Cryopreservation and resuscitation method for induced differentiation metaphase cells of pluripotent stem cells and application of cryopreservation and resuscitation method

Abstract

The invention provides a cryopreservation and resuscitation method for induced differentiation of pluripotent stem cells to metaphase cells and application, and the cryopreservation method comprises the following steps: (1) the initial cells are pluripotent stem cells, and are induced and differentiated to the metaphase; (2) recovering the cells by using a mild digestive enzyme, counting, and resuspending the cells in a cryopreservation solution; and (3) carrying out programmed cooling to-80 DEG C, and then transferring into liquid nitrogen for preservation for later use. According to the cryopreservation method and the cryopreservation liquid provided by the invention, the cryopreservation survival rate of the differentiated metaphase cells is obviously improved, apoptosis or irreversible stress injury induced by a traditional cryopreservation liquid is avoided, and the problems of large cell injury and low survival rate caused by using a general cryopreservation liquid in the prior art are obviously improved. The resuscitated cells keep good differentiation potential, can smoothly form a mature renal unit structure, and solves the problem of subsequent differentiation failure in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a freezing and recovery method of pluripotent stem cell induced mid-stage cells and application thereof. BACKGROUND

[0002] With the rapid development of regenerative medicine and tissue engineering, the use of pluripotent stem cells (PSCs) to induce differentiation in vitro to form organoids has become a research hotspot. In particular, human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have shown great potential in the construction of kidney organoids, providing a new technical platform for kidney disease research, drug screening, and personalized medicine. However, the induction and differentiation process of kidney organoids usually requires 18-30 days, and the process is continuous and cannot be interrupted, which poses a serious challenge to the flexible arrangement of experimental operations and industrial production.

[0003] Currently, there have been many studies on the freezing technology of pluripotent stem cells. CN105087472B discloses an induced pluripotent stem cell freezing solution and its application. The freezing solution uses IMDM / F12 basic medium as the matrix, contains DMSO, dextran 40, albumin, and Thiazovivin, etc., does not use animal serum, and avoids the risk of serum transmission of animal-derived pathogens.

[0004] However, the existing freezing and recovery methods are mainly aimed at undifferentiated pluripotent stem cells or terminally differentiated mature cells. The freezing method for cells in the mid-stage of differentiation, especially the primitive streak cells in the induction process of kidney organoids, is not mature. The existing general freezing solution (such as CryoStor, CellBanker, etc.) has limited protection ability for cells in the intermediate stage of differentiation, resulting in large cell damage, low survival rate, growth retardation after recovery, and subsequent differentiation failure or unstable differentiation rate. These problems seriously hinder the progress and application of kidney organoid research.

[0005] Therefore, it is urgent to develop a freezing and recovery method for cells in the mid-stage of differentiation in the induction process of kidney organoids, to solve the problems existing in the prior art and promote the development of kidney organoid research and application. SUMMARY

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a method for cryopreservation and recovery of pluripotent stem cell induced differentiation mid-stage cells and application, which is used to solve the problems of continuous non-interruptible kidney organoid induction differentiation process, immature cryopreservation method of primitive streak stage cells, and limited protection ability of general cryopreservation solution for differentiation mid-stage cells, etc., aiming at the key node of Day 4 differentiation mid-stage cells, realizing efficient cryopreservation and recovery, which helps to break the continuity limitation of kidney organoid induction process, realizes the modularization and standardization of production process, and lays a foundation for large-scale preparation and clinical application of kidney organoids.

[0007] To achieve the above-mentioned purpose and other related purposes, the present application provides the following technical solutions.

[0008] In a first aspect of the present application, a method for cryopreservation of pluripotent stem cell induced differentiation mid-stage cells is provided, comprising the following steps:

[0009] (1) The starting cells are pluripotent stem cells (PSCs) induced to the mid-stage;

[0010] (2) The cells are recovered and counted using a mild digestion enzyme, and resuspended in a cryopreservation solution;

[0011] (3) After programmed cooling to-80℃, it is transferred to liquid nitrogen for storage and standby.

[0012] Further, in step (1), the pluripotent stem cells are selected from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).

[0013] Further, in step (1), the pluripotent stem cells are induced to the primitive streak stage.

[0014] Specifically, the pluripotent stem cells are cultured using a culture medium containing a WNT agonist and a Noggin factor to induce primitive streak differentiation. The culture medium is a cell differentiation culture medium, and the cell differentiation culture medium is selected from Advanced RPMI 1640 + 1X GlutaMAX culture medium; the WNT agonist is selected from CHIR99021, and the concentration is 8 μM; the concentration of the Noggin factor is 5 ng / mL.

[0015] Further, in step (1), the cells after induction differentiation have high cell density under a microscope and are arranged in a single layer adherent.

[0016] Further, in step (2), the induced differentiation cells are recovered using a mild digestion enzyme, counted and resuspended in a cryopreservation solution.

[0017] Further, the resuspension density is 2×10 6 ~3×106 cells / mL.

[0018] Further, the mild digestion enzyme is selected from Accutase®.

[0019] Further, in step (2), the cryopreservation solution comprises basal medium, serum replacement, cryoprotectant, and key growth factor. Specifically, the basal medium is selected from APEL medium; the serum replacement is selected from KnockOut™ Serum Replacement (KOSR); the cryoprotectant is selected from dimethyl sulfoxide (DMSO); and the key growth factor is selected from fibroblast growth factor FGF9.

[0020] Further, in terms of volume percentage, the APEL medium accounts for 50-70%, the KOSR accounts for 20-30%, and the DMSO accounts for 5-10% in the cryopreservation solution; and the final concentration of the FGF9 factor in the cryopreservation solution is 20-30 ng / mL.

[0021] Further, in terms of volume percentage, the APEL medium accounts for 60%, the KOSR accounts for 30%, and the DMSO accounts for 10% in the cryopreservation solution; and the final concentration of the FGF9 factor in the cryopreservation solution is 20 ng / mL.

[0022] Compared with the conventional PSC cryopreservation solution which focuses more on maintaining pluripotency, the cryopreservation solution formula focuses more on protecting the characteristics of cells in the differentiation stage by using the differentiation-friendly APEL medium and the differentiation stage factor FGF9, thereby avoiding differentiation to abnormal lineages or dedifferentiation after resuscitation. Compared with the general cryopreservation solution, the components of the formula are more compatible with the differentiation system, which can reduce the “environmental mutation” of cells from the cryopreservation solution to the resuscitation medium, and improve the survival rate and state stability of cells after resuscitation.

[0023] Further, the cryopreservation solution further comprises zwitterionic magnetic nanoparticles (ZMNPs) and betaine. The concentration of the ZMNPs is 0.05-0.1 mg / mL, and the concentration of the betaine is 50-100 mM.

[0024] The ZMNPs are composed of a ferroferric oxide core layer and a zwitterionic polymer shell layer, and the shell layer can form hydration, reduce the diffusion coefficient of water molecules, and inhibit ice crystal growth and recrystallization. Moreover, the ZMNPs can generate a magneto-thermal effect under an external alternating magnetic field, thereby achieving uniform and rapid rewarming of cells during the resuscitation process. Meanwhile, the betaine as an osmotic regulator can control the change of intracellular osmotic pressure in the range of 5-8 mOsm / ℃ during the programmed cooling stage, thereby protecting the stability of the cell membrane. Through the combination of the ZMNPs and the betaine, the DMSO concentration can be reduced, the toxic effect can be reduced, and higher cell protection effect can be provided.

[0025] Further, the cryopreservation solution further comprises 1-5 mM glutathione (GSH), which can scavenge free radicals and reduce oxidative damage.

[0026] Further, the cryopreservation solution further comprises 5-10 ng / mL of low-concentration Wnt3a, and the original stripe formation of the cells depends on Wnt signal, and the low-concentration Wnt3a cooperates with FGF9 to maintain the mesoderm differentiation direction.

[0027] Further, in step (3), the programmed cooling rate is-1℃ / min.

[0028] Further, in step (3), the programmed cooling is to-80℃, and after 4 hours, the cells are transferred into liquid nitrogen for preservation.

[0029] In a second aspect of the present application, a method for resuscitating pluripotent stem cell-induced differentiation mid-stage cells is provided, comprising the following steps:

[0030] (4) When the cells need to be resuscitated, the cryopreserved cells are taken out from the liquid nitrogen and quickly put into a 37℃ water bath to completely melt the cryopreservation solution within 1-2 minutes;

[0031] (5) The melted cells are slowly diluted by adding DMEM culture solution, and then centrifuged to remove DMSO;

[0032] (6) The cells are inoculated into a suspension culture system to continue the induction and differentiation of kidney organoids.

[0033] In a third aspect of the present application, the application of the cryopreservation and resuscitation method in the preparation of kidney organoids is provided.

[0034] Further, the preparation of kidney organoids comprises the following steps:

[0035] S1, Day-1 stage: pluripotent stem cells are inoculated into a 24-well plate coated with Matrigel, and adherent culture is carried out using mTeSR1 medium containing a ROCK inhibitor;

[0036] S2, Day0-4 stage: cell differentiation medium containing WNT agonist and Noggin factor is used for culture to induce the differentiation of original stripes;

[0037] S3, Day4 stage: the cells are inoculated into a low-adhesion U-shaped bottom plate to form spheres, and the medium of Day4-7 stage is used for culture;

[0038] S4, Day4-7 stage: cell differentiation medium containing FGF-9 and Activin A factors is used for culture to induce mesoderm;

[0039] S5, Day 7-11 stage: replace with FGF-9 factor-containing cell differentiation medium for culture to induce metanephric mesenchyme;

[0040] S6, Day 11-18 stage: replace with factor-free cell differentiation medium for culture to promote nephron formation.

[0041] Among them, the freezing and recovery method is special for the original striped cells in the Day 4 stage; that is, it is applied after step S2 and before S3.

[0042] As described above, the freezing and recovery method of pluripotent stem cell-induced differentiation mid-stage cells and the application of the present application have the following beneficial effects:

[0043] 1. The freezing method provided by the present application significantly improves the freezing survival rate of differentiation mid-stage cells. Compared with directly freezing 3D-shaped kidney organoids, the structure survival rate of the organoids after recovery is significantly improved (>90%), avoiding the apoptosis or irreversible stress damage induced by traditional freezing solutions. Compared with the cell damage and low survival rate caused by the use of general freezing solutions in the prior art, the present application has obvious improvement. The differentiation potential of the recovered cells is good, and mature nephron structures can be successfully formed, solving the problem of subsequent differentiation failure in the prior art.

[0044] 2. By adding specific components such as APEL culture solution, KOSR, appropriate amount of DMSO and FGF9 factor in the freezing solution, the freezing solution is more compatible with the differentiation system, reducing the "environmental mutation" of the cells from the freezing solution to the recovery culture solution, and improving the survival rate and state stability of the recovered cells.

[0045] 3. The method promotes the standardization and modularization of organoid induction, so that the process which originally needs to be operated continuously for 18-30 days can be paused and frozen at the Day 4 stage, which is suitable for industrialized mass production requirements. Compared with directly freezing 3D-shaped kidney organoids, the method of the present application significantly improves the structure survival rate of the organoids after recovery of the differentiation mid-stage cells.

[0046] 4. The operation process provided by the present application is standardized and suitable for automatic culture system, which is beneficial to the modularization and high standardization of kidney organoid production. The method is also suitable for hESC system and hiPSC system. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The whole flow chart of the freezing and recovery method of pluripotent stem cell-induced differentiation mid-stage cells disclosed in the present application.

[0048] Figure 2This 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.

[0049] Figure 3 Fluorescence images of organoid structures from the non-cryopreserved, Day 4 cryopreserved, and Day 18 directly cryopreserved groups in Example 3 of this invention.

[0050] 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

[0051] 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.

[0052] Example 1: Preparation of cryopreservation solution

[0053] 1. Take 6 mL of APEL solution into a sterile bottle;

[0054] 2. Add 3 mL of KOSR and mix well;

[0055] 3. Slowly add DMSO to a final concentration of 10%, and gently mix to prevent local osmotic pressure shocks;

[0056] 4. Add FGF9 to a final concentration of 20 ng / mL;

[0057] 5. After aseptic filtration at 0.22µm, dispense into 1mL tubes and store in the dark.

[0058] Example 2: Cryopreservation of cells in mid-differentiation stage

[0059] (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.)

[0060] (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 cells / year. 6 After being reduced to cells / mL, the cells were resuspended in the cryopreservation solution prepared in Example 1.

[0061] (3) Use the program cooling box to reduce to -80°C at a speed of -1°C / min, and after 4 h, transfer into liquid nitrogen for preservation, ready for use.

[0062] Example 3: Resuscitation and kidney organoid induction

[0063] (4) Resuscitate the cells in Example 2 after being frozen for one month. Take the frozen cells out of the liquid nitrogen and quickly put them into a 37°C water bath to completely melt the frozen solution within 1-2 min;

[0064] (5) Add DMEM culture solution to the melted cells to dilute slowly, and then centrifuge to remove the frozen solution, repeat twice;

[0065] (6) Seed the cells in a low-adhesion 96-well U-bottom plate, 1x10 5 cells, 200 μL / well; replace the culture medium with Advanced RPMI 1640 + 1X GlutaMAX medium, and add FGF-9 (concentration of 20 ng / mL) and Activin A (concentration of 10 ng / mL); centrifuge the 96-well plate at 200g for 30 seconds to make the cells aggregate in the U-bottom to form spheres, and spontaneously form organoid precursor structures.

[0066] Then continue the differentiation process from Day4 to Day18:

[0067] Day 4-7 is the intermediate mesoderm (IM) induction stage: no medium replacement is required during the Day 4-7 stage, and PAX2, LHX1, etc. IM marker expression is detected on the 7th day.

[0068] Day 7-11 is the metanephric mesenchyme (MM) induction stage: from the 7th day to the 11th day, replace the culture medium with Advanced RPMI 1640 + 1X GlutaMAX medium, and only add FGF9 (concentration of 20 ng / mL), 200 μL / well, and continue to suspend culture. Remove Activin A during this stage and only use FGF-9 to induce intermediate mesoderm to further differentiate into metanephric mesenchyme. Change the medium once on Day 9 to induce the cells to differentiate into the metanephric lineage. SIX2, WT1, etc. MM marker expression is detected on the 11th day.

[0069] Day 11-18 is the nephron formation stage: from day 11 to day 18, the culture medium is replaced with Advanced RPMI 1640 + 1X GlutaMAX basal medium without any growth factors, 200 μL / well, 2-3 days of liquid exchange, and continue to culture. In this stage, cells spontaneously differentiate to form nephron structures. The whole differentiation process lasts for 18 days, and the expression of LRP2, PODXL, ECAD and the like is detected on the 18th day.

[0070] Results: The detection results show that, as Figure 3 , the morphology and marker expression of the organoids are complete, and are basically consistent with the non-frozen control. Compared with the 3D structure kidney organoids directly frozen at Day 18, the renal parenchymal cell expression area is increased by more than 50%.

[0071] Example 4: Comparison of the present freezing solution with traditional and commercial freezing solutions

[0072] The freezing solution in Example 2 is replaced with a traditional freezing solution (FBS + DMSO) or a commercial freezing solution (CryoStor CS10), and then the method of Example 3 is used for recovery and continued differentiation. Multiple experiments are performed, and the survival rates of cells recovered in the three ways are shown in Table 1. It can be seen that the survival rate of the present freezing solution is much higher than that of the traditional and commercial freezing solutions. The present freezing solution can effectively maintain the activity and differentiation potential of cells at the middle stage of induced differentiation of pluripotent stem cells, and solves the technical problems of low cell recovery rate and impaired function in the traditional freezing method.

[0073] Table 1. Survival rate of cells recovered using the present freezing solution and traditional and commercial freezing solutions

[0074] Freeze solution Successful resuscitation batches / total experimental batches Success rate The present freeze solution 12 / 21 57% Conventional freeze solution 2 / 21 10% Commercially available freeze solution 1 / 13 8%

[0075] Among them, the comparison chart of the Day 18 organoid markers and structure formed by the present freezing solution and the commercial freezing solution is shown in Figure 4 . The results show that: the expression levels of LRP2, PODXL, E-cadherin and the like of the present freezing solution have no significant difference with the non-frozen control, and compared with the commercial freezing solution CryoStor CS10 control, the renal parenchymal structure formation area is increased by > 90%.

[0076] Example 5: Preparation of freezing solution

[0077] 1. Take 6.5 mL of APEL solution in a sterile bottle;

[0078] 2. Add 3 mL of KOSR and mix well;

[0079] 3. Slowly add DMSO to a final concentration of 5%, and gently mix to prevent local osmotic shock;

[0080] 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;

[0081] 5. After 0.22 pm sterile filtration, aliquot 1 mL per tube and store in the dark.

[0082] Example 6: Cryopreservation and recovery

[0083] (1) The starting cells are hiPSCs, which are seeded into a 24-well plate and cultured in Advanced RPMI 1640 + 1X GlutaMAX medium with the addition of WNT agonist CHIR99021 (at a concentration of 8 mM) and Noggin factor (at a concentration of 5 ng / mL), with 500 pL / well of medium changed daily for a total of 4 days. (Day 4 detection of primitive streak markers TBXT, MIXL1 high expression to confirm stage differentiation.)

[0084] (2) The hiPSC primitive streak cells induced to differentiate to Day 4 are then digested with Accutase enzyme for 7 min, gently dispersed, and recovered and counted. After adjusting the cell concentration to 2 x 10 6 cells / mL, resuspend in the cryopreservation solution prepared in Example 5;

[0085] (3) Use a programmed cooling box to reduce to -80°C at a rate of -1°C / min, and after 4 h, transfer to liquid nitrogen for storage.

[0086] (4) After one month of cryopreservation, the cells are recovered by removing the cryopreserved cells from the liquid nitrogen and quickly placing them in a 37°C water bath, with the addition of an alternating magnetic field (20-50 kHz, 10-15 kA / m) to completely melt the cryopreservation solution within 1-2 min.

[0087] (5) Add DMEM culture solution to the melted cells to slowly dilute, and then centrifuge to remove the cryopreservation solution, repeating the operation twice.

[0088] (6) Seed the cells in a low-adhesion 96-well U-bottom plate at a concentration of 1 x 10 5 cells per well, 200 pL / well; change the medium to Advanced RPMI 1640 + 1X GlutaMAX medium with the addition of FGF-9 (at a concentration of 20 ng / mL) and Activin A (at a concentration of 10 ng / mL); centrifuge the 96-well plate at 200 g for 30 seconds to make the cells aggregate in the U-bottom to form spheres, and spontaneously form organoid precursor structures.

[0089] Then continue the differentiation procedure from Day 4 to Day 18, consistent with the differentiation procedure in Example 3.

[0090] Results: The detection results show that the cell recovery activity can reach 55%; the expression levels of LRP2, PODXL, E-cadherin, etc. have no significant difference with the non-frozen control.

[0091] Example 7: Preparation of cryopreservation solution

[0092] 1. Take 6 mL of APEL solution in a sterile bottle;

[0093] 2. Add 3 mL of KOSR and mix well;

[0094] 3. Slowly add DMSO to a final concentration of 10%, and gently mix to prevent local osmotic shock;

[0095] 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;

[0096] 5. After 0.22 µm sterile filtration, aliquot 1 mL per tube and store in the dark.

[0097] Example 8: Cryopreservation and recovery

[0098] (1) The starting cells are hESCs, which are seeded into a 24-well plate and cultured in Advanced RPMI 1640 + 1X GlutaMAX medium with the addition of WNT agonist CHIR99021 (concentration of 8 µM) and Noggin factor (concentration of 5 ng / mL), with 500 µL / well of medium changed daily for a total of 4 days. (Day 4 detection of high expression of primitive streak markers TBXT and MIXL1 to confirm stage differentiation.)

[0099] (2) Then the hESC-derived primitive streak cells induced to differentiate to Day 4 are digested with Accutase enzyme for 7 min, gently dispersed, recovered and counted, and the cell concentration is adjusted to 2×10 6 cells / mL, and then resuspended in the cryopreservation solution prepared in Example 7;

[0100] (3) Use a programmed cooling box to reduce to -80°C at a rate of -1°C / min, and after 4 h, transfer to liquid nitrogen for storage, ready for use.

[0101] (4) After one month of cryopreservation, the cells are recovered by taking the cryopreserved cells out of the liquid nitrogen and quickly placing them in a 37°C water bath, and the cryopreservation solution is completely melted within 1-2 min.

[0102] (5) After thawing, slowly dilute the cells with DMEM medium, then centrifuge to remove the cryopreservation solution, repeat twice.

[0103] (6) Seed the cells in low attachment 96-well U-bottom plates at 1x10 5 cells, 200 μL / well; replace the medium with Advanced RPMI 1640 + 1X GlutaMAX medium, and add FGF-9 (at a concentration of 20 ng / mL) and Activin A (at a concentration of 10 ng / mL); centrifuge the 96-well plate at 200g for 30 seconds to make the cells aggregate in the U-bottom to form spheroids, and spontaneously form organoid precursor structures.

[0104] Then continue the differentiation process from Day4~18, which is consistent with the differentiation process in Example 3.

[0105] Results: The test results show that the cell recovery activity can reach 52%; the expression levels of LRP2, PODXL, E-cadherin, etc. have no significant difference with the non-frozen control.

[0106] In summary, the freezing method and freezing solution provided by the present application significantly improve the freezing survival rate of cells in the differentiation medium, avoid the apoptosis or irreversible stress damage induced by traditional freezing solution, and significantly improve the problem of cell damage and low survival rate caused by the use of general freezing solution in the prior art. The differentiation potential of the recovered cells is good, and mature kidney unit structures can be successfully formed, solving the problem of subsequent differentiation failure in the prior art. Therefore, the present application effectively overcomes the shortcomings of the prior art and has high industrial utilization value.

[0107] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for cryopreservation of multipotent stem cell-induced differentiation mid-stage cells, characterized by, Comprising the following steps: (1) the starting cells are pluripotent stem cells, which are induced to differentiate to the middle stage; (2) the cells are recovered and counted using mild digestive enzymes, and resuspended in a cryopreservation solution; (3) after programmed cooling to-80℃, the cells are stored in liquid nitrogen for standby.

2. The cryopreservation method of claim 1, wherein, In step (1), the pluripotent stem cells are selected from human embryonic stem cells or human induced pluripotent stem cells.

3. The cryopreservation method of claim 1, wherein, In step (2), the recovered cells after induction differentiation were counted and resuspended in freezing medium at a resuspension density of 2 x 10 6 ~3 x 10 6 cells / mL.

4. The cryopreservation method of claim 1, wherein, In step (2), the cryopreservation solution comprises the following components: APEL culture solution, KnockOutTM serum replacement, dimethyl sulfoxide, fibroblast growth factor FGF9.

5. The cryopreservation method of claim 1, wherein, In terms of volume percentage, the APEL culture solution accounts for 50-70%, the KnockOutTM serum replacement accounts for 20-30%, and the dimethyl sulfoxide accounts for 5-10% in the cryopreservation solution; the final concentration of FGF9 factor in the cryopreservation solution is 20-30 ng / mL.

6. The cryopreservation method of claim 1, wherein, The cryopreservation solution also contains zwitterionic magnetic nanoparticles and betaine.

7. The cryopreservation method of claim 1, wherein, The concentration of zwitterionic magnetic nanoparticles is 0.05-0.1 mg / mL, and the concentration of betaine is 50-100 mM.

8. The cryopreservation method of claim 1, wherein, In step (3), the programmed cooling rate is-1℃ / min.

9. A method for resuscitating pluripotent stem cell-induced mid-differentiation cells, characterized in that, Comprising the following steps: (4) when the cells need to be recovered, the cryopreserved cells are taken out from the liquid nitrogen and quickly put into a 37℃ water bath to completely melt the cryopreservation solution within 1-2 min; (5) after melting, the cells are slowly diluted by adding DMEM culture solution, and then centrifuged to remove DMSO; (6) the cells are inoculated in a suspension culture system to continue the induction and differentiation of kidney organoids.

10. The application of the cryopreservation method according to any one of claims 1-8 or the recovery method according to claim 9 in the preparation of kidney organoids.

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