Resuscitation culture and passage method of human induced pluripotent stem cells

Abstract

The invention relates to the technical field of biological cells, in particular to a recovery culture and passage method of human induced multifunctional stem cells, the adopted culture technical means is no longer rough, more specific and refined, the success rate of obtaining the multifunctional stem cells is high, the probability that the multifunctional stem cells cannot survive is reduced, and the survival rate of the multifunctional stem cells is increased. The efficient and high-success-rate culture of the multifunctional stem cells is facilitated, and large-scale recovery and subculture can be realized.

Description

Technical Field

[0001] This application relates to the field of biological cell technology, and in particular to a method for the resuscitation, culture, and passage of human induced pluripotent stem cells. Background Technology

[0002] Induced pluripotent stem cell (iPSC) technology refers to the reprogramming of terminally differentiated somatic cells into pluripotent stem cells by introducing specific transcription factors. Cell reprogramming is the process by which differentiated cells, under specific conditions, are reversed, returning to a pluripotent state, forming embryonic stem cell lines, or further developing into new individuals. Differentiation is the result of selective gene expression and does not alter the genetic material; reprogramming, in a sense, is a reversal of differentiation. Unlike classic embryonic stem cell technology and somatic cell nuclear transfer technology, iPSC technology does not use embryonic cells or egg cells, thus avoiding ethical issues. Furthermore, iPSC technology allows for the preparation of proprietary stem cells using the patient's own somatic cells, significantly reducing the possibility of immune rejection. The emergence of iPSCs has generated a strong response in stem cell, epigenetics, and biomedical research fields, providing a groundbreaking new understanding of the regulatory mechanisms of pluripotency and further bridging the gap between stem cells and clinical disease treatment. iPSCs have enormous potential value in cell replacement therapy, pathogenesis research, new drug screening, and clinical treatment of diseases such as nervous system diseases and cardiovascular diseases.

[0003] For example, Chinese patent application number CN201110036898.4 discloses a method for culturing human induced pluripotent stem cells using human bone marrow mesenchymal stem cells as a trophoblast, achieved through the following technical solution:

[0004] (1) Preparation of trophoblast cells: Human bone marrow mesenchymal stem cells of the third to fifth generation after passage culture and cryopreservation and thawing were used as trophoblast cells. The cells were seeded into six-well plates treated with 0.1% gelatin, with a cell confluence of 80%-90%, and inactivated by treatment with 10 μg / ml mitomycin C for 2 hours. The inactivated cells were used within 24 hours.

[0005] (2) Obtaining human induced pluripotent stem cells: Foreskin fibroblasts from children aged 2-3 years were transfected twice with a retrovirus carrying transcription factors Klf4, Sox2, Oct4 and c-Myc. On the second day, vitamin C 25 μg / ml was added to the culture system. On the fifth day, VPA 2 μM was added. On the sixth day, the transfected fibroblasts were seeded onto feeder cells. On the seventh day, the culture medium containing bFGF and serum replacement was changed to human ESCs culture medium. The medium was changed daily until ESC-like clones appeared. The clones were selected and expanded for culture.

[0006] (3) Expansion culture of human induced pluripotent stem cells: Select ESC-like clones under a microscope, digest them with type IV collagenase for 5-10 minutes, gently blow them into small clumps, and inoculate them onto the prepared feeder cells. Change the medium every day and passage them every 7-8 days. The expanded cells are passaged mechanically.

[0007] (4) Biological characteristics and maintenance of pluripotency of human induced pluripotent stem cells: After 14 generations of expansion culture on human bone marrow mesenchymal stem cells as trophoblast, the expression of ESCs-specific genes of human induced pluripotent stem cells after expansion culture was identified by RT-PCR. At the same time, the EB formation method and the immunodeficient mouse teratoma formation method were used to detect whether they could differentiate into the three germ layers in vivo and in vitro.

[0008] Regarding the aforementioned technologies, the inventors believe that the following technical defects exist and require improvement:

[0009] Existing cell culture techniques are crude, lack specificity and precision, and have a relatively low success rate in obtaining pluripotent stem cells. Often, pluripotent stem cells fail to survive, which is not conducive to the efficient and high-success-rate culture of pluripotent stem cells, and makes it impossible to achieve large-scale resuscitation and passage culture. Summary of the Invention

[0010] This application provides a method for the resuscitation, culture, and passage of human induced pluripotent stem cells to improve the following technical problems:

[0011] Existing cell culture techniques are crude, lack specificity and precision, and have a relatively low success rate in obtaining pluripotent stem cells. Often, pluripotent stem cells fail to survive, which is not conducive to the efficient and high-success-rate culture of pluripotent stem cells, and makes it impossible to achieve large-scale resuscitation and passage culture.

[0012] In a first aspect, this application provides a method for the resuscitation, culture, and passage of human induced pluripotent stem cells, employing the following technical solution:

[0013] A method for the resuscitation, culture, and passage of human induced pluripotent stem cells, comprising the following steps:

[0014] Step a: Prepare a six-well plate A and a water bath. Spread 20% Matrigel solution into 1-3 wells of the six-well plate A, and label the cell line information, passage number and date on the lid of the six-well plate A. Add 10 μM of Y-27632 inhibitor to E8 medium, mix thoroughly by inverting to form E8-10Y medium, and preheat E8-10Y medium at room temperature for 15-25 minutes.

[0015] Step b: Aspirate the pre-coated Matrigel solution from the wells of six-well plate A and add 1.5 mL of E8-10Y medium to each well of six-well plate A;

[0016] Step c: Remove the frozen cell tubes from the dry ice, add 500 μL of preheated E8-10Y culture medium, and then thaw and sterilize.

[0017] Step d: Gently pipette the cell suspension using a 1000μL wide-mouth pipette tip and transfer it to a new 15mL centrifuge tube. Rinse the cell tube with 1mL of E8-10Y medium, ensuring that all residual cells are collected. Transfer the rinsing solution to the 15mL centrifuge tube as well. Add E8-10Y medium to the centrifuge tube to bring the total volume to 5.3-5.8mL.

[0018] Step e: Centrifuge to precipitate the cells, then carefully aspirate the supernatant, add 750 μL of E8-10Y medium, gently pipette the cell pellet with a 1000 μL wide-mouth pipette tip, add another 750 μL of E8-10Y medium to bring the total volume to 1.5 mL, and gently pipette to mix.

[0019] Step f: Add the cell suspension dropwise into the wells of six-well plate A, and gently shake six-well plate A to distribute the cells evenly;

[0020] Step g: Incubate the cells in an incubator for 24 hours. Preheat the E8 medium at room temperature for 15-20 minutes and replace it with fresh E8 medium without Y-27632 inhibitor. Then replace the E8 medium every other day.

[0021] Step h: Every 4-5 days, when the cell confluence reaches 75-80%, the cells can be passaged or used for differentiation experiments;

[0022] Step i: Prepare a six-well plate B, coat the wells of the six-well plate B with Vitronectin solution, prepare 17 mL of E8-10Y medium and preheat at room temperature for 20 minutes, and prepare cell dissociation enzymes and equilibrate at room temperature or preheat at 37°C for 12-18 minutes.

[0023] Step j: Aspirate the pre-coated Vitronectin solution from the wells of the six-well plate B and add 1.5 mL of E8-10Y medium to each well of the six-well plate B;

[0024] Step k: Carefully aspirate the old culture medium from one of the wells that has reached 75-80% cell confluence, wash the well, add 500 μL of preheated cell dissociation enzyme to the center of the well, and gently shake the six-well plate B to cover the entire surface of the well with the solution.

[0025] Step 1: Incubate in an incubator for 5-10 minutes;

[0026] Step m: After all the cells have detached from the six-well plate B, collect all the detached cells into a 15mL centrifuge tube;

[0027] Step n: Slowly add 5 mL of E8-10Y culture medium to a 15 mL centrifuge tube using a 5 mL serum pipette, keeping the total volume of liquid in the centrifuge tube at 5-6 mL. Centrifuge to precipitate the cells, and carefully aspirate the supernatant without disturbing the cell pellet.

[0028] Step o: Add 1 mL of E8-10Y culture medium using a p1000 wide pipette tip and gently pipette the cell pellet.

[0029] Step p: Then slowly add an additional 5 mL of LE8-10Y culture medium using a 5 mL serum pipette to bring the total volume to 5.8-6.2 mL. Gently pipette to mix thoroughly. Then add 5 drops of cell suspension to the first well of a 6-well plate B pre-coated with Vitronectin solution using a 5 mL serum pipette. Gently shake the 6-well plate B to distribute the cells evenly.

[0030] Step q: Examine under a microscope to see if the cell seeding density in the first well reaches 50%, and observe whether the cell clusters are all the same size of 6 cells. After observing that the standard is met, gently shake the six-well plate B to distribute the cells evenly, and then put the cells into the incubator to continue culturing.

[0031] Step r: After 24 hours, replace with fresh E8 medium without Y-27632 inhibitor, and preheat the E8 medium at room temperature for 15-20 minutes. Replace the E8 medium every other day and continue to culture and maintain the cells until they reach cell confluence suitable for passage or experimentation.

[0032] In one feasible technical solution of this application, steps b and j are both completed quickly within 30-60 seconds to avoid drying of the hole surface.

[0033] In one feasible technical solution of this application, in step c, the thawing and disinfection process is as follows: gently shake the cell tube in a water bath at 36.6-37.3°C until only small pieces of ice remain in the cell tube, and then take the cell tube to a biosafety cabinet and spray it with 75% ethanol for disinfection.

[0034] In one feasible technical solution of this application, the centrifugation conditions in steps e and n are as follows: treatment at room temperature, centrifugal force between 280-320g, and treatment time of 4-6 minutes.

[0035] In one feasible technical solution of this application, in step f, the cell density is checked under a microscope to ensure that the cell density of each well is not less than 7 × 10⁻⁶. 5 If the cell density is insufficient, increase the volume of the cell suspension.

[0036] In one feasible technical solution of this application, in steps g, l and q, the environment inside the incubator is set as follows: temperature 37℃, CO2 concentration level 5.0%, pH value 7.2-7.4, and relative saturation humidity 95%.

[0037] In one feasible technical solution of this application, in steps g and r, the E8 culture medium is replaced by: aspirating the old culture medium and adding fresh E8 culture medium, using a 5 mL pipette to add 2 mL of E8 culture medium to each well, and slowly adding the E8 culture medium along the well wall.

[0038] In one feasible technical solution of this application, in step k, the washing process is as follows: gently wash twice with 2.5 mL of 1XDPBS buffer, and then aspirate the 1XDPBS buffer.

[0039] In one feasible technical solution of this application, in step 1, the cells are checked every 2-3 minutes during incubation to see if they begin to round out and detach from the surface of the six-well plate. If the cells are still tightly attached, incubation continues, with the incubation time increasing by 20 seconds each time. The separated cell clumps will be passaged in tiny cell clusters, with an average of 6 cells per cell cluster.

[0040] In one feasible technical solution of this application, in step m, the method of collecting cells is as follows: tilt the six-well plate B at 45 degrees, gently blow the six-well plate B with a p1000 wide pipette tip, and gently blow the cell clumps to disperse them into uniformly sized clumps, with an average of 5-7 cells per clump.

[0041] In summary, this application includes at least one of the following beneficial technical effects:

[0042] Firstly, compared to conventional culture medium, E8-10Y medium contains the Y-27632 inhibitor, which can significantly reduce cell apoptosis. At the same time, the preheated E8-10Y medium is more suitable for cell survival. During the rapid incubation of cells in the incubator, the E8-10Y medium is replaced with fresh E8 medium without the Y-27632 inhibitor. That is, the Y-27632 inhibitor is removed within 24 hours to promote normal cell proliferation.

[0043] Secondly, during cell passage culture, the 20% matrigel solution was replaced with the Vitronectin solution. Similarly, in the early stage of passage, E8-10Y medium was used for culture. Within 24 hours, it was replaced again with fresh E8 medium without Y-27632 inhibitor to promote rapid cell proliferation.

[0044] The entire method and steps of this application employ more specific and refined culture techniques, resulting in a higher success rate in obtaining pluripotent stem cells and reducing the probability of pluripotent stem cells failing to survive. This facilitates efficient and high-success-rate culture of pluripotent stem cells, enabling large-scale resuscitation and passage culture. Detailed Implementation

[0045] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0046] This application discloses a method for the resuscitation, culture, and passage of human induced pluripotent stem cells, comprising the following steps:

[0047] Step a: Prepare a six-well plate A and a water bath. Spread 20% Matrigel solution into 1-3 wells of the six-well plate A, and label the cell line information, passage number and date on the lid of the six-well plate A. Add 10 μM of Y-27632 inhibitor to E8 medium, mix thoroughly by inverting to form E8-10Y medium, and preheat E8-10Y medium at room temperature for 15-25 minutes.

[0048] Step b: Aspirate the pre-coated Matrigel solution from the wells of the six-well plate A, and add 1.5 mL of E8-10Y medium to each well of the six-well plate A. The above operation should be completed quickly within 30-60 seconds to avoid drying out the surface of the wells.

[0049] Step c: Remove the frozen cell tubes from the dry ice, add 500 μL of preheated E8-10Y medium, and then thaw and sterilize. The thaw and sterilization process is as follows: gently shake the cell tubes in a water bath at 36.6-37.3℃ until only small pieces of ice remain in the cell tubes. Then take the cell tubes to the biosafety cabinet and spray them with 75% ethanol for sterilization.

[0050] Step d: Gently pipette the cell suspension using a 1000μL wide-mouth pipette tip and transfer it to a new 15mL centrifuge tube. Rinse the cell tube with 1mL of E8-10Y medium, ensuring that all residual cells are collected. Transfer the rinsing solution to the 15mL centrifuge tube as well. Add E8-10Y medium to the centrifuge tube to bring the total volume to 5.3-5.8mL.

[0051] Step e: Centrifuge to pellet the cells. The centrifugation conditions are as follows: at room temperature, centrifuge at a force between 280-320g for 4-6 minutes. Then carefully aspirate the supernatant, add 750μL of E8-10Y medium, gently pipette the cell pellet using a 1000μL wide-mouth pipette tip, add another 750μL of E8-10Y medium to bring the total volume to 1.5mL, and gently pipette to mix.

[0052] Step f: Add the cell suspension dropwise to the wells of six-well plate A, gently shake plate A to distribute the cells evenly, and check the cell density under a microscope to ensure that the cell density in each well is not less than 7 × 10⁻⁶. 5 If the cell density is insufficient, increase the volume of the cell suspension.

[0053] Step g: Incubate the cells in an incubator with the following environmental settings: temperature 37℃, CO2 concentration 5.0%, pH 7.2-7.4, and relative humidity 95%. After 24 hours, preheat the E8 medium at room temperature for 15-20 minutes and replace it with fresh E8 medium without Y-27632 inhibitor. Then, replace the E8 medium every other day. The method for replacing the E8 medium is as follows: aspirate the old medium and add fresh E8 medium. Use a 5mL pipette to add 2mL of E8 medium to each well, slowly adding the E8 medium along the well wall.

[0054] Step h: Every 4-5 days, when the cell confluence reaches 75-80%, the cells can be passaged or used for differentiation experiments;

[0055] Step i: Prepare a six-well plate B, coat the wells of the six-well plate B with Vitronectin solution, prepare 17 mL of E8-10Y medium and preheat at room temperature for 20 minutes, and prepare cell dissociation enzymes and equilibrate at room temperature or preheat at 37°C for 12-18 minutes.

[0056] Step j: Aspirate the pre-coated Vitronectin solution from the wells of the six-well plate B, and add 1.5 mL of E8-10Y medium to each well of the six-well plate B. The above operation should be completed quickly within 30-60 seconds to avoid drying out the surface of the wells.

[0057] Step k: Carefully aspirate the old culture medium from one of the wells that has reached 75-80% cell confluence and wash the well by gently washing it twice with 2.5 mL of 1X DPBS buffer, then aspirate the 1X DPBS buffer; add 500 μL of preheated cell dissociation enzyme to the center of the well, and gently shake the six-well plate B to cover the entire surface of the well.

[0058] Step 1: Incubate in an incubator for 5-10 minutes. The incubator environment should be set as follows: temperature 37℃, CO2 concentration 5.0%, pH 7.2-7.4, and relative humidity 95%. During incubation, check every 2-3 minutes to see if the cells begin to round out and detach from the surface of the six-well plate. If the cells are still tightly attached, continue incubation, increasing the incubation time by 20 seconds each time. The separated cell clumps will be passaged in tiny cell clusters, with an average of 6 cells per cell cluster.

[0059] Step m: After all the cells have detached from the six-well plate B, collect all the detached cells into a 15mL centrifuge tube. The method of cell collection is as follows: tilt the six-well plate B at a 45-degree angle and gently blow the six-well plate B with a p1000 wide pipette tip to disperse the cell clumps into uniformly sized clumps, with an average of 5-7 cells per clump.

[0060] Step n: Slowly add 5 mL of E8-10Y culture medium to a 15 mL centrifuge tube using a 5 mL serum pipette, maintaining a total volume of 5-6 mL in the centrifuge tube. Centrifuge to precipitate the cells. The centrifugation conditions are as follows: room temperature, centrifugal force between 280-320 g, and processing time of 4-6 minutes. Carefully aspirate the supernatant without disturbing the cell pellet.

[0061] Step o: Add 1 mL of E8-10Y culture medium using a p1000 wide pipette tip and gently pipette the cell pellet.

[0062] Step p: Then slowly add an additional 5 mL of LE8-10Y culture medium using a 5 mL serum pipette to bring the total volume to 5.8-6.2 mL. Gently pipette to mix thoroughly. Then add 5 drops of cell suspension to the first well of a 6-well plate B pre-coated with Vitronectin solution using a 5 mL serum pipette. Gently shake the 6-well plate B to distribute the cells evenly.

[0063] Step q: Examine under a microscope to see if the cell seeding density in the first well reaches 50%, and whether the cell clusters are of average size 6 cells. After observing that the standard is met, gently shake the six-well plate B to distribute the cells evenly. Then place the cells in an incubator for further culture. The incubator environment is set as follows: temperature 37℃, CO2 concentration 5.0%, pH 7.2-7.4, and relative humidity 95%.

[0064] Step r: After 24 hours, replace with fresh E8 medium without Y-27632 inhibitor, and preheat the E8 medium at room temperature for 15-20 minutes. Replace the E8 medium every other day. The method for replacing the E8 medium is as follows: aspirate the old medium and add fresh E8 medium. Using a 5 mL pipette, add 2 mL of E8 medium to each well, slowly adding the E8 medium along the well wall. Continue to culture and maintain the cells until they reach the cell confluence suitable for passage or experimentation.

[0065] The beneficial technical effects of the method for resuscitation, culture, and passage of human induced pluripotent stem cells according to the embodiments of this application are roughly as follows:

[0066] Firstly, compared to conventional culture medium, E8-10Y medium contains the Y-27632 inhibitor, which can significantly reduce cell apoptosis. At the same time, the preheated E8-10Y medium is more suitable for cell survival. During the rapid incubation of cells in the incubator, the E8-10Y medium is replaced with fresh E8 medium without the Y-27632 inhibitor. That is, the Y-27632 inhibitor is removed within 24 hours to promote normal cell proliferation.

[0067] Secondly, during cell passage culture, the 20% matrigel solution was replaced with the Vitronectin solution. Similarly, in the early stage of passage, E8-10Y medium was used for culture. Within 24 hours, it was replaced again with fresh E8 medium without Y-27632 inhibitor to promote rapid cell proliferation.

[0068] The entire method and steps of this application employ more specific and refined culture techniques, resulting in a higher success rate in obtaining pluripotent stem cells and reducing the probability of pluripotent stem cells failing to survive. This facilitates efficient and high-success-rate culture of pluripotent stem cells, enabling large-scale resuscitation and passage culture.

[0069] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for the resuscitation, culture, and passage of human induced pluripotent stem cells, characterized in that, Includes the following steps: Step a: Prepare a six-well plate A and a water bath. Spread 20% Matrigel solution into 1-3 wells of the six-well plate A, and label the cell line information, passage number and date on the lid of the six-well plate A. Add 10 μM of Y-27632 inhibitor to E8 medium, mix thoroughly by inverting to form E8-10Y medium, and preheat E8-10Y medium at room temperature for 15-25 minutes. Step b: Aspirate the pre-coated Matrigel solution from the wells of six-well plate A and add 1.5 mL of E8-10Y medium to each well of six-well plate A; Step c: Remove the frozen cell tubes from the dry ice, add 500 μL of preheated E8-10Y culture medium, and then thaw and sterilize. Step d: Gently pipette the cell suspension using a 1000μL wide-mouth pipette tip and transfer it to a new 15mL centrifuge tube. Rinse the cell tube with 1mL of E8-10Y medium, ensuring that all residual cells are collected. Transfer the rinsing solution to the 15mL centrifuge tube as well. Add E8-10Y medium to the centrifuge tube to bring the total volume to 5.3-5.8mL. Step e: Centrifuge to precipitate the cells, then carefully aspirate the supernatant, add 750 μL of E8-10Y medium, gently pipette the cell pellet with a 1000 μL wide-mouth pipette tip, add another 750 μL of E8-10Y medium to bring the total volume to 1.5 mL, and gently pipette to mix. Step f: Add the cell suspension dropwise into the wells of six-well plate A, and gently shake six-well plate A to distribute the cells evenly; Step g: Incubate the cells in an incubator for 24 hours. Preheat the E8 medium at room temperature for 15-20 minutes and replace it with fresh E8 medium without Y-27632 inhibitor. Then replace the E8 medium every other day. Step h: Every 4-5 days, when the cell confluence reaches 75-80%, the cells can be passaged or used for differentiation experiments; Step i: Prepare a six-well plate B, coat the wells of the six-well plate B with Vitronectin solution, prepare 17 mL of E8-10Y medium and preheat at room temperature for 20 minutes, and prepare cell dissociation enzymes and equilibrate at room temperature or preheat at 37°C for 12-18 minutes. Step j: Aspirate the pre-coated Vitronectin solution from the wells of the six-well plate B and add 1.5 mL of E8-10Y medium to each well of the six-well plate B; Step k: Carefully aspirate the old culture medium from one of the wells that has reached 75-80% cell confluence, wash the well, add 500 μL of preheated cell dissociation enzyme to the center of the well, and gently shake the six-well plate B to cover the entire surface of the well with the solution. Step 1: Incubate in an incubator for 5-10 minutes; Step m: After all the cells have detached from the six-well plate B, collect all the detached cells into a 15mL centrifuge tube; Step n: Slowly add 5 mL of E8-10Y culture medium to a 15 mL centrifuge tube using a 5 mL serum pipette, keeping the total volume of liquid in the centrifuge tube at 5-6 mL. Centrifuge to precipitate the cells, and carefully aspirate the supernatant without disturbing the cell pellet. Step o: Add 1 mL of E8-10Y culture medium using a p1000 wide pipette tip and gently pipette the cell pellet. Step p: Then slowly add an additional 5 mL of LE8-10Y culture medium using a 5 mL serum pipette to bring the total volume to 5.8-6.2 mL. Gently pipette to mix thoroughly. Then add 5 drops of cell suspension to the first well of a 6-well plate B pre-coated with Vitronectin solution using a 5 mL serum pipette. Gently shake the 6-well plate B to distribute the cells evenly. Step q: Examine under a microscope to see if the cell seeding density in the first well reaches 50%, and observe whether the cell clusters are all the same size of 6 cells. After observing that the standard is met, gently shake the six-well plate B to distribute the cells evenly, and then put the cells into the incubator to continue culturing. Step r: After 24 hours, replace with fresh E8 medium without Y-27632 inhibitor, and preheat the E8 medium at room temperature for 15-20 minutes. Replace the E8 medium every other day and continue to culture and maintain the cells until they reach cell confluence suitable for passage or experimentation.

2. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, Both steps b and j should be completed quickly within 30-60 seconds to avoid drying out the surface of the hole.

3. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In step c, the thawing and sterilization process is as follows: gently shake the cell tubes in a water bath at 36.6-37.3°C until only small pieces of ice remain in the cell tubes, then take the cell tubes to a biosafety cabinet and spray them with 75% ethanol for sterilization.

4. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In steps e and n, the centrifugation conditions are as follows: treatment at room temperature, centrifugal force between 280-320g, and treatment time of 4-6 minutes.

5. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In step f, the cell density is checked under a microscope to ensure that the cell density in each well is not less than 7 × 10⁻⁶. 5 If the cell density is insufficient, increase the volume of the cell suspension.

6. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In steps g, l, and q, the environment inside the incubator is set as follows: temperature 37°C, CO2 concentration 5.0%, pH 7.2-7.4, and relative saturation humidity 95%.

7. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In steps g and r, the E8 medium is replaced as follows: the old medium is aspirated and fresh E8 medium is added. Using a 5 mL pipette, 2 mL of E8 medium is added to each well, and the E8 medium is slowly added along the well wall.

8. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In step k, the washing process is as follows: gently wash twice with 2.5 mL of 1XDPBS buffer, and then aspirate the 1XDPBS buffer.

9. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In step 1, the cells are checked every 2-3 minutes during incubation to see if they begin to round out and detach from the surface of the six-well plate. If the cells are still tightly attached, incubation continues, with the incubation time increasing by 20 seconds each time. The separated cell clumps will be passaged in tiny cell clusters, with an average of 6 cells per cell cluster.

10. The method for resuscitation, culture, and passage of human induced pluripotent stem cells according to claim 1, characterized in that, In step m, the cells are collected as follows: tilt the six-well plate B at a 45-degree angle, and gently blow the six-well plate B with a p1000 wide pipette tip to disperse the cell clumps into uniformly sized clumps, with an average of 5-7 cells per clump.