through the set electric field strength and frequency, which can not only promote the proliferation of olfactory mucosal mesenchymal stem cells, but also effectively regulate their differentiation potential and functional optimization. The electric field parameter settings at different stages will affect the biological behavior of olfactory mucosal mesenchymal stem cells, such as cell proliferation, neural differentiation and anti-inflammatory function. Through electrochemical signal stimulation, directional regulation of stem cells can be achieved, improving their operability in clinical applications.

[0116] In this embodiment, the culture of olfactory mucosal mesenchymal stem cells is carried out under electrochemical stimulation with set electric field strength and frequency. First, the electrode system is installed in a culture dish and connected to an external electrical signal generator through a sterile interface. The electrode material is a platinum-coated electrode, which has good conductivity and biocompatibility to avoid toxicity or interference of the electrode material to the cells.

[0117] The electrode spacing is set to 3mm-7mm to ensure the uniformity of the electric field distribution. The electrode thickness is 0.05mm-0.2mm to ensure the stability of the electric field and the conductivity.

[0118] In this embodiment, the electrochemical signal has different parameter settings at different stages such as cell proliferation, neural differentiation and optimization of anti-inflammatory function, including control parameters such as electric field strength, frequency and electrical stimulation time.

[0119] Electrochemical signal stimulation during the proliferation phase:

[0120] The electric field strength is set at 0.3V / cm-0.7V / cm, which can activate the voltage-gated ion channels on the cell membrane and promote cell proliferation and metabolism.

[0121] The frequency is set to 30Hz-70Hz. The alternating current within this frequency range can regulate the electrical activity within the cells and promote the acceleration of the cell cycle.

[0122] Each electrical stimulation lasts for 15 minutes to 30 minutes, and electrical stimulation is performed 1 to 3 times a day for 3 days to 4 days to ensure that appropriate electric field stimulation is given during the cell expansion process and no cell stress response is caused.

[0123] Electrochemical signal stimulation during neural differentiation stage:

[0124] The electric field strength was adjusted to 0.2V / cm-0.5V / cm. A lower electric field strength was helpful in inducing mesenchymal stem cells to differentiate into neural cell-like directions.

[0125] The frequency is 10Hz-30Hz, and the low-frequency current is suitable for promoting the electrical activity of neuron-like cells, thereby supporting the generation of neuron-like cells.

[0126] Each electrical stimulation lasts for 10-20 minutes, with 1-2 electrical stimulations per day for 3-4 days, ensuring long-term regulation of the differentiation process by the electric field while avoiding damage to the differentiated neuron-like cells by the electric field.

[0127] The effect of electrochemical stimulation on the direction of cell differentiation during neural differentiation can be regulated and calculated by the following formula:

[0128]

[0129] Among them, I d (t) represents the influence index of neural differentiation, E(t) is the electric field strength, f(t) is the frequency, and α and β are coefficients related to the cell differentiation potential. The efficiency of neural differentiation can be further controlled by adjusting the electric field strength and frequency.

[0130] Electrochemical signal stimulation during the anti-inflammatory function optimization phase:

[0131] The electric field strength is set at 0.1V / cm-0.4V / cm. The lower electric field strength helps maintain cell activity while enhancing the secretion of anti-inflammatory factors in cells.

[0132] The frequency is 20Hz-40Hz, which can regulate cell signaling pathways and enhance the immunoregulatory function of mesenchymal stem cells.

[0133] Each electrical stimulation lasts for 5-15 minutes, once a day, for 3-4 days. This frequency of electrical stimulation helps olfactory mucosal mesenchymal stem cells release more anti-inflammatory factors, such as IL-10 and TGF-β, and improves their effectiveness in anti-inflammatory applications.

[0134] The secretion of anti-inflammatory factors can be estimated by the following formula:

[0135] Q cytokine (t) = Q 0 ·e λ(E·t)

[0136] Among them, Q cytokine (t) is the secretion amount of anti-inflammatory factors at a certain time point, Q 0 is the initial secretion level, λ is the correlation coefficient between the electric field and the cell secretion response, E is the electric field strength, and t is the stimulation duration. By controlling the electric field strength and time, the secretion efficiency of anti-inflammatory factors can be optimized.

[0137] In this embodiment, the application of electrochemical signals is carried out through a precise control system to ensure that the parameters of each stage can be automatically adjusted according to the growth and functional requirements of the cells to avoid over-stimulation or under-stimulation. At the same time, the real-time regulation of the electric field parameters enables the olfactory mucosa mesenchymal stem cells to maintain high activity and functionality during proliferation and functional optimization, thus providing a good basis for their application in nerve repair and anti-inflammatory treatment.

[0138] S5. Use a real-time monitoring system to monitor cell proliferation status, metabolic levels, and membrane potential changes, and dynamically adjust electrochemical signal parameters based on the monitoring results through an intelligent feedback mechanism;

[0139] Step S5 specifically includes the following steps:

[0140] S5.1. Microscopic imaging monitoring: The frequency of cell image acquisition is once every 6-12 hours, and the microscopic imaging analysis system monitors cell morphology and density in real time;

[0141] S5.2 Metabolic monitoring: monitor glucose, lactate and amino acid concentrations, and record metabolic levels in real time, with each test interval of 2h-6h;

[0142] S5.3. Electrophysiological monitoring: real-time monitoring of changes in cell membrane potential and dynamic adjustment of electric field strength and frequency;

[0143] S5.4. Intelligent feedback system adjustment: The system performs real-time data processing based on AI algorithm and adjusts electrochemical parameters according to the following formula.

[0144] The calculation formula for adjusting the electrochemical parameters in step S5.4 is as follows: P(t) = α·E(t) + β·f(t) + γ·ΔVm (t), where P(t) represents the cell proliferation rate, E(t) is the electric field strength, f(t) is the frequency, and ΔV m (t) is the change of cell membrane potential, α, β and γ are the preset feedback parameters, the electric field strength range is 0.1V / cm-1V / cm, and the frequency range is 1Hz-100Hz.

[0145] Specifically, for step S5, the advanced monitoring system can track cell proliferation, metabolic status, and electrophysiological changes in real time to ensure that the cells are in the best culture conditions. Based on the real-time collected data, the intelligent feedback system dynamically adjusts the electrochemical signal parameters through the AI ​​algorithm to ensure that the stimulation intensity and frequency can be optimized according to the growth status of the cells, thereby improving the proliferation efficiency and functional performance of the cells. This closed-loop control mechanism ensures the accuracy and stability of cell culture.

[0146] In this embodiment, the culture process of olfactory mucosal mesenchymal stem cells is equipped with a high-precision real-time monitoring system, which can continuously track cell proliferation, metabolite concentration and membrane potential changes to ensure that the cells are always in the optimal growth environment. The monitoring system mainly includes a microscopic imaging system, a metabolic monitoring module and an electrophysiological monitoring system, which automatically adjusts the culture conditions and the application of electrochemical signals in conjunction with an intelligent feedback mechanism.

[0147] Microscopic imaging monitoring:

[0148] In this embodiment, the microscopic imaging system automatically collects cell images every 6h-12h. The image analysis software embedded in the system can analyze the density, morphological changes and proliferation of cells in real time. By quantitatively analyzing the morphological characteristics of cells, the health status and growth trend of cells can be judged, especially in the expansion stage of olfactory mucosal mesenchymal stem cells, the culture conditions can be discovered and adjusted in time.

[0149] Metabolic monitoring:

[0150] In this embodiment, the metabolic monitoring module can detect the concentration of key metabolites in the culture medium, including glucose, lactic acid, and amino acids. Metabolic data is automatically recorded every 2h-6h to generate a metabolic curve. Changes in metabolic levels can reflect the proliferation activity and metabolic needs of cells.

[0151] The consumption rate of metabolites in the culture medium was calculated by the following formula to predict the time point of the next culture medium change:

[0152]

[0153] Among them, R metabolite(t) is the consumption rate of a metabolite, ΔC is the change in metabolite concentration, and Δt is the time interval. According to this formula, the time for culture medium replacement can be determined to ensure that the cells are adequately supplied with nutrients and metabolic waste does not accumulate.

[0154] Electrophysiological monitoring:

[0155] In this embodiment, the membrane potential changes of the cells are monitored in real time by an electrophysiological monitoring system. Electrophysiological monitoring is essential for understanding the response of cells under electrochemical signal stimulation. The changes in the cell membrane potential can reflect the degree of cell response to the electric field, and then determine whether the electric field stimulation is within an appropriate range.

[0156] Electrophysiological signal monitoring uses the following formula to express the change of membrane potential with electrochemical stimulation time:

[0157]

[0158] Among them, V m (t) is the membrane potential at time t, V m0 is the initial membrane potential, E(t) is the electric field strength, and f(t) is the frequency. This formula can help adjust the electric field parameters in real time to ensure that the cell's electrical activity is maintained at an appropriate level.

[0159] Smart Feedback System Adjustments:

[0160] In this embodiment, the intelligent feedback system processes the data obtained from microscopic imaging, metabolic monitoring and electrophysiological monitoring in real time based on AI algorithms. The system dynamically adjusts the parameters of the electrochemical signal to adapt to the actual needs of the cells by learning the response curves of the cells under different conditions.

[0161] The adjustment of electrochemical parameters can be optimized by the following formula:

[0162] P(t)=α·E(t)+β·f(t)+γ·ΔV m (t)

[0163] Among them, P(t) represents the cell proliferation rate, E(t) is the electric field strength, f(t) is the frequency, ΔV m (t) is the change in membrane potential, and α, β, and γ are coefficients adjusted according to actual culture conditions. Through this formula, the system can adjust the electric field strength and frequency according to real-time data to ensure that the stimulation intensity is moderate, which can promote cell proliferation without causing cell damage.

[0164] In this embodiment, the intelligent feedback mechanism greatly improves the automation level of the cell culture process. The system automatically optimizes the electrochemical signal parameters according to the real-time response of the cells to ensure that the cell growth environment is always in the best state. This closed-loop control system avoids the uncertainty in human operation and improves the stability and consistency of cell culture. At the same time, intelligent feedback can automatically adjust the electrical stimulation parameters according to the cell needs at different stages, so that the olfactory mucosal mesenchymal stem cells can obtain the most suitable electrochemical stimulation at different stages of proliferation, differentiation and functional optimization, thereby maximizing their potential clinical application value.

[0165] S6. Perform phenotypic identification and multidirectional differentiation capacity testing on the expanded olfactory mucosal mesenchymal stem cells, and freeze or use qualified cells for clinical applications.

[0166] Step S6 specifically includes the following steps:

[0167] S6.1. Phenotypic identification: Each batch was tested at least 3 times, using 1×10 5 cells-5×10 5 cells, and the positive expression of surface markers CD73, CD90, and CD105, and the negative expression of CD34 and CD45 were detected by flow cytometry;

[0168] S6.2. Multidirectional differentiation test: Olfactory mucosal mesenchymal stem cells were cultured in neural differentiation induction medium and the concentrations of IL-10 and TGF-β in the culture supernatant were detected by ELISA;

[0169] S6.3. Cell cryopreservation: The cell concentration is controlled at 0.5×10 6 cells / mL-5×10 6 cells / mL, the freezing solution contained 5%-15% DMSO, the cooling rate was -0.5℃ / min--2℃ / min, and the cells were finally stored in a liquid nitrogen tank.

[0170] The multidirectional differentiation test in step S6.2 specifically includes the following steps:

[0171] Neural differentiation test: Olfactory mucosal mesenchymal stem cells were cultured in neural differentiation induction medium. After 10-20 days of differentiation, the expression of Nestin and MAP2 was detected by immunofluorescence.

[0172] Anti-inflammatory ability test: The concentrations of IL-10 and TGF-β in the culture supernatant were detected by ELISA method, with a sample volume of 50μL-200μL.

[0173] Specifically, for step S6, by phenotypic identification of the expanded olfactory mucosal mesenchymal stem cells, it can be confirmed that they retain the characteristics of mesenchymal stem cells, and their differentiation potential can be further verified through multidirectional differentiation tests. Qualified olfactory mucosal mesenchymal stem cells need to be frozen for long-term storage and clinical application. Phenotypic and functional testing of cells is crucial to ensure that the cells meet quality standards before application, and optimization of cell freezing technology can ensure that the cells remain highly active after recovery.

[0174] In this example, phenotypic identification was performed by flow cytometry to ensure that the expanded olfactory mucosal mesenchymal stem cells maintained their surface markers of mesenchymal stem cells. The surface markers detected included positive expression of CD73, CD90, and CD105, and negative expression of CD34 and CD45, which were consistent with the characteristics of mesenchymal stem cells. In the specific operation, the test of each batch was repeated at least 3 times, using 1×10 5 cells to 5×10 5 cells to ensure data reliability and consistency.

[0175] During flow cytometry analysis, the expression rate of the marker was calculated using the following formula:

[0176]

[0177] Among them, P marker is the positive expression rate of a marker, N positive is the number of positive cells, N total is the total cell number. This formula ensures the accuracy and quantification of phenotypic identification.

[0178] In this embodiment, the multidirectional differentiation ability test is a key step in the quality verification of olfactory mucosal mesenchymal stem cells. First, a neural differentiation test is performed. Olfactory mucosal mesenchymal stem cells are inoculated into a neural differentiation induction medium, and after culturing for 10-20 days, the expression levels of neural differentiation markers Nestin and MAP2 are detected by immunofluorescence.

[0179] When detecting neural differentiation markers by immunofluorescence, the differentiation efficiency was calculated by the following formula:

[0180]

[0181] Among them, D neural represents the differentiation rate of neuron-like cells, N Nestinpositive represents the number of Nestin-positive cells, N total is the total cell number.

[0182] In the anti-inflammatory function test, the ELISA method was used to detect the concentration of anti-inflammatory factors IL-10 and TGF-β in the cell culture supernatant. The test sample volume was 50μL-200μL, and quantitative analysis was performed using the standard curve method to ensure that the amplified olfactory mucosal mesenchymal stem cells had stable anti-inflammatory function. The detection principle of ELISA is based on antigen-antibody reaction, and the concentration of the target protein in the sample is determined by colorimetry.

[0183] The concentration of anti-inflammatory factors can be calculated by the following formula:

[0184]

[0185] Among them, C cytokine is the factor concentration in the sample, A sample is the absorbance of the sample, A standard is the absorbance of the standard, C standard is the known concentration of the standard. This formula ensures the accuracy of quantitative detection of anti-inflammatory function.

[0186] In this embodiment, after the olfactory mucosa mesenchymal stem cells pass the phenotypic identification and functional test, qualified cells will be cryopreserved. The purpose of cell cryopreservation is to preserve them for a long time and ensure that the cells can still maintain their activity after recovery. The key to cell cryopreservation is to control the cooling rate to avoid the formation of ice crystals in the cells to damage the cell structure.

[0187] The cell concentration was controlled at 0.5×10 6 cells / mL-5×10 6 cells / mL, and the freezing solution contains 5%-15% DMSO as a protective agent to prevent cells from being damaged during the freezing process.

[0188] The cooling rate was set to -0.5°C / min--2°C / min, and the temperature was gradually reduced by a programmed cooling device, and the cells were finally stored in a liquid nitrogen tank (-196°C).

[0189] During cell freezing, cell survival rate and activity after recovery were evaluated by the following formula:

[0190]

[0191] Among them, S cell Represents the cell survival rate, N livepost-thaw N is the number of cells that survived after recovery. totalpre-freeze is the total number of cells before freezing. This formula was used to evaluate changes in cell activity during freezing and thawing.

[0192] In this embodiment, standardized phenotypic identification, multidirectional differentiation testing and optimized cell cryopreservation process are used to ensure that olfactory mucosal mesenchymal stem cells maintain their functionality and high activity after long-term storage and resuscitation, providing a stable and reliable cell source for subsequent clinical applications.

[0193] Please see attached Figure 2 , a device for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells, used in conjunction with the above-mentioned process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells, comprising:

[0194] Closed sterile bioreactor system for controlling temperature, CO 2 Concentration, O 2 concentration and pH value;

[0195] An electrochemical signal generating device equipped with platinum-coated electrodes for applying electric signals with an electric field strength ranging from 0.1V / cm to 1V / cm and a frequency ranging from 1Hz to 100Hz to regulate cell proliferation, differentiation and function;

[0196] Real-time monitoring system for integrated cell morphology monitoring, metabolic detection, and electrophysiological signal detection;

[0197] Intelligent feedback control module, used to dynamically adjust electric field parameters through AI algorithm.

[0198] Embodiment 1:

[0199] 1. Description of technical solution:

[0200] This embodiment provides a basic hospital-grade olfactory mucosa mesenchymal stem cell preparation and aseptic culture process. The process completes the separation, culture and function optimization of olfactory mucosa mesenchymal stem cells through the following steps:

[0201] S1. Tissue collection and cold chain transportation: 1 mm tissue was collected from the olfactory mucosa of the donor's nasal cavity through minimally invasive surgery. 3 Up to 5mm 3 The tissue should be immediately placed in a cold chain transport medium containing DMEM / F12 basal medium and 1% penicillin-streptomycin. The transportation temperature should be kept below 4°C for no more than 30 minutes.

[0202] S2. Enzyme digestion and single cell suspension preparation: Cut the olfactory mucosa tissue into 1 mm pieces under sterile conditions. 3 -2mm 3 , add 0.2% collagenase I and 0.05% hyaluronidase, digest at 37°C for 30 minutes, terminate the reaction with an equal amount of 10% fetal bovine serum, filter and centrifuge, then resuspend the single cell suspension, and the centrifugation condition is 150g for 5 minutes.

[0203] S3. Serum-free culture and environmental control: The isolated cells were placed in serum-free culture medium and cultured in a bioreactor with a controlled temperature of 37°C and CO 2 The concentration is 5%, and the oxygen concentration is 5%.

[0204] S4. Electrochemical signal regulation: During the cell proliferation stage, the electric field strength applied was 0.5 V / cm, the frequency was 50 Hz, each stimulation was 20 min, twice a day, for 3 days; during the neural differentiation stage, the electric field strength was set to 0.3 V / cm, the frequency was 20 Hz, the stimulation was 15 min, once a day, for 3 days; during the anti-inflammatory stage, the electric field strength was set to 0.2 V / cm, the frequency was 30 Hz, the stimulation was 10 min, once a day, for 3 days.

[0205] S5. Real-time monitoring and feedback control: The cell density and morphological changes are monitored every 6 hours through the microscopic imaging system, the metabolite concentration is detected every 6 hours, and the cell membrane potential is monitored electrophysiologically. Combined with the AI ​​feedback mechanism, the electric field strength and frequency are adjusted according to the real-time data.

[0206] S6. Phenotypic identification and cryopreservation: Flow cytometry was performed (CD73, CD90, CD105 positive, CD34, CD45 negative). After the cells passed the neural differentiation test and ELISA test, the cell cryopreservation concentration was 1×10 6 cells / mL, the freezing solution contained 10% DMSO, and the cooling rate was -1℃ / min.

[0207] Summarize:

[0208] This embodiment provides a standardized hospital-grade olfactory mucosa mesenchymal stem cell preparation process, which can ensure efficient cell proliferation and functional optimization and is suitable for basic production needs.

[0209] Example 2: Improved electrochemical signal control process

[0210] 1. Description of technical solution

[0211] This embodiment optimizes the setting of electrochemical signals based on the first embodiment, and improves the efficiency of cell proliferation and differentiation. The specific steps are as follows:

[0212] S1. Tissue collection and cold chain transportation: Same as in Example 1, the transportation temperature is maintained at 2°C-4°C.

[0213] S2. Enzyme digestion and single cell suspension preparation: The digestion conditions were adjusted to 37° C. for 25 minutes. The other steps were the same as those in Example 1.

[0214] S3, serum-free culture and environmental control: same as in Example 1, the temperature was controlled at 37°C, CO 2 The concentration is 5%, and the oxygen concentration is 5%.

[0215] S4. Electrochemical signal regulation:

[0216] Proliferation stage: the electric field strength was 0.6 V / cm, the frequency was 45 Hz, each stimulation was 20 min, twice a day, and continued for 3 days.

[0217] Neural differentiation stage: the electric field strength is 0.35 V / cm, the frequency is 25 Hz, each stimulation is 15 min, once a day, and lasts for 4 days.

[0218] Anti-inflammatory optimization stage: the electric field strength was 0.25 V / cm, the frequency was 35 Hz, each stimulation was 10 min, once a day, and continued for 4 days.

[0219] S5. Real-time monitoring and feedback control: same as in the first embodiment.

[0220] S6. Phenotypic identification and cryopreservation: Same as Example 1, with unchanged cryopreservation parameters.

[0221] Summarize:

[0222] By further optimizing the electrochemical signal parameters, this embodiment improves the proliferation rate and differentiation efficiency of cells, and is suitable for higher quality cell preparation requirements.

[0223] Embodiment three:

[0224] 1. Description of technical solution

[0225] This embodiment introduces an intelligent feedback system of AI algorithm based on the second embodiment, which can adjust the electrochemical signal parameters according to the cell state in real time. The specific steps are as follows:

[0226] S1. Tissue collection and cold chain transportation: Same as Example 2, the transportation temperature is 2°C-4°C.

[0227] S2. Enzyme digestion and single-cell suspension preparation: The digestion temperature was adjusted to 36.5°C and the time was 25 minutes.

[0228] S3. Serum-free culture and environmental control: same as in Example 2.

[0229] S4. Electrochemical signal regulation:

[0230] Proliferation stage: electric field strength is 0.55 V / cm and frequency is 48 Hz.

[0231] Neural differentiation stage: electric field strength is 0.32 V / cm and frequency is 22 Hz.

[0232] Anti-inflammatory optimization stage: the electric field strength is 0.22V / cm and the frequency is 32Hz.

[0233] S5. Real-time monitoring and feedback control: The AI ​​feedback system automatically adjusts the electric field parameters based on the monitoring data updated every 3 hours to ensure that the cells are always in the best growth state.

[0234] S6. Phenotypic identification and cryopreservation: same as Example 2.

[0235] Summarize:

[0236] Through the AI ​​intelligent feedback mechanism, this embodiment realizes real-time dynamic adjustment of electrochemical signal parameters, thereby improving the automation and accuracy of cell culture.

[0237] Embodiment 4:

[0238] 1. Description of technical solution

[0239] This embodiment enhances the real-time monitoring function based on the third embodiment, and further improves the accuracy of cell culture. The specific steps are as follows:

[0240] S1. Tissue collection and cold chain transportation: Same as Example 3, except that the transportation temperature is 2°C.

[0241] S2. Enzyme digestion and single-cell suspension preparation: The digestion time is 20 minutes.

[0242] S3. Serum-free culture and environmental control: Same as Example 3.

[0243] S4. Electrochemical signal regulation:

[0244] Proliferation stage: electric field strength is 0.58V / cm and frequency is 46Hz.

[0245] Neural differentiation stage: electric field strength is 0.33 V / cm and frequency is 24 Hz.

[0246] Anti-inflammatory optimization stage: the electric field strength is 0.23V / cm and the frequency is 34Hz.

[0247] S5. Real-time monitoring and feedback control: The monitoring system collects cell images and metabolic data every 6 hours, and works with the AI ​​system to adjust the electric field parameters.

[0248] S6. Phenotypic identification and freezing: The freezing solution contains 10% DMSO, and the cooling rate is -1.1°C / min.

[0249] Summarize:

[0250] By strengthening real-time monitoring and data integration, this embodiment improves the refined management of cell culture and enhances the consistency of cell proliferation and differentiation.

[0251] Embodiment five:

[0252] 1. Description of technical solution

[0253] This embodiment optimizes the cell freezing and thawing process based on the fourth embodiment to ensure that the cells maintain high activity after freezing and thawing. The specific steps are as follows:

[0254] S1. Tissue collection and cold chain transportation: Same as Example 4, except that the transportation temperature is 2°C.

[0255] S2. Enzyme digestion and single-cell suspension preparation: The digestion temperature is 36.5°C and the time is 20 minutes.

[0256] S3. Serum-free culture and environmental control: Same as Example 4.

[0257] S4. Electrochemical signal regulation:

[0258] Proliferation stage: electric field strength is 0.57 V / cm and frequency is 47 Hz.

[0259] Neural differentiation stage: electric field strength is 0.31 V / cm and frequency is 23 Hz.

[0260] Anti-inflammatory optimization stage: the electric field strength is 0.22V / cm and the frequency is 33Hz.

[0261] S5. Real-time monitoring and feedback control: same as in Example 4.

[0262] S6. Phenotypic identification and cryopreservation: The cryopreserved cell concentration was 1×10 6 cells / mL, the freezing solution contained 10% DMSO, the cooling rate was optimized to -0.8℃ / min, and the cell survival rate after recovery was greater than 85%.

[0263] Summarize:

[0264] By optimizing the freezing and thawing processes, this embodiment ensures that the cells maintain high activity and functionality after long-term storage, making them suitable for long-term storage and clinical applications.

[0265] Experimental design:

[0266] 1. Purpose of the experiment

[0267] The purpose of this experiment is to verify the advantages of the hospital-grade olfactory mucosa mesenchymal stem cells (OM-MSCs) preparation and aseptic culture process of the present invention in improving cell activity, proliferation efficiency, neural differentiation potential, anti-inflammatory function and cell survival rate after cryopreservation.

[0268] 2. Experimental Design

[0269] The experimental design will test multiple indicators on the role of electrochemical signals, the regulation effect of the intelligent feedback system, and the efficiency of the real-time monitoring system. To ensure the objectivity and effectiveness of the experimental results, a controlled experimental design is adopted, including a control group and multiple experimental groups for comparison.

[0270] Experimental group design:

[0271] Experimental Group A: The basic process of the present invention, including basic electrochemical signal regulation and sterile bioreactor control, refer to Example 1.

[0272] Experimental Group B: Optimized electrochemical signal regulation, referring to Example 2, focusing on testing the effects of electrochemical signals on cell proliferation and differentiation at different stages.

[0273] Experimental group C: an intelligent feedback mechanism was added to dynamically regulate electrochemical signals through real-time data monitoring and AI feedback, refer to Example 3.

[0274] Experimental group D: enhanced real-time monitoring system, combined with higher frequency cell morphology and metabolism detection, refer to Example 4.

[0275] Experimental group E: Optimized cryopreservation and thawing process to ensure that the cell survival rate and function remain in the best state after cryopreservation, refer to Example 5.

[0276] Control group: traditional process without electrochemical signal and intelligent feedback mechanism.

[0277] Experimental samples:

[0278] Donor: Olfactory mucosal tissues were harvested from the same donor to ensure that differences between donors were minimized.

[0279] Initial cell volume: The cell concentration of each group is unified as 1×10 6 cells / mL to maintain experimental consistency.

[0280] 3. Experimental steps

[0281] 1. Tissue collection and cold chain transportation

[0282] Olfactory mucosal tissue (1-5 mm) was collected from the same donor by minimally invasive surgery. 3 ) and immediately place in cold chain transport fluid and maintain transportation at 2-4℃.

[0283] All experimental and control groups were treated in the same way to ensure consistent basic operations.

[0284] 2. Cell Isolation and Culture

[0285] Each experimental group and control group were separated and cultured according to their own processes:

[0286] Experimental group A: using basic electrochemical signal control, refer to the electric field strength and frequency of Example 1.

[0287] Experimental Group B: Optimized electrochemical signals were used to evaluate the effects of electric field strength and frequency at different stages (proliferation, differentiation, and anti-inflammatory function).

[0288] Experimental Group C: An intelligent feedback system was introduced to dynamically adjust the electric field parameters based on real-time monitoring data of cells.

[0289] Experimental Group D: Enhanced real-time monitoring system, which records data such as cell morphology, metabolic changes, membrane potential, etc. in real time, and dynamically adjusts culture conditions.

[0290] Experimental Group E: Optimize the freezing and thawing process, focusing on the survival rate and functional recovery of cells after freezing.

[0291] Control group: No electrochemical signal and intelligent feedback system were used, and environmental conditions were artificially controlled.

[0292] 3. Experimental observation indicators

[0293] The key observation indicators in the experiment are: cell proliferation rate, neural differentiation efficiency, anti-inflammatory factor secretion, cell survival rate after freezing and functional recovery.

[0294] 4. Experimental evaluation and detection methods

[0295] Cell proliferation rate: The cell proliferation rate was determined by MTT method or CCK-8 method. Samples were taken on the 3rd and 4th day of culture to detect the cell proliferation in each group.

[0296] Neural differentiation ability: On day 4, the neural cell markers Nestin and MAP2 were detected by immunofluorescence to evaluate the neural differentiation potential of cells in each group.

[0297] Anti-inflammatory function: The concentrations of anti-inflammatory factors (such as IL-10 and TGF-β) secreted by cells were detected by ELISA. The test samples were the cell culture supernatant on the 3rd and 4th days.

[0298] Cell freezing and recovery: Cryopreservation was performed on the 4th day of culture and thawed after 30 days. The survival rate of the frozen cells was determined by trypan blue staining or flow cytometry, and the cells were cultured for another 2 days to observe the recovery of cell function (secretion level of anti-inflammatory factors and neural differentiation potential) after recovery.

[0299] 4. Data Recording and Analysis

[0300] 1. Cell proliferation rate

[0301] Group Cell proliferation rate on day 3 (%) Cell proliferation rate on day 4 (%) Experimental Group A 125 170 Experimental Group B 135 180 Experimental Group C 145 190 Experimental Group D 150 200 Experimental Group E 147 195 Control group 100 130

[0302] 2. Neural Differentiation Efficiency

[0303] Group Nestin positive rate (%) MAP2 positive rate (%) Experimental Group A 55 45 Experimental Group B 60 50 Experimental Group C 65 55 Experimental Group D 70 60 Experimental Group E 68 58 Control group 50 40

[0304] 3. Secretion level of anti-inflammatory factors

[0305]

[0306] 4. Survival rate after cryopreservation and functional recovery after resuscitation

[0307]

[0308] V. Experimental Summary

[0309] Cell proliferation: The cell proliferation rates of experimental groups A to E were significantly higher than those of the control group, especially the experimental groups C, D, and E, which had the highest proliferation rates on the 4th day, proving that electrochemical signal regulation, intelligent feedback mechanism, and real-time monitoring system have a significant promoting effect on cell proliferation.

[0310] Neural differentiation potential: The neural differentiation efficiency of experimental groups B to E was significantly higher than that of experimental group A and the control group, especially experimental groups D and E, with the highest Nestin and MAP2 positivity rates, indicating that the optimized regulation and intelligent feedback of electrochemical signals greatly improved the neural differentiation ability of olfactory mucosa mesenchymal stem cells.

[0311] Anti-inflammatory function: The secretion levels of anti-inflammatory factors (IL-10 and TGF-β) in experimental groups C, D, and E were significantly higher than those in experimental group A and the control group, indicating that the optimized electrochemical signals and intelligent feedback significantly enhanced the anti-inflammatory ability of cells.

[0312] Survival rate after cryopreservation: The survival rate after cryopreservation and functional recovery after thawing in experimental group E were the best, which further verified that the optimized cryopreservation process can maintain the activity and functionality of cells to the greatest extent.

[0313] Comprehensive conclusion: This experiment verified the significant advantages of the process of the present invention in improving the proliferation efficiency, neural differentiation potential, anti-inflammatory function and post-cryostorage activity of olfactory mucosal mesenchymal stem cells, and proved its innovation and effectiveness in automated control and functional optimization.

[0314] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for preparing and aseptically culturing hospital-grade olfactory mucosal mesenchymal stem cells, characterized in that: The following steps are involved: S1. Collect olfactory mucosal tissue from the donor's olfactory mucosa area through minimally invasive surgery, and place the collected tissue in cold chain transport fluid and maintain it below 4°C during transportation; S2. Under sterile conditions, the olfactory mucosa tissue was minced and enzymatically digested, the obtained tissue fragments were digested with an enzyme digestion solution, and then a single cell suspension was collected by centrifugation and filtration; S3, culturing the isolated olfactory mucosa mesenchymal stem cells in a serum-free culture medium, and controlling the culture environment by an automated bioreactor system, including the regulation of temperature, CO2 concentration, and O2 concentration; S4, applying electrochemical signals to the olfactory mucosal mesenchymal stem cells to stimulate cell proliferation, differentiation or function optimization by setting the electric field strength and frequency; S5. Use a real-time monitoring system to monitor cell proliferation status, metabolic levels, and membrane potential changes, and dynamically adjust electrochemical signal parameters based on the monitoring results through an intelligent feedback mechanism; S6. Perform phenotypic identification and multidirectional differentiation capacity testing on the expanded olfactory mucosal mesenchymal stem cells, and freeze or use qualified cells for clinical applications.

2. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 1, characterized in that: The S2 step specifically includes the following steps: S2.

1. Aseptic processing: Cut the olfactory mucosa tissue into pieces in a biological safety cabinet. The size of the tissue fragments should be 1 mm. 3 -2mm 3 ; S2.2, enzyme digestion treatment: prepare a solution with a mass concentration of 0.2% collagenase I and 0.05% hyaluronidase, the digestion temperature is 35°C-37°C, and the digestion time is 20min-40min; S2.3, stop enzyme digestion: when stopping the enzyme reaction, add an equal volume of 10% fetal bovine serum culture medium, the termination time is 1min-3min; S2.4, Filtration and centrifugation: The mesh size used for filtration is 100μm-150μm, the centrifugal speed is 100g-300g, and the centrifugal time is 3min-7min; S2.

5. Cell resuspension: The cell resuspension density is controlled at 1×10 6 cells / mL-5×10 6 cells / mL.

3. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 1, characterized in that: The S3 step specifically includes the following steps: S3.

1. Initial cell culture: The cell density was 1×10 6 cells / mL-5×10 6 cells / mL; S3.

2. Bioreactor Setup: The temperature is controlled at 35℃-38℃; CO2 concentration is set at 3%-7%; O2 concentration is set at 3%-10%; S3.

3. Automated culture medium replacement: The frequency of culture medium replacement is once every 12h-24h, depending on the cell density.

4. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 1, characterized in that: The S4 step specifically includes the following steps: S4.

1. Electrode installation and connection: The spacing between platinum-coated electrodes is 3mm-7mm, and the electrode thickness is 0.05mm-0.2mm; S4.

2. Electrochemical signal stimulation during the proliferation stage: the electric field strength ranges from 0.3V / cm to 0.7V / cm, the frequency is 30Hz to 70Hz, the duration of each electrical stimulation is 15min to 30min, and the electrical stimulation is 1 to 3 times a day for 3 to 4 days; S4.

3. Electrochemical signal stimulation during the neural differentiation stage: the electric field strength ranges from 0.2V / cm to 0.5V / cm, the frequency ranges from 10Hz to 30Hz, the duration of each electrical stimulation is 10min to 20min, and the electrical stimulation is performed once or twice a day for 3d to 4d. S4.

4. Electrochemical signal stimulation in the anti-inflammatory function optimization stage: the electric field strength range is 0.1V / cm-0.4V / cm, the frequency is 20Hz-40Hz, each electrical stimulation lasts for 5min-15min, and the electrical stimulation is performed once a day for 2d-4d.

5. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 1, characterized in that: The step S5 specifically includes the following steps: S5.

1. Microscopic imaging monitoring: The frequency of cell image acquisition is once every 6-12 hours, and the microscopic imaging analysis system monitors cell morphology and density in real time; S5.2 Metabolic monitoring: monitor glucose, lactate and amino acid concentrations, and record metabolic levels in real time, with each test interval of 2h-6h; S5.

3. Electrophysiological monitoring: real-time monitoring of changes in cell membrane potential and dynamic adjustment of electric field strength and frequency; S5.

4. Intelligent feedback system adjustment: The system performs real-time data processing based on AI algorithm and adjusts electrochemical parameters according to the following formula.

6. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 5, characterized in that: The calculation formula for adjusting the electrochemical parameters in step S5.4 is as follows: P(t) = α·E(t) + β·f(t) + γ·ΔV m (t), where P(t) represents the cell proliferation rate, E(t) is the electric field strength, f(t) is the frequency, and ΔV m (t) is the change of cell membrane potential, α, β and γ are the preset feedback parameters, the electric field strength range is 0.1V / cm-1V / cm, and the frequency range is 1Hz-100Hz.

7. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 1, characterized in that: The step S6 specifically includes the following steps: S6.

1. Phenotypic identification: Each batch was tested at least 3 times, using 1×10 5 cells-5×10 5 cells, and the positive expression of surface markers CD73, CD90, and CD105, and the negative expression of CD34 and CD45 were detected by flow cytometry; S6.

2. Multidirectional differentiation test: Olfactory mucosal mesenchymal stem cells were cultured in neural differentiation induction medium and the concentrations of IL-10 and TGF-β in the culture supernatant were detected by ELISA; S6.

3. Cell cryopreservation: The cell concentration is controlled at 0.5×10 6 cells / mL-5×10 6 cells / mL, the freezing solution contained 5%-15% DMSO, the cooling rate was -0.5℃ / min--2℃ / min, and the cells were finally stored in a liquid nitrogen tank.

8. The process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells according to claim 6, characterized in that: The multidirectional differentiation test in step S6.2 specifically includes the following steps: Neural differentiation test: Olfactory mucosal mesenchymal stem cells were cultured in neural differentiation induction medium. After 10-20 days of differentiation, the expression of Nestin and MAP2 was detected by immunofluorescence. Anti-inflammatory ability test: The concentrations of IL-10 and TGF-β in the culture supernatant were detected by ELISA method, with a sample volume of 50μL-200μL.

9. A device for preparing and aseptically culturing hospital-grade olfactory mucosal mesenchymal stem cells, characterized in that: Used in conjunction with the process for preparing and aseptically culturing hospital-grade olfactory mucosa mesenchymal stem cells as described in any one of claims 1 to 8, comprising: Closed sterile bioreactor system for controlling temperature, CO2 concentration, O2 concentration and pH value; An electrochemical signal generating device equipped with platinum-coated electrodes for applying electric signals with an electric field strength ranging from 0.1V / cm to 1V / cm and a frequency ranging from 1Hz to 100Hz to regulate cell proliferation, differentiation and function; Real-time monitoring system for integrated cell morphology monitoring, metabolic detection, and electrophysiological signal detection; Intelligent feedback control module, used to dynamically adjust electric field parameters through AI algorithm.

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