A pretreatment method for improving the success rate of freezing and recovery of human embryonic stem cell differentiation middle stage cells and application thereof

Abstract

The application provides a pretreatment method for improving the freezing-recovery success rate of human embryonic stem cell differentiation middle stage cells and application, and comprises the following steps: before starting kidney organoid induction differentiation, culturing human embryonic stem cells in a composite culture system for a period of time; wherein the composite culture system comprises a basic culture medium and an additive, and the additive comprises a PD0332991 reagent. Through pretreatment of human embryonic stem cells before starting induction differentiation, the PD0332991 improves the tolerance of differentiation middle stage cells to freezing stress through a double mechanism of cell cycle regulation and stress defense enhancement, can obviously improve the freezing-recovery survival rate of Day4 differentiation middle stage cells under the premise of not affecting the differentiation potential of hESC, and ensures that most of the recovered cells continue to differentiate into kidney organoids with complete structure and normal marker expression.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture and organoid manufacturing technology, and in particular to a pretreatment method and its application for improving the success rate of cryopreservation and thawing of human embryonic stem cells in the mid-differentiation stage. Background Technology

[0002] As organoid biomanufacturing moves towards automation and modularization, researchers are gradually introducing a "staged cryopreservation" strategy into the PSC differentiation process. For example, mid-differentiation cells are cryopreserved during induction Day 4 (the primitive stripe stage) to facilitate flexible transitions to the kidney organoid maturation process from Day 5 to Day 18. However, the current survival rate of Day 4 cryopreserved cells after thawing is low, their condition is unstable, and their differentiation potential is easily impaired, becoming a bottleneck affecting downstream yield and consistency.

[0003] Currently, although cryopreservation solution formulations have been optimized, few studies have systematically improved the tolerance of hESCs to subsequent cryopreservation stress starting from their initial cell state. Therefore, it is necessary to develop methods to enhance the subsequent cryopreservation-thaw performance of hESCs through pretreatment before induction. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a pretreatment method and application for improving the success rate of cryopreservation-thawing of human embryonic stem cells in the mid-differentiation stage, in order to solve the problems of low survival rate after cryopreservation and thawing, unstable state, and easily damaged differentiation potential in the prior art, which affect the downstream yield and consistency.

[0005] To achieve the above and other related objectives, the present invention provides the following technical solutions:

[0006] A first aspect of the present invention provides a pretreatment method for improving the success rate of cryopreservation-thawing of human embryonic stem cells in the mid-differentiation stage, comprising the following steps: culturing human embryonic stem cells (hESCs) in a composite culture system for a period of time before the induction of kidney organoid differentiation, to obtain pretreated human embryonic stem cells. The composite culture system includes a basal culture medium and an additive, wherein the additive includes PD0332991 reagent.

[0007] On the one hand, PD0332991 is a known cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor. By specifically inhibiting CDK4 / 6 activity, PD0332991 can arrest hESCs in the G1 phase. Cells in the G1 phase have relatively low metabolic activity and less DNA replication pressure, making them more resistant to DNA damage and organelle destruction caused by ice crystal formation and osmotic pressure changes during cryopreservation, thereby reducing apoptosis during cryopreservation-thawing. On the other hand, PD0332991 enhances cellular stress tolerance by inducing the expression of various stress-protective molecules, thereby enhancing cellular resistance. Therefore, through a dual mechanism of cell cycle regulation and enhanced stress defense, PD0332991 improves the tolerance of mid-differentiation cells to cryopreservation stress without affecting the differentiation potential of hESCs, thus increasing cryopreservation-thawing survival rates.

[0008] Furthermore, the final concentration of the PD0332991 reagent in the composite culture system is 1~5 μM.

[0009] Furthermore, the basal culture medium is selected from E8 medium or mTeSR1 medium.

[0010] Furthermore, the additive also includes coenzyme Q10 (CoQ10). As a potent antioxidant and a key component of the mitochondrial electron transport chain, CoQ10 can protect cell membranes by scavenging free radicals and lipid peroxidation products, and reduce mitochondrial damage during cryopreservation-thawing. The combined use of CoQ10 and PD0332991 can form a multi-protective mechanism, reducing oxidative stress and protecting mitochondrial function.

[0011] Furthermore, the final concentration of coenzyme Q10 in the co-culture system is 1~20 μM.

[0012] Furthermore, the additive also includes a co-antioxidant selected from NAD(P)H-related compounds, including nicotinamide (NAM), nicotinamide mononucleotide (NMN), or nicotinamide ribose (NR). NAD(P)H-related compounds enhance the antioxidant effect of CoQ10 and mitochondrial function by increasing intracellular NAD+ levels, thereby achieving significant cellular protective effects at the same or lower concentrations.

[0013] Furthermore, the final concentration of the auxiliary antioxidant in the composite culture system is 0.1~2.5mM.

[0014] Furthermore, before initiating the kidney organoid induction differentiation, human embryonic stem cells were cultured in basal medium to a confluence of 50-60%, then additives were added, and the culture was continued for 12-24 hours to obtain pretreated human embryonic stem cells. The organoid induction differentiation program was then initiated on the pretreated human embryonic stem cells.

[0015] Furthermore, after initiating the differentiation induction procedure, suspension-induced differentiation was carried out until day 18.

[0016] Furthermore, the cells were cryopreserved on day 4 of induced differentiation. After thawing, the cryopreserved cells were induced again until day 18 to form kidney organoids.

[0017] A second aspect of the present invention provides the application of the above-described pretreatment method in the preparation of kidney organoids.

[0018] A third aspect of the present invention provides the application of the above-described pretreatment method in improving the cryopreservation-resuscitation survival rate of mid-differentiation cells.

[0019] Specifically, the process involves inducing pretreated human embryonic stem cells to differentiate to the intermediate stage, followed by cryopreservation and thawing. This method is particularly effective in improving the survival rate of cryopreserved and thawed human embryonic stem cells on day 4 of induced differentiation, achieving a survival rate exceeding 90%.

[0020] Furthermore, the cryopreservation-thawing process includes the following steps:

[0021] (1) The starting cells were pretreated human embryonic stem cells, which were induced to differentiate to the intermediate stage;

[0022] (2) Use mild digestive enzymes to recover and count the cells, and resuspend the cells in cryopreservation solution;

[0023] (3) After the temperature is cooled to -80℃, it is transferred to liquid nitrogen for storage and future use;

[0024] (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.

[0025] (5) After thawing, slowly dilute the cells with DMEM culture medium, and then centrifuge to remove the cryopreservation solution;

[0026] (6) The cells were seeded into a suspension culture system to continue the induction and differentiation of kidney organoids.

[0027] Furthermore, the cryopreservation solution comprises the following components: APEL culture medium, KnockOut™ serum substitute, dimethyl sulfoxide, and fibroblast growth factor FGF9.

[0028] Furthermore, by volume percentage, the cryopreservation solution contains 50-70% APEL culture medium, 20-30% KnockOut™ serum substitute, and 5-10% dimethyl sulfoxide; the final concentration of FGF9 factor in the cryopreservation solution is 20-30 ng / mL.

[0029] As described above, the pretreatment method and its application for improving the success rate of cryopreservation and thawing of human embryonic stem cells in the mid-differentiation stage according to the present invention have the following beneficial effects:

[0030] 1. This invention can significantly improve the cryopreservation-resuscitation survival rate of Day 4 differentiated mid-differentiation cells (>90%, 40% higher than the control group) by pretreating human embryonic stem cells before the start of induced differentiation, ensuring that the vast majority of resuscitated cells continue to differentiate into kidney organoids with intact structure and normal biomarker expression.

[0031] 2. The pretreatment operation of the present invention is simple and does not affect the pluripotency of hESCs and the subsequent differentiation process. It can be seamlessly connected with existing cryopreservation solution formulations and induction differentiation processes, and is suitable for industrial production systems.

[0032] 3. PD0332991 improves the tolerance of mid-differentiation cells to cryopreservation stress through a dual mechanism of cell cycle regulation and stress defense enhancement without affecting the differentiation potential of hESCs. The combined use of CoQ10 and PD0332991 can form a multi-protective mechanism, reduce oxidative stress and protect mitochondrial function, and further improve cryopreservation-thaw survival rate. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the application of the pretreatment method of the present invention in the organoid differentiation process.

[0034] Figure 2 This is an immunofluorescence image of organoids after cell resuscitation following pretreatment in Example 1, taken on Day 18.

[0035] Figure 3 This is a statistical graph showing the expression levels of organoid Day 18 marker RNA after cell resuscitation following pretreatment in Example 1. Detailed Implementation

[0036] 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.

[0037] Example 1

[0038] Cryopreservation of pretreated mid-differentiation cells

[0039] (1) Initial cell preparation: Human embryonic stem cells (hESC) were used and cultured in mTeSR1 medium in 6-well plates until the confluence reached 50-60%.

[0040] (2) Cell pretreatment (Day-2): Add PD0332991 reagent to the culture medium of the above hESCs to a final concentration of 1 μM, and continue culturing for 24 hours to obtain pretreated hESCs.

[0041] (3) Induction initiation (Day-1): The pretreated hESCs were recovered from the 6-well plate and digested using a mild digestive enzyme, and resuspended in mTeSR1 medium + 10 μM Y27632; then incubated at approximately 2.5 × 10⁻⁶ ppm. 4 The cells / cm² were seeded at a density of 500 μL / well in 24-well plates coated with hESC-qualified-Matrigel and incubated for 24 h.

[0042] (4) Primary stripe differentiation stage (Day 0-4): From day 0 to day 4, Advanced RPMI 1640 + 1X GlutaMAX medium was used, with the addition of WNT agonist CHIR99021 (concentration of 8 μM) and Noggin factor (concentration of 5 ng / mL). The medium was changed daily at 500 μL / well for a total of 4 days.

[0043] (5) Cryopreservation (Day 4): On the 4th day after the cells were seeded into 24-well plates, mid-differentiation cells were cryopreserved: hESC-derived primitive striped cells were digested with Accutase for 7 min, gently dispersed, collected and counted, and the cell concentration was adjusted to 2×10⁻⁶. 6 After reaching a cell / mL concentration, the cells were resuspended in cryopreservation solution (60% APEL medium, 30% KOSR, 10% DMSO; final concentration of FGF9 factor 20 ng / mL); the temperature was lowered to -80℃ at a rate of -1℃ / min using a programmed cooling box, and after 4 h, the cells were transferred to liquid nitrogen for storage and later use.

[0044] Example 2

[0045] Cryopreservation of pretreated mid-differentiation cells

[0046] (1) Initial cell preparation: Human embryonic stem cells (hESC) were used and cultured in mTeSR1 medium in 6-well plates until the confluence reached 50-60%.

[0047] (2) Cell pretreatment (Day-2): Add PD0332991 reagent to the culture medium of the above hESCs to a final concentration of 1 μM, culture for 12 hours, then add coenzyme Q10 to a final concentration of 5 μM and nicotinamide mononucleotide to a final concentration of 1 mM, and continue to culture for 12 hours to obtain pretreated hESCs.

[0048] (3) Induction initiation (Day-1): The pretreated hESCs were recovered from the 6-well plate and digested using a mild digestive enzyme, and resuspended in mTeSR1 medium + 10 μM Y27632; then incubated at approximately 2.5 × 10⁻⁶ ppm. 4 The cells / cm² were seeded at a density of 500 μL / well in 24-well plates coated with hESC-qualified-Matrigel and incubated for 24 h.

[0049] (4) Primary stripe differentiation stage (Day 0-4): From day 0 to day 4, Advanced RPMI 1640 + 1X GlutaMAX medium was used, with the addition of WNT agonist CHIR99021 (concentration of 8 μM) and Noggin factor (concentration of 5 ng / mL). The medium was changed daily at 500 μL / well for a total of 4 days.

[0050] (5) Cryopreservation (Day 4): On the 4th day after the cells were seeded into 24-well plates, mid-differentiation cells were cryopreserved: hESC-derived primitive striped cells were digested with Accutase for 7 min, gently dispersed, collected and counted, and the cell concentration was adjusted to 2×10⁻⁶. 6 After reaching a cell / mL concentration, the cells were resuspended in cryopreservation solution (60% APEL medium, 30% KOSR, 10% DMSO; final concentration of FGF9 factor 20 ng / mL); the temperature was lowered to -80℃ at a rate of -1℃ / min using a programmed cooling box, and after 4 h, the cells were transferred to liquid nitrogen for storage and later use.

[0051] Comparative Example 1

[0052] This comparative example is hESC mid-differentiation cells directly cryopreserved without pretreatment, that is, compared with Examples 1 and 2, the cell pretreatment step (2) is missing.

[0053] Example 3: Effect Verification

[0054] Cells from Examples 1, 2, and Comparative Example 1 were cryopreserved for one month and then thawed. Survival rate and morphological scores were recorded 24 hours after thawing. The thawing success rate is shown in Table 1. The thawing procedure was as follows: the cryopreserved cells were removed from liquid nitrogen and quickly placed in a 37°C water bath to completely thaw the cryopreservation solution within 1–2 minutes; after thawing, DMEM culture medium was added to slowly dilute the cells, and then the cryopreservation solution was removed by centrifugation.

[0055] Table 1. Comparison of Recovery Success Rates

[0056] Number of successes / Total number of experiments Success rate Example 1 12 / 13 92% Example 2 17 / 18 94% Comparative Example 1 5 / 11 45%

[0057] The mid-differentiation cells from Example 1, after being revived, were further induced to Day 18 to form kidney organoids. The induction and differentiation steps from Day 4 to Day 18 are as follows:

[0058] Days 4-7 are the intercellular mesodermal (IM) induction phase: Cells are seeded in 96-well U-shaped plates with low adhesion, with 1 × 10⁶ cells per well. 5 Cells, 200 μL / well; culture medium replaced with Advanced RPMI 1640 + 1X GlutaMAX medium, with added FGF-9 (20 ng / mL) and Activin A (10 ng / mL); 96-well plates were centrifuged at 200 g for 30 seconds to allow cells to aggregate in a U-shaped bottom and form spheroids, spontaneously forming organoid precursor structures. No medium change was required during Days 4-7. Expression of IM markers such as PAX2 and LHX1 was detected on Day 7.

[0059] 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.

[0060] Days 11-18 are 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, and the medium is changed every 2-3 days, and the culture continues.

[0061] Immunofluorescence image of organoid Day 18 in Example 1 is shown below. Figure 2As shown, the expression levels of Day 18 marker RNA in Example 1 are as follows: Figure 3 As shown, the control group consisted of cells that had been normally induced to differentiate to Day 18 without pretreatment or cryopreservation.

[0062] The results showed that the organoid formation rate and structural marker expression of Example 1 Day 18 did not decrease. This indicates that the cryopreservation-thaw survival rate of pretreated cells was more than 40% higher than that of untreated cells, without affecting their subsequent differentiation into kidney organoids.

[0063] In summary, this invention, by pretreating human embryonic stem cells before the onset of induced differentiation, utilizes PD0332991 through a dual mechanism of cell cycle regulation and enhanced stress defense. This improves the tolerance of mid-differentiation cells to cryopreservation stress without affecting the differentiation potential of hESCs, significantly increasing the cryopreservation-resuscitation survival rate of Day 4 mid-differentiation cells. This ensures that the vast majority of resuscitated cells continue to differentiate into structurally intact kidney organoids with normal biomarker expression. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and possesses high industrial application value.

[0064] 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 pretreatment method for improving the success rate of cryopreservation-thawing of human embryonic stem cells in the mid-differentiation stage, characterized in that, Used in the differentiation process of human embryonic stem cells to induce kidney organoids, wherein the mid-differentiation cells are primitive stripe stage cells; The pretreatment method includes the following steps: before the induction of kidney organoid differentiation, human embryonic stem cells are cultured in basal culture medium to a confluence of 50-60%, then additives are added and cultured for another 24 hours to obtain pretreated human embryonic stem cells. The basal culture medium was selected from mTeSR1 medium; The additive includes reagent PD0332991.

2. The pretreatment method according to claim 1, characterized in that, The final concentration of the PD0332991 reagent in the composite culture system is 1~5 μM.

3. The pretreatment method according to claim 1, characterized in that, The additive also includes coenzyme Q10, and the final concentration of coenzyme Q10 in the composite culture system is 1~20 μM.

4. The pretreatment method according to claim 1, characterized in that, The additive also includes an auxiliary antioxidant, which includes at least one of nicotinamide, nicotinamide mononucleotide, or nicotinamide ribose.

5. The application of the pretreatment method as described in any one of claims 1-4 in the preparation of kidney organoids.

6. The application of the pretreatment method according to any one of claims 1-4 in improving the cryopreservation-thaw survival rate of primitive stripe stage cells of human embryonic stem cell-induced kidney organoids.

7. The application according to claim 6, characterized in that, Pretreated human embryonic stem cells were induced to differentiate to the primitive stripe stage, and then cryopreserved and thawed.

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