A method for preparing stem cells using a fully automated cell preparation instrument

Abstract

This invention discloses a method for preparing stem cells using a fully automated cell preparation instrument, relating to the field of stem cell preparation technology. The method includes the following steps: S1, cell collection; S2, stem cell isolation; S3, induction of differentiation or activation; S4, expansion culture; S5, washing and concentration; and S6, quality detection. This method for preparing stem cells using a fully automated, closed-loop stem cell preparation process relies on standardized instrument operation and fluid flow to complete all stages of cell isolation, induction, culture, and purification. Appropriate induction factors are used to achieve targeted induction or stemness enhancement of stem cells. Combined with a sterile gas environment that meets the physiological needs of cells, this method completely avoids the differences and contamination risks associated with manual operation, making the stem cell preparation process simpler and more efficient. It effectively ensures the stability and safety of the preparation process, meeting the standardized preparation requirements for clinical-grade stem cells.

Description

Technical Field

[0001] This invention relates to the field of stem cell preparation technology, specifically a method for preparing stem cells using a fully automated cell preparation instrument. Background Technology

[0002] Stem cells possess self-renewal and multi-directional differentiation capabilities, showing broad application prospects in tissue repair, immune regulation, and disease treatment; however, traditional stem cell preparation methods have the following problems:

[0003] The operation process is cumbersome, requiring manual operation on multiple platforms and in multiple steps, resulting in low efficiency and long cycle time.

[0004] Open-type operations are prone to contamination and cannot meet the requirements for clinical-grade cell preparation.

[0005] Significant differences exist between different operators and laboratories, resulting in poor product consistency. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing stem cells using a fully automated cell preparation instrument, thus solving the problems mentioned in the background section.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing stem cells using a fully automated cell preparation instrument, comprising the following steps:

[0008] S1. Cell Collection: Obtaining biological samples containing stem cells;

[0009] S2. Stem cell isolation: The biological sample is added to the separation module, and stem cells are obtained by density gradient centrifugation or magnetic bead sorting.

[0010] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0011] Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; the Ficoll solution and whole blood are centrifuged in the centrifuge cup until PBMCs are separated; the intermediate layer in the separation cup is extracted from valve 16 through valve 4 into an intermediate liquid bag for later use; the washing solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup and is mixed to clean the centrifuge cup; the remaining solution flows from valve 16 through pump 1 and then through valve 10 to clean the contents of the centrifuge cup. The waste washing liquid is discharged; the reserved intermediate layer liquid flows from valve 4 through pump 1 and valve 16 into the centrifuge cup; the washing liquid flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and is mixed to wash the cells in the centrifuge cup; during centrifugation, the supernatant is removed from valve 16 through pump 1 and valve 10; during centrifugation, the supernatant is further removed from valve 17 through pump 1 and valve 10, achieving concentration; the resuspension flows from valve 6 through pump 1 and valve 17 into the centrifuge cup to resuspend the cells.

[0012] S3. Induced differentiation or activation: In the reaction module, inducing factors or activators are added to induce stem cells to differentiate into the target lineage or enhance their stemness.

[0013] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0014] The induction reagent flows from valve 5 through pump 1 and then through valve 16 into the centrifuge cup; and is incubated at a constant temperature; during the culture process, the required gas is supplied to the centrifuge cup at the set concentration through valve 18.

[0015] S4. Amplification Culture: Stem cell amplification is carried out in the culture module under preset temperature, gas concentration and nutrient conditions;

[0016] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0017] The culture medium flows from valve 2, through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the replenishment. When the culture volume in the centrifuge cup exceeds the preset value, a medium change is required: During centrifugation, the medium supernatant is removed by flowing from valve 16 through pump 1 and through valve 10. Then, the culture medium flows from valve 2, through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the medium change. Under a constant temperature of 37 degrees Celsius, the medium change is repeated according to the set time points for amplification culture. During the culture process, the required gas is supplied to the centrifuge cup at the set concentration through valve 18.

[0018] S5. Washing and Concentration: The purification module removes fragments, impurities, and residual reagents, and concentrates the stem cells to obtain stem cells.

[0019] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0020] The washing solution flows from valve 1 through pump 1, then through valve 17, into the centrifuge cup and is mixed to wash the cells inside. During centrifugation, it flows from valve 16 through pump 1, then through valve 10 to remove the supernatant after washing. During centrifugation, it flows from valve 17 through pump 1, then through valve 10 to further remove the supernatant and achieve concentration. The resuspension solution flows from valve 6 through pump 1, then through valve 17, into the centrifuge cup, is mixed, and resuspends the cells. It then flows from valve 16 through pump 1, then through valve 7 to collect the cells from the centrifuge cup into a liquid bag.

[0021] S6. Quality testing: Testing the purity, activity, phenotype, and sterility indicators of stem cells;

[0022] Steps S1 to S6 are all completed in a fully automated cell preparation instrument.

[0023] Furthermore, in step S2, the stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, induced pluripotent stem cells, or embryonic stem cells.

[0024] Furthermore, in step S1, the biological sample includes bone marrow, umbilical cord blood, peripheral blood, adipose tissue, placental tissue, or skin fibroblasts.

[0025] Furthermore, in step S3, the inducing factor includes growth factors, cytokines, small molecule compounds, or gene editing reagents.

[0026] Furthermore, in steps S3 and S4, the required gas is a ternary mixed gas filtered through a 0.22μm sterile filter membrane, with a composition of 5%. High purity balance .

[0027] Furthermore, in step S4, the device system identifies the stem cell type and automatically loads the corresponding in vivo physiological microenvironment baseline parameters to lock the target regulation range; the built-in sensor in the culture module collects data every 30 seconds.

[0028] Further data collection included fluid shear force, dissolved oxygen, oxygen partial pressure, and culture temperature. Concentration, cell density, glucose concentration, lactate concentration.

[0029] Furthermore, based on a pre-set professional formula algorithm and combined with real-time sensor data, the optimal control parameters for the current cell growth state are calculated.

[0030] Therefore, based on the optimized control parameters, each precision peristaltic pump, oxygen partial pressure regulating valve, PID temperature control module, etc. Precision control valve synchronous adjustment.

[0031] Furthermore, the formula for the accurate calculation model of three-dimensional fluid shear force is as follows:

[0032]

[0033] in, Fluid shear force; : Dynamic viscosity of the culture medium; : Fluid flow rate in the cultivation module; Culture chamber height; : Width of the culture chamber; : Fluid velocity gradient;

[0034] The oxygen partial pressure dynamic control algorithm includes dissolved oxygen concentration calculation and oxygen partial pressure dynamic balance equation. The dissolved oxygen concentration calculation formula is as follows:

[0035]

[0036] in, Dissolved oxygen concentration in the culture system; Henry coefficient; Oxygen mass transfer coefficient;

[0037] The dynamic equilibrium equation for oxygen partial pressure is as follows:

[0038]

[0039] in, Oxygen mass transfer coefficient; : Oxygen consumption rate of stem cells; Oxygen saturation concentration in the culture medium; Real-time density of stem cells; Dissolved oxygen concentration in the culture system Over time The rate of change;

[0040] The formula for the precise PID temperature control algorithm required for the incubation temperature is as follows:

[0041]

[0042] in, : Output power of the temperature control module; Temperature deviation; : Proportional coefficient; Integral coefficient; Differential coefficients; : Indicates temperature deviation Regarding time The first derivative, i.e., temperature deviation Over time The rate of change;

[0043] The formula for the nutrient mass transfer regulation algorithm is as follows:

[0044]

[0045] in, : Nutrient mass transfer flux; Nutrient diffusion coefficient; Nutrient concentration gradient; the control target is to maintain glucose concentration at 5.5~6.0 mmol / L;

[0046] The formula for the microenvironment comprehensive adaptation algorithm is as follows:

[0047]

[0048] in, : Microenvironmental comprehensive adaptability, with a value of 0~100, and ≥90 is the optimal state; , , , Shear force, oxygen partial pressure, temperature, and nutrient single parameter compatibility; , , , The weighting coefficients are 0.25, 0.35, 0.20, and 0.20, respectively.

[0049] This invention provides a method for preparing stem cells using a fully automated cell preparation instrument, which has the following beneficial effects:

[0050] 1. This method for preparing stem cells using a fully automated cell preparation instrument employs a fully closed and automated stem cell preparation process. It is adaptable to various biological samples and stem cell types, and relies on standardized instrument operation and fluid circuit operation to complete all stages of cell separation, induction, culture, and purification. Appropriate inducing factors are used to achieve targeted induction or stemness enhancement of stem cells. Combined with a sterile gas environment that meets the physiological needs of cells, it completely avoids the differences and contamination risks caused by manual operation, making the stem cell preparation process simpler and more efficient. It effectively ensures the stability and safety of the preparation process and meets the standardized preparation requirements of clinical-grade stem cells.

[0051] 2. This method for preparing stem cells using a fully automated cell preparation instrument automatically matches appropriate physiological microenvironment parameters according to the stem cell type. With the help of multi-dimensional real-time sensing and professional algorithm models, it achieves dynamic optimization and adjustment of culture parameters, accurately simulates the natural microenvironment for in vivo stem cell growth, significantly improves the stem cell maintenance capacity and expansion efficiency, stabilizes the differentiation potential of cells, ensures the consistency of stem cell product quality across different batches, and comprehensively improves the quality and clinical application value of stem cell preparation. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the steps of the present invention;

[0053] Figure 2 This is a schematic diagram of the device's external structure according to the present invention;

[0054] Figure 3 This is a schematic diagram of the fluid circuit of the device of the present invention;

[0055] Figure 4 This is a schematic diagram of an umbilical cord mesenchymal stem cell image taken on day 1 according to the present invention.

[0056] Figure 5 This is a schematic diagram of an umbilical cord mesenchymal stem cell image taken on day 2 according to the present invention.

[0057] Figure 6 This is a schematic diagram of an umbilical cord mesenchymal stem cell on day 2.5 according to the present invention. Detailed Implementation

[0058] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0059] like Figures 1-3 As shown, where, Figure 3 In the diagram, 1-22 refer to control valves. This invention provides a technical solution: a method for preparing stem cells using a fully automated cell preparation instrument, comprising the following steps:

[0060] S1. Cell Collection: Obtaining biological samples containing stem cells; biological samples include bone marrow, umbilical cord blood, peripheral blood, adipose tissue, placental tissue, or skin fibroblasts;

[0061] S2. Stem cell isolation: The biological sample is added to the isolation module, and stem cells are obtained by density gradient centrifugation or magnetic bead sorting; stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, induced pluripotent stem cells or embryonic stem cells.

[0062] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0063] Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; the Ficoll solution and whole blood are centrifuged in the centrifuge cup until PBMCs are separated; the intermediate layer in the separation cup is extracted from valve 16 through valve 4 into an intermediate liquid bag for later use; the washing solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup and is mixed to clean the centrifuge cup; the remaining solution flows from valve 16 through pump 1 and then through valve 10 to clean the contents of the centrifuge cup. The waste washing liquid is discharged; the reserved intermediate layer liquid flows from valve 4 through pump 1 and valve 16 into the centrifuge cup; the washing liquid flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and is mixed to wash the cells in the centrifuge cup; during centrifugation, the supernatant is removed from valve 16 through pump 1 and valve 10; during centrifugation, the supernatant is further removed from valve 17 through pump 1 and valve 10, achieving concentration; the resuspension flows from valve 6 through pump 1 and valve 17 into the centrifuge cup to resuspend the cells.

[0064] S3. Induced Differentiation or Activation: In the reaction module, inducing factors or activators are added to induce stem cells to differentiate into the target lineage or enhance their stemness; inducing factors include growth factors, cytokines, small molecule compounds or gene editing reagents;

[0065] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0066] The induction reagent flows from valve 5 through pump 1 and then through valve 16 into the centrifuge cup; and is incubated at a constant temperature; during the culture process, the required gas is supplied to the centrifuge cup at the set concentration through valve 18.

[0067] S4. Amplification Culture: Stem cell amplification is carried out in the culture module under preset temperature, gas concentration and nutrient conditions;

[0068] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0069] The culture medium flows from valve 2, through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the replenishment. When the culture volume in the centrifuge cup exceeds the preset value, a medium change is required: During centrifugation, the medium supernatant is removed by flowing from valve 16 through pump 1 and through valve 10. Then, the culture medium flows from valve 2, through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the medium change. Under a constant temperature of 37 degrees Celsius, the medium change is repeated according to the set time points for amplification culture. During the culture process, the required gas is supplied to the centrifuge cup at the set concentration through valve 18.

[0070] In steps S3 and S4, the required gas is a ternary gas mixture filtered through a 0.22 μm sterile filter membrane, with a composition of 5%. High purity balance ;

[0071] S5. Washing and Concentration: The purification module removes fragments, impurities, and residual reagents, and concentrates the stem cells to obtain stem cells.

[0072] The specific procedure for this step within the fully automated cell preparation instrument is as follows:

[0073] The washing solution flows from valve 1 through pump 1, then through valve 17, into the centrifuge cup and is mixed to wash the cells inside. During centrifugation, it flows from valve 16 through pump 1, then through valve 10 to remove the supernatant after washing. During centrifugation, it flows from valve 17 through pump 1, then through valve 10 to further remove the supernatant and achieve concentration. The resuspension solution flows from valve 6 through pump 1, then through valve 17, into the centrifuge cup, is mixed, and resuspends the cells. It then flows from valve 16 through pump 1, then through valve 7 to collect the cells from the centrifuge cup into a liquid bag.

[0074] S6. Quality testing: Testing the purity, activity, phenotype, and sterility indicators of stem cells;

[0075] Steps S1 to S6 are all completed in a fully automated cell preparation instrument.

[0076] In step S4, the device system identifies the stem cell type and automatically loads the corresponding baseline parameters of the in vivo physiological microenvironment, locking in the target regulation range; the built-in sensors in the culture module collect data every 30 seconds; the collected data includes fluid shear force, dissolved oxygen, oxygen partial pressure, culture temperature, etc. Concentration, cell density, glucose concentration, lactate concentration; based on a preset professional formula algorithm and combined with real-time sensor data, calculate the optimal control parameters for the current cell growth state;

[0077] Therefore, based on the optimized control parameters, each precision peristaltic pump, oxygen partial pressure regulating valve, PID temperature control module, etc. Precision control valve synchronous adjustment;

[0078] The formula for the accurate calculation model of three-dimensional fluid shear force is as follows:

[0079]

[0080] in, Fluid shear force; : Dynamic viscosity of the culture medium; : Fluid flow rate in the cultivation module; Culture chamber height; : Width of the culture chamber; : Fluid velocity gradient;

[0081] The oxygen partial pressure dynamic control algorithm includes dissolved oxygen concentration calculation and oxygen partial pressure dynamic balance equation. The dissolved oxygen concentration calculation formula is as follows:

[0082]

[0083] in, Dissolved oxygen concentration in the culture system; Henry coefficient; Oxygen mass transfer coefficient;

[0084] The dynamic equilibrium equation for oxygen partial pressure is as follows:

[0085]

[0086] in, Oxygen mass transfer coefficient; : Oxygen consumption rate of stem cells; Oxygen saturation concentration in the culture medium; Real-time density of stem cells; Dissolved oxygen concentration in the culture system Over time The rate of change;

[0087] The formula for the precise PID temperature control algorithm required for the incubation temperature is as follows:

[0088]

[0089] in, : Output power of the temperature control module; Temperature deviation; : Proportional coefficient; Integral coefficient; Differential coefficients; : Indicates temperature deviation Regarding time The first derivative, i.e., temperature deviation Over time The rate of change;

[0090] The formula for the nutrient mass transfer regulation algorithm is as follows:

[0091]

[0092] in, : Nutrient mass transfer flux; Nutrient diffusion coefficient; Nutrient concentration gradient; the control target is to maintain glucose concentration at 5.5~6.0 mmol / L;

[0093] The formula for the microenvironment comprehensive adaptation algorithm is as follows:

[0094]

[0095] in, : Microenvironmental comprehensive adaptability, with a value of 0~100, and ≥90 is the optimal state; , , , Shear force, oxygen partial pressure, temperature, and nutrient single parameter compatibility; , , , The weighting coefficients are 0.25, 0.35, 0.20, and 0.20, respectively.

[0096] Example: 10g of adipose tissue was collected, mechanically sheared, digested with collagenase, and filtered to obtain a single-cell suspension of adipose tissue.

[0097] Cell suspension was added to the separation module, and a mononuclear cell layer was obtained by density gradient centrifugation, followed by magnetic bead sorting. Mesenchymal stem cells;

[0098] Adding TGF-β and bFGF to the reaction module promotes the maintenance of stemness in mesenchymal stem cells;

[0099] The physiological microenvironment parameters of mesenchymal stem cells were loaded, and the shear force was set to 0.01~0.05 Pa, oxygen partial pressure to be 4% (4.2 kPa), temperature to be 37℃±0.1℃, and glucose concentration to be 5.5~6.0 mmol / L.

[0100] The sensor collects data on shear force, oxygen partial pressure, temperature, and glucose / lactic acid every 30 seconds;

[0101] The peristaltic pump flow rate was adjusted according to the three-dimensional fluid shear force formula to stabilize the shear force at 0.03 Pa; dissolved oxygen was maintained according to the oxygen partial pressure balance formula; the temperature was precisely controlled at 37.0℃ according to the PID algorithm; and the glucose concentration was maintained at 5.8 mmol / L according to Fick's law.

[0102] After 7 days of continuous culture, the overall microenvironmental fit (S) was ≥95, and the cell number increased approximately 12-fold. Figure 4-6 As shown, Figures 4-6 This is a schematic diagram of umbilical cord mesenchymal stem cell imaging on days 1, 2, and 2.5.

[0103] The purification module removes cell debris, dead cells, and residual reagents, concentrating the stem cells to... ;

[0104] Test results: Cell viability 96%, With a purity of 98%, stable osteogenic / adipogenic / chondrogenic differentiation potential, a 30% improvement in stemness maintenance rate compared to traditional culture, and passing sterility testing.

[0105] In summary, this method for preparing stem cells using a fully automated cell preparation instrument employs a closed-loop, fully automated stem cell preparation process. It is adaptable to various biological samples and stem cell types, and relies on standardized instrument operation and fluid flow to complete all stages of cell separation, induction, culture, and purification. Appropriate induction factors are used to achieve targeted induction or stemness enhancement of stem cells. Combined with a sterile gas environment that meets the physiological needs of cells, it completely avoids the differences and contamination risks caused by manual operation, making the stem cell preparation process simpler and more efficient. It effectively ensures the stability and safety of the preparation process, meeting the standardized preparation requirements for clinical-grade stem cells.

[0106] Based on the stem cell type, the system automatically matches and adapts the physiological microenvironment parameters. With the help of multi-dimensional real-time sensing and professional algorithm models, it achieves dynamic optimization and adjustment of culture parameters, accurately simulates the natural microenvironment for stem cell growth in vivo, significantly improves the stemness maintenance ability and expansion efficiency of stem cells, stabilizes the differentiation potential of cells, ensures the consistency of quality of different batches of stem cell products, and comprehensively improves the quality of stem cell preparation and its clinical application value.

[0107] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for preparing stem cells using a fully automated cell preparation instrument, characterized in that: Includes the following steps: S1. Cell Collection: Obtaining biological samples containing stem cells; S2. Stem cell isolation: The biological sample is added to the separation module, and stem cells are obtained by density gradient centrifugation or magnetic bead sorting. The specific procedure for this step within the fully automated cell preparation instrument is as follows: Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup. Ficoll solution and whole blood were centrifuged in a centrifuge cup until PBMCs were separated. The intermediate layer in the separation cup was extracted from valve 16 through valve 4 and placed in an intermediate liquid bag for later use. The washing solution flowed from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and was mixed to clean the centrifuge cup. The washing waste liquid in the centrifuge cup was discharged from valve 16 through pump 1 and valve 10. The reserved intermediate layer liquid flowed from valve 4 through pump 1 and valve 16 into the centrifuge cup. The washing solution flowed from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and was mixed to clean the cells in the centrifuge cup. During centrifugation, the supernatant was removed from valve 16 through pump 1 and valve 10. During centrifugation, the supernatant was further removed from valve 17 through pump 1 and valve 10 to achieve concentration. The resuspension solution flowed from valve 6 through pump 1 and valve 17 into the centrifuge cup to resuspend the cells. S3. Induced differentiation or activation: In the reaction module, inducing factors or activators are added to induce stem cells to differentiate into the target lineage or enhance their stemness. The specific procedure for this step within the fully automated cell preparation instrument is as follows: The induction reagent flows from valve 5 through pump 1 and then through valve 16 into the centrifuge cup; and is incubated at a constant temperature; during the culture process, the required gas is supplied to the centrifuge cup at the set concentration through valve 18. S4. Amplification Culture: Stem cell amplification is carried out in the culture module under preset temperature, gas concentration and nutrient conditions; The specific procedure for this step within the fully automated cell preparation instrument is as follows: The culture medium flows from valve 2, through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the replenishment. When the culture volume in the centrifuge cup exceeds the preset value, a medium change is required: During centrifugation, the medium supernatant is removed by flowing from valve 16 through pump 1 and through valve 10. Then, the culture medium flows from valve 2, through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the medium change. Under a constant temperature of 37 degrees Celsius, the medium change is repeated according to the set time points for amplification culture. During the culture process, the required gas is supplied to the centrifuge cup at the set concentration through valve 18. S5. Washing and Concentration: The purification module removes fragments, impurities, and residual reagents, and concentrates the stem cells to obtain stem cells. The specific procedure for this step within the fully automated cell preparation instrument is as follows: The washing solution flows from valve 1 through pump 1, then through valve 17, into the centrifuge cup and is mixed to wash the cells inside. During centrifugation, it flows from valve 16 through pump 1, then through valve 10 to remove the supernatant after washing. During centrifugation, it flows from valve 17 through pump 1, then through valve 10 to further remove the supernatant and achieve concentration. The resuspension solution flows from valve 6 through pump 1, then through valve 17, into the centrifuge cup, is mixed, and resuspends the cells. It then flows from valve 16 through pump 1, then through valve 7 to collect the cells from the centrifuge cup into a liquid bag. S6. Quality testing: Testing the purity, activity, phenotype, and sterility indicators of stem cells; Steps S1 to S6 are all completed in a fully automated cell preparation instrument.

2. The method for preparing stem cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S2, the stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, induced pluripotent stem cells, or embryonic stem cells.

3. The method for preparing stem cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S1, the biological samples include bone marrow, umbilical cord blood, peripheral blood, adipose tissue, placental tissue, or skin fibroblasts.

4. The method for preparing stem cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S3, the inducing factors include growth factors, cytokines, small molecule compounds, or gene editing reagents.

5. The method for preparing stem cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In steps S3 and S4, the required gas is a ternary mixed gas filtered through a 0.22μm sterile filter membrane, with a composition of 5%. High purity balance .

6. The method for preparing stem cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S4, the device system identifies the stem cell type and automatically loads the corresponding baseline parameters of the in vivo physiological microenvironment, locking the target range for regulation; the built-in sensor in the culture module collects data every 30 seconds.

7. A method for preparing stem cells using a fully automated cell preparation instrument according to claim 6, characterized in that: The collected data includes fluid shear force, dissolved oxygen, oxygen partial pressure, and culture temperature. Concentration, cell density, glucose concentration, lactate concentration.

8. A method for preparing stem cells using a fully automated cell preparation instrument according to claim 7, characterized in that: Based on a pre-set professional formula algorithm and combined with real-time sensor data, the optimal control parameters for the current cell growth state are calculated. Therefore, based on the optimized control parameters, each precision peristaltic pump, oxygen partial pressure regulating valve, PID temperature control module, etc. Precision control valve synchronous adjustment.

9. A method for preparing stem cells using a fully automated cell preparation instrument according to claim 8, characterized in that: The formula for the accurate calculation model of three-dimensional fluid shear force is as follows: ； in, Fluid shear force; : Dynamic viscosity of the culture medium; : Fluid flow rate in the cultivation module; Culture chamber height; : Width of the culture chamber; : Fluid velocity gradient; The oxygen partial pressure dynamic control algorithm includes dissolved oxygen concentration calculation and oxygen partial pressure dynamic balance equation. The dissolved oxygen concentration calculation formula is as follows: ； in, Dissolved oxygen concentration in the culture system; Henry coefficient; Oxygen mass transfer coefficient; The dynamic equilibrium equation for oxygen partial pressure is as follows: ； in, Oxygen mass transfer coefficient; : Oxygen consumption rate of stem cells; Oxygen saturation concentration in the culture medium; Real-time density of stem cells; Dissolved oxygen concentration in the culture system Over time The rate of change; The formula for the precise PID temperature control algorithm required for the incubation temperature is as follows: ； in, : Output power of the temperature control module; Temperature deviation; : Proportional coefficient; Integral coefficient; Differential coefficients; : Indicates temperature deviation Regarding time The first derivative, i.e., temperature deviation Over time The rate of change; The formula for the nutrient mass transfer regulation algorithm is as follows: ； in, : Nutrient mass transfer flux; Nutrient diffusion coefficient; Nutrient concentration gradient; the control target is to maintain glucose concentration at 5.5~6.0 mmol / L; The formula for the microenvironment comprehensive adaptation algorithm is as follows: ； in, : Microenvironmental comprehensive adaptability, with a value of 0~100, and ≥90 is the optimal state; , , , Shear force, oxygen partial pressure, temperature, and nutrient single parameter compatibility; , , , The weighting coefficients are 0.25, 0.35, 0.20, and 0.20, respectively.

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