A method for cryopreservation and recovery of pluripotent stem cell induced mesoderm cells and applications thereof

Abstract

The application provides a freezing and recovery method and application of pluripotent stem cell induced differentiation middle stage cells, and the freezing method comprises the following steps: (1) the starting cells are pluripotent stem cells, which are induced to the middle stage; (2) the cells are recovered and counted by using a mild digestion enzyme, and the cells are resuspended in a freezing solution; (3) after programmed cooling to-80 DEG C, the cells are transferred into liquid nitrogen for storage, and are ready for use. The freezing method and the freezing solution provided by the application significantly improve the freezing survival rate of the differentiation middle stage cells, avoid the apoptosis or irreversible stress damage induced by the traditional freezing solution, and obviously improve the problems of cell damage and low survival rate caused by the general freezing solution in the prior art. The differentiation potential of the recovered cells is good, and the cells can successfully form mature kidney unit structures, so that the problem of subsequent differentiation failure in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for cryopreservation and thawing of pluripotent stem cell cells in the mid-differentiation stage induced by differentiation. Background Technology

[0002] With the rapid development of regenerative medicine and tissue engineering, the in vitro differentiation of pluripotent stem cells (PSCs) into organoids has become a research hotspot. In particular, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have shown great potential in the construction of kidney organoids, providing a new technological platform for kidney disease research, drug screening, and personalized medicine. However, the induction and differentiation process of kidney organoids typically requires 18-30 days and must be continuous and uninterrupted, posing significant challenges to the flexible scheduling of experimental procedures and industrial-scale production.

[0003] Currently, there has been considerable research on cryopreservation technology for pluripotent stem cells. CN105087472B discloses a cryopreservation solution for induced pluripotent stem cells and its application. This cryopreservation solution uses IMDM / F12 basal medium as a matrix and contains components such as DMSO, dextran 40, albumin, and Thiazovin. It does not use animal serum, thus avoiding the risk of serum-borne pathogens.

[0004] However, current cryopreservation and thawing methods primarily target undifferentiated pluripotent stem cells or terminally differentiated mature cells. Cryopreservation methods for cells in the mid-differentiation stage, particularly the primitive streak stage cells in the kidney organoid induction process, are still immature. Existing universal cryopreservation solutions (such as CryoStor and CellBanker) have limited protective capabilities for cells in the intermediate differentiation stage, leading to significant cell damage, low survival rates, delayed growth after thawing, and subsequent differentiation failure or unstable differentiation rates. These problems severely restrict the progress and widespread application of kidney organoid research.

[0005] Therefore, there is an urgent need to develop a cryopreservation and thawing method specifically for mid-differentiation cells in the kidney organoid induction process, in order to solve the problems existing in the current technology and promote the development of kidney organoid research and application. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method and application for cryopreservation and thawing of pluripotent stem cell-induced mid-differentiation cells. This method addresses the problems in the prior art, such as the uninterrupted continuous differentiation process of kidney organoids, the immature cryopreservation method for primitive stripe stage cells, and the limited protective ability of universal cryopreservation solutions for mid-differentiation cells. Targeting the critical node of Day 4 mid-differentiation cells, this invention achieves efficient cryopreservation and thawing, which helps to break the continuity limitation of the kidney organoid induction process, realize the modularization and standardization of the production process, and lay the foundation for the large-scale preparation and clinical application of kidney organoids.

[0007] To achieve the above and other related objectives, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for cryopreservation and thawing of pluripotent stem cell cells in the mid-stage of induced differentiation, comprising the following steps:

[0009] (1) The starting cells are pluripotent stem cells, which are induced to differentiate into the intermediate stage;

[0010] (2) Use mild digestive enzymes to recover and count the cells, and resuspend the cells in cryopreservation solution;

[0011] (3) After the temperature is cooled to -80℃, it is transferred to liquid nitrogen for storage and later use.

[0012] (4) When it is necessary to thaw the cells, remove the frozen cells from the liquid nitrogen and quickly put them into a 37°C water bath to allow the cryopreservation solution to thaw completely within 1 to 2 minutes.

[0013] (5) After melting, add DMEM culture medium to slowly dilute the cells, and then centrifuge to remove DMSO;

[0014] (6) The cells were seeded into a suspension culture system to continue the induction and differentiation of kidney organoids.

[0015] In step (2), the cryopreservation solution comprises the following components: APEL culture medium, KnockOut™ serum substitute, dimethyl sulfoxide, and fibroblast growth factor FGF9.

[0016] Furthermore, in step (1), pluripotent stem cells are induced to differentiate into the primitive stripe stage.

[0017] Specifically, pluripotent stem cells were cultured in a medium containing WNT agonist and Noggin to induce primitive stripe differentiation. The medium was a cell differentiation medium selected from AdvancedRPMI 1640 + 1X GlutaMAX medium; the WNT agonist was selected from CHIR99021 at a concentration of 8 μM; and the Noggin concentration was 5 ng / mL.

[0018] Furthermore, in step (1), the induced differentiated cells have a high cell density under a microscope and are arranged in a monolayer adherent arrangement.

[0019] Furthermore, in step (2), the induced differentiated cells are recovered using a mild digestive enzyme, counted, and resuspended in cryopreservation solution.

[0020] Furthermore, the resuspension density is 2×10 6 ~3×10 6 cells / mL.

[0021] Furthermore, the mild digestive enzyme is selected from Accutase®.

[0022] Furthermore, by volume percentage, APEL culture medium accounts for 50-70%, KOSR for 20-30%, and DMSO for 5-10% in the cryopreservation solution; the final concentration of FGF9 factor in the cryopreservation solution is 20-30 ng / mL.

[0023] Furthermore, by volume percentage, APEL culture medium accounted for 60%, KOSR for 30%, and DMSO for 10% in the cryopreservation solution; the final concentration of FGF9 factor in the cryopreservation solution was 20 ng / mL.

[0024] Compared to conventional PSC cryopreservation solutions, which prioritize maintaining pluripotency, this cryopreservation solution formula, through its differentiation-friendly APEL medium and differentiation-stage factor FGF9, focuses more on protecting the characteristics of cells during differentiation, preventing differentiation into abnormal lineages or dedifferentiation after thawing. Compared to universal cryopreservation solutions, this formula's components are more compatible with differentiation systems, reducing the "environmental mutation" of cells from cryopreservation to thawing culture, and improving cell survival rate and stability after thawing.

[0025] Furthermore, the cryopreservation solution also contains zwitterionic magnetic nanoparticles (ZMNPs) and betaine. The concentration of ZMNPs is 0.05~0.1 mg / mL, and the concentration of betaine is 50~100 mM.

[0026] ZMNPs consist of a magnetite core and a zwitterionic polymer shell. The shell can form hydration, reducing the water molecule diffusion coefficient and inhibiting ice crystal growth and recrystallization. Furthermore, ZMNPs can generate a magnetocaloric effect under an applied alternating magnetic field, achieving uniform and rapid rewarming during cell resuscitation. Simultaneously, betaine, as an osmotic regulator, controls intracellular osmotic pressure changes within the range of 5-8 mOsm / ℃ during the programmed cooling phase, protecting cell membrane stability. The combined use of ZMNPs and betaine can reduce DMSO concentration, decrease toxic effects, and provide a higher level of cell protection.

[0027] Furthermore, the cryopreservation solution also includes 1-5 mM glutathione (GSH), which can scavenge free radicals and reduce oxidative damage.

[0028] Furthermore, the cryopreservation solution also includes a low concentration of Wnt3a (5-10 ng / mL). The formation of primitive stripes in cells depends on Wnt signaling, and the low concentration of Wnt3a works synergistically with FGF9 to maintain the direction of mesodermal differentiation.

[0029] Furthermore, in step (3), the program cooling rate is -1℃ / min.

[0030] Furthermore, in step (3), the program cools down to -80°C and is then transferred to liquid nitrogen for storage after 4 hours.

[0031] A second aspect of the invention provides the application of this cryopreservation and thawing method in the preparation of kidney organoids.

[0032] Furthermore, the preparation of kidney organoids includes the following steps:

[0033] S1 and Day-1 phases: Pluripotent stem cells were seeded into matrix gel-coated 24-well plates and cultured in mTeSR1 medium containing ROCK inhibitors for adherent culture.

[0034] S2 and Day 0-4 phases: Cells were cultured in a cell differentiation medium containing WNT agonist and Noggin factor to induce primitive stripe differentiation;

[0035] S3 and Day 4 phases: Cells are seeded in a low-adhesion U-shaped substrate to form spheres and cultured using the culture medium from Day 4 to Day 7.

[0036] S4 and Day 4-7 phases: Cells were cultured in a culture medium containing FGF-9 and Activin A to induce interstitial mesoderm.

[0037] S5 and Day 7-11: The cells were cultured in a culture medium containing FGF-9 to induce renal mesenchymal tissue differentiation.

[0038] S6, Day 11-18: The cells were cultured in a cell differentiation medium without added factors to promote nephron formation.

[0039] This cryopreservation and thawing method is specifically designed for the original striped cells in Day 4; that is, it is applied after step S2 and before step S3.

[0040] As described above, the method and application of cryopreservation and thawing of pluripotent stem cell cells in the mid-differentiation stage of the present invention have the following beneficial effects:

[0041] 1. The cryopreservation method provided by this invention significantly improves the cryopreservation survival rate of mid-differentiation cells. Compared with directly cryopreserving 3D morphological kidney organoids, it significantly improves the survival rate of organoid structures after thawing mid-differentiation cells (>90%), avoiding apoptosis or irreversible stress damage induced by traditional cryopreservation solutions. This method significantly improves upon the problems of large cell damage and low survival rate caused by using universal cryopreservation solutions in existing technologies. The differentiation potential of thawed cells is well maintained, and they can successfully form mature nephron structures, solving the problem of subsequent differentiation failure in existing technologies.

[0042] 2. This invention makes the cryopreservation solution more compatible with the differentiation system by adding specific components such as APEL culture medium, KOSR, appropriate amount of DMSO and FGF9 factor to the cryopreservation solution, reducing the "environmental mutation" of cells from cryopreservation solution to thawing culture medium, and improving the survival rate and state stability of cells after thawing.

[0043] 3. This method promotes the standardization and modularization of organoid induction processes, allowing procedures that previously required continuous operation for 18-30 days to be paused and cryopreserved at Day 4, making it suitable for large-scale industrial production. Compared to directly cryopreserving 3D morphological kidney organoids, this method significantly improves the survival rate of organoid structures after cell resuscitation during mid-differentiation.

[0044] 4. The standardized operating procedure provided by this invention is applicable to automated culture systems, which is beneficial for the modularization and high standardization of kidney organoid production. Furthermore, this method is applicable to both hESC and hiPSC systems. Attached Figure Description

[0045] Figure 1 This is an overall flowchart of the method for cryopreservation and resuscitation of pluripotent stem cell cells in the mid-stage of induced differentiation disclosed in this invention.

[0046] Figure 2 This is a flowchart illustrating the steps of the method for cryopreservation and resuscitation of pluripotent stem cell-induced mid-differentiation cells disclosed in this invention.

[0047] Figure 3Fluorescence images of organoid structures from the non-cryopreserved, Day 4 cryopreserved, and Day 18 directly cryopreserved groups in Example 3 of this invention.

[0048] Figure 4 This is a comparison diagram of the organoid markers and formation structures of Day 18 after successful resuscitation of the cryopreservation solution of this invention and commercially available cryopreservation solutions in Example 4 of this invention. Detailed Implementation

[0049] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0050] Example 1: Preparation of cryopreservation solution

[0051] 1. Take 6 mL of APEL solution into a sterile bottle;

[0052] 2. Add 3 mL of KOSR and mix well;

[0053] 3. Slowly add DMSO to a final concentration of 10%, and gently mix to prevent local osmotic pressure shocks;

[0054] 4. Add FGF9 to a final concentration of 20 ng / mL;

[0055] 5. After sterile filtration at 0.22µm, dispense into 1mL tubes and store in the dark.

[0056] Example 2: Cryopreservation of cells in mid-differentiation stage

[0057] (1) hESCs were used as the starting cells. The starting cells were seeded into 24-well plates and cultured in Advanced RPMI 1640 + 1X GlutaMAX medium supplemented with the WNT agonist CHIR99021 (8 μM) and Noggin (5 ng / mL). The medium was changed daily at 500 μL / well for 4 days. (Day 4 was used to detect high expression of the original stripe markers TBXT and MIXL1 to confirm the stage of differentiation.)

[0058] (2) Then, the hESC-derived primitive striped cells induced to differentiate to Day 4 were digested with Accutase for 7 min, gently dispersed, collected, and counted. The cell concentration was adjusted to 2 × 10⁻⁶. 6 After being reduced to cells / mL, the cells were resuspended in the cryopreservation solution prepared in Example 1;

[0059] (3) Use a programmable cooling box to cool to -80℃ at a rate of -1℃ / min, and transfer to liquid nitrogen for storage after 4 hours for later use.

[0060] Example 3: Resuscitation and Kidney Organoid Induction

[0061] (4) After freezing the cells in Example 2 for one month, the frozen cells were taken out of liquid nitrogen and quickly placed in a 37°C water bath to completely thaw the cryopreservation solution within 1 to 2 minutes.

[0062] (5) After thawing, slowly dilute the cells with DMEM culture medium, then centrifuge to remove the cryopreservation solution. Repeat the operation twice.

[0063] (6) Seed cells in a 96-well U-shaped plate with low adhesion, seeding 1 × 10⁶ cells per well. 5 Cells, 200 μL / well; the culture medium was changed to Advanced RPMI 1640 + 1X GlutaMAX medium, with the addition of FGF-9 (concentration of 20 ng / mL) and Activin A (concentration of 10 ng / mL); 96-well plates were centrifuged at 200g for 30 seconds to allow the cells to aggregate in the U-shaped bottom to form spheres and spontaneously form organoid precursor structures.

[0064] Then continue the differentiation process from Day 4 to Day 18:

[0065] Days 4-7 are the interstitial mesodermal (IM) induction phase: no medium change is required during Days 4-7. On day 7, the expression of IM markers such as PAX2 and LHX1 is detected.

[0066] Days 7-11 represent the metanephric mesenchymal (MM) induction phase: From day 7 to day 11, the culture medium was replaced with Advanced RPMI 1640 + 1X GlutaMAX medium, with only FGF9 (20 ng / mL) added at 200 μL / well, and the cells were continued in suspension culture. Activin A was removed during this phase, and only FGF-9 was used to induce further differentiation of the mesenchymal mesoderm into metanephric mesenchymal tissue. The medium was changed once on day 9 to induce cell differentiation into the metanephric lineage. On day 11, the expression of MM markers such as SIX2 and WT1 was detected.

[0067] Days 11-18 represent the nephron formation stage: From day 11 to day 18, the culture medium was replaced with Advanced RPMI 1640 + 1X GlutaMAX basal medium (200 μL / well) without any growth factors, and the medium was changed every 2-3 days. During this stage, cells spontaneously differentiated to form nephron structures. The entire differentiation process lasted 18 days. On day 18, organoid expression of LRP2, PODXL, ECAD, etc., was detected.

[0068] Results: The test results show that, Figure 3The organoid morphology and biomarker expression were intact and basically consistent with the non-frozen control. Compared with the 3D structured kidney organoid at Day 18 of direct cryopreservation, the expression area of ​​renal parenchymal cells increased by more than 50%.

[0069] Example 4: Comparison of this cryopreservation solution with traditional and commercially available cryopreservation solutions

[0070] The cryopreservation solution in Example 2 was replaced with a traditional cryopreservation solution (FBS+DMSO) or a commercially available cryopreservation solution (CryoStor CS10), and then the cells were thawed and redifferentiated using the method in Example 3. Multiple experiments were conducted, and the cell survival rates after thawing using the three methods are shown in Table 1. It is evident that the survival rate after thawing with this cryopreservation solution is significantly higher than that with traditional and commercially available cryopreservation solutions. This cryopreservation solution can effectively maintain the activity and differentiation potential of pluripotent stem cells in the mid-stage of induced differentiation, solving the technical problems of low cell thawing rate and functional impairment in traditional cryopreservation methods.

[0071] Table 1. Cell survival rates after thawing using this cryopreservation solution compared to conventional and commercially available cryopreservation solutions.

[0072] cryopreservation solution Survival batches / total experimental batches Survival rate This cryopreservation solution 12 / 21 57% Traditional cryopreservation solution 2 / 21 10% Commercially available cryopreservation solutions 1 / 13 8%

[0073] The comparison chart of organoid markers and formation structures on Day 18 of successful resuscitation using this cryopreservation solution and commercially available cryopreservation solutions is shown below. Figure 4 As shown in the figure. The results showed that the expression levels of LRP2, PODXL, and E-cadherin in this cryopreservation solution were not significantly different from those in the non-cryopreservation control. Compared with the commercially available cryopreservation solution CryoStor CS10, the renal parenchymal structure formation area was increased by >90%.

[0074] Example 5: Preparation of cryopreservation solution

[0075] 1. Take 6.5 mL of APEL solution into a sterile bottle;

[0076] 2. Add 3 mL of KOSR and mix well;

[0077] 3. Slowly add DMSO to a final concentration of 5%, and gently mix to prevent local osmotic pressure shocks;

[0078] 4. Add FGF9 to a final concentration of 20 ng / mL; add ZMNPs to a final concentration of 0.05 mg / mL; add betaine to a final concentration of 50 mM;

[0079] 5. After sterile filtration at 0.22µm, dispense into 1mL tubes and store in the dark.

[0080] Example 6: Cryopreservation and Thawing

[0081] (1) The starting cells were hiPSCs. The starting cells were seeded into 24-well plates and cultured in Advanced RPMI 1640 + 1X GlutaMAX medium supplemented with WNT agonist CHIR99021 (8 μM) and Noggin (5 ng / mL). The medium was changed daily at 500 μL / well for 4 days. (Day 4 was used to detect high expression of the original stripe markers TBXT and MIXL1 to confirm the stage of differentiation.)

[0082] (2) Then, the hiPSC primitive striped cells induced to differentiate to Day 4 were digested with Accutase for 7 min, gently dispersed, collected and counted, and the cell concentration was adjusted to 2×10⁻⁶. 6 After being reduced to cells / mL, the cells were resuspended in the cryopreservation solution prepared in Example 5;

[0083] (3) Use a programmable cooling box to cool to -80℃ at a rate of -1℃ / min, and transfer to liquid nitrogen for storage after 4 hours for later use.

[0084] (4) After freezing the above cells for one month, thaw them by taking the frozen cells out of liquid nitrogen and quickly putting them into a 37°C water bath, and applying an alternating magnetic field (20-50 kHz, 10-15 kA / m) to completely thaw the cryopreservation solution within 1-2 minutes.

[0085] (5) After thawing, add DMEM culture medium to slowly dilute the cells, then centrifuge to remove the cryopreservation solution. Repeat the operation twice.

[0086] (6) Seed cells in a 96-well U-shaped plate with low adhesion, seeding 1 × 10⁶ cells per well. 5 Cells, 200 μL / well; the culture medium was changed to Advanced RPMI 1640 + 1X GlutaMAX medium, with the addition of FGF-9 (concentration of 20 ng / mL) and Activin A (concentration of 10 ng / mL); 96-well plates were centrifuged at 200g for 30 seconds to allow the cells to aggregate in the U-shaped bottom to form spheres and spontaneously form organoid precursor structures.

[0087] Then continue the differentiation process from Day 4 to Day 18, which is consistent with the differentiation process in Example 3.

[0088] Results: The test results showed that the cell recovery viability rate reached 55%; the expression levels of LRP2, PODXL, E-cadherin, etc. were not significantly different from those of the unfrozen control.

[0089] Example 7: Preparation of cryopreservation solution

[0090] 1. Take 6 mL of APEL solution into a sterile bottle;

[0091] 2. Add 3 mL of KOSR and mix well;

[0092] 3. Slowly add DMSO to a final concentration of 10%, and gently mix to prevent local osmotic pressure shocks;

[0093] 4. Add FGF9 to a final concentration of 20 ng / mL; add Wnt3a to a final concentration of 5 ng / mL; add glutathione to a final concentration of 2 mM;

[0094] 5. After sterile filtration at 0.22µm, dispense into 1mL tubes and store in the dark.

[0095] Example 8: Cryopreservation and Thawing

[0096] (1) hESCs were used as the starting cells. The starting cells were seeded into 24-well plates and cultured in Advanced RPMI 1640 + 1X GlutaMAX medium supplemented with the WNT agonist CHIR99021 (8 μM) and Noggin (5 ng / mL). The medium was changed daily at 500 μL / well for 4 days. (Day 4 was used to detect high expression of the original stripe markers TBXT and MIXL1 to confirm the stage of differentiation.)

[0097] (2) Then, the hESC-derived primitive striped cells induced to differentiate to Day 4 were digested with Accutase for 7 min, gently dispersed, collected, and counted. The cell concentration was adjusted to 2 × 10⁻⁶. 6 After being reduced to cells / mL, the cells were resuspended in the cryopreservation solution prepared in Example 7;

[0098] (3) Use a programmable cooling box to cool to -80℃ at a rate of -1℃ / min, and transfer to liquid nitrogen for storage after 4 hours for later use.

[0099] (4) After freezing the above cells for one month, thaw them by taking the frozen cells out of liquid nitrogen and quickly putting them into a 37°C water bath to completely thaw the cryopreservation solution within 1 to 2 minutes.

[0100] (5) After thawing, add DMEM culture medium to slowly dilute the cells, then centrifuge to remove the cryopreservation solution. Repeat the operation twice.

[0101] (6) Seed cells in a 96-well U-shaped plate with low adhesion, seeding 1 × 10⁶ cells per well. 5Cells, 200 μL / well; the culture medium was changed to Advanced RPMI 1640 + 1X GlutaMAX medium, with the addition of FGF-9 (concentration of 20 ng / mL) and Activin A (concentration of 10 ng / mL); 96-well plates were centrifuged at 200g for 30 seconds to allow the cells to aggregate in the U-shaped bottom to form spheres and spontaneously form organoid precursor structures.

[0102] Then continue the differentiation process from Day 4 to Day 18, which is consistent with the differentiation process in Example 3.

[0103] Results: The test results showed that the cell recovery viability rate reached 52%; the expression levels of LRP2, PODXL, E-cadherin, etc. were not significantly different from those of the unfrozen control.

[0104] In summary, the cryopreservation method and cryopreservation solution provided by this invention significantly improve the cryopreservation survival rate of mid-differentiation cells, avoiding apoptosis or irreversible stress damage induced by traditional cryopreservation solutions. This represents a significant improvement compared to the problems of large cell damage and low survival rates caused by using universal cryopreservation solutions in existing technologies. The revived cells retain good differentiation potential and can successfully form mature nephron structures, solving the problem of subsequent differentiation failure in existing technologies. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.

[0105] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for cryopreservation and resuscitation of mid-stage cells induced from pluripotent stem cell differentiation, characterized in that, Includes the following steps: (1) The starting cells are pluripotent stem cells, which are induced to differentiate into the intermediate stage; (2) Use mild digestive enzymes to recover and count the cells, and resuspend the cells in cryopreservation solution; (3) After the temperature is cooled to -80℃, it is transferred to liquid nitrogen for storage and future use; (4) When it is necessary to thaw the cells, remove the frozen cells from the liquid nitrogen and quickly put them into a 37°C water bath to allow the cryopreservation solution to thaw completely within 1 to 2 minutes. (5) After melting, add DMEM culture medium to slowly dilute the cells, and then centrifuge to remove DMSO; (6) The cells were seeded into a suspension culture system to continue the induction and differentiation of kidney organoids; In step (2), the cryopreservation solution comprises the following components: APEL culture medium, KOSR serum substitute, dimethyl sulfoxide, and fibroblast growth factor FGF9; By volume percentage, APEL culture medium accounts for 50-70% of the cryopreservation solution, KOSR serum substitute accounts for 20-30%, dimethyl sulfoxide accounts for 5-10%, and the final concentration of FGF9 factor in the cryopreservation solution is 20-30 ng / mL, with the sum of the contents of each component being 100%.

2. The cryopreservation and thawing method according to claim 1, characterized in that, In step (1), the pluripotent stem cells are selected from human embryonic stem cells or human induced pluripotent stem cells.

3. The cryopreservation and thawing method according to claim 1, characterized in that, In step (2), the induced differentiated cells were recovered using a mild digestive enzyme, counted, and resuspended in cryopreservation solution at a density of 2 × 10⁻⁶. 6 ~3×10 6 cells / mL.

4. The cryopreservation and thawing method according to claim 1, characterized in that, In step (3), the cooling rate is -1℃ / min.

5. The application of the cryopreservation and thawing method according to any one of claims 1 to 4 in the preparation of kidney organoids.

Images