Dopaminergic neural precursor cell cryo- and cryopreservation methods

Abstract

The application provides a dopamine nerve precursor cell freezing solution which is free of serum and animal-derived components, and contains a clinical-grade freezing base solution dextran sodium chloride injection, a non-osmotic cryoprotectant injection-grade human serum albumin (HSA) and an osmotic cryoprotectant injection-grade DMSO, wherein the mass concentration of the non-osmotic cryoprotectant is less than 10% and the mass concentration of the osmotic cryoprotectant is less than 15%, and the dopamine nerve precursor cell freezing solution further contains an additive capable of improving the recovery rate of dopamine nerve precursor cells. The dopamine nerve precursor cell freezing solution provided by the application has simple and clear components, can greatly reduce the damage to cells during the freezing process, significantly improve the recovery rate of cells after resuscitation, maintain the purity and the potential for continuous differentiation of the cells, and is suitable for the storage of clinical-grade dopamine nerve precursor cells at different differentiation stages.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation technology, specifically to a cryopreservation solution and method for dopaminergic neural progenitor cells. Background Technology

[0002] Parkinson's disease (PD) is one of the most common neurodegenerative diseases, affecting approximately 6 million people worldwide, with China having the largest number of PD patients globally. The prevalence of PD among people aged 65 and above in my country is about 1.7%, with nearly 100,000 new cases diagnosed annually. World Health Organization experts predict that the number of PD patients in China will reach 5 million by 2030. With the increasing number of PD cases, the market for PD medications is expanding. Current treatments primarily focus on alleviating symptoms by supplementing with dopamine, but these methods cannot cure or reverse the disease progression and have serious side effects, thus necessitating the introduction of new treatment methods. A typical pathological feature of PD is the loss of dopaminergic neurons in the substantia nigra region of the midbrain; therefore, cell transplantation is currently the most promising method for curing and controlling the disease.

[0003] Human pluripotent stem cells have unlimited proliferative capacity and can differentiate into almost all functional cells in vitro, including nerve cells. Therefore, human pluripotent stem cells can be used to induce differentiation into dopaminergic neural progenitor cells, which can then be applied in clinical research to treat Parkinson's disease (PD) through cell transplantation.

[0004] Cell cryopreservation, as an effective method of cell storage, is one of the main research directions in the preservation of cell-based drugs in regenerative medicine. Cryopreservation allows for timely use of cells at any time while reducing the risk of microbial contamination. For neural cells, especially dopaminergic neural progenitor cells derived from pluripotent stem cells, considerations must be given not only to the viability and recovery rate after thawing, but also to the purity and differentiation status of the cells after thawing, and whether they still possess the potential for further maturation and differentiation. After thawing using existing cell cryopreservation solutions, cell viability and recovery rates significantly decrease compared to before cryopreservation, resulting in a smaller number of viable cells after thawing, making them unsuitable for direct cell transplantation. Most existing cryopreservation solutions are based on cell culture media, with complex compositions, which not only significantly increase cryopreservation costs but also hinder clinical applications. Some cryopreservation solutions contain fetal bovine serum, whose composition is unclear and varies greatly between batches, making them unsuitable for storing clinical-grade cells and hindering clinical applications. Furthermore, the complex composition of existing cryopreservation solutions requires repeated washing after cell thawing to be suitable for clinical use, leading to significant cell loss. Currently, an ideal cryopreservation solution for neural cells has not yet been developed.

[0005] Therefore, developing a serum-free cryopreservation solution with simple and clearly defined components that can simultaneously ensure the viability and recovery rate of revived neural cells and maintain cell function is an important topic for expanding the clinical application of neural stem cells. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a cryopreservation solution for dopaminergic neural progenitor cells that is free of serum and animal-derived components. The cryopreservation solution comprises a clinical-grade cryopreservation base solution, a non-permeable cryoprotectant, and a permeable cryoprotectant. The non-permeable cryoprotectant has a mass concentration of less than 10%, and the permeable cryoprotectant has a mass concentration of less than 15%. The non-permeable cryoprotectant is injectable-grade human serum albumin (HSA), and the permeable cryoprotectant is injectable-grade DMSO. The clinical-grade cryopreservation base solution is dextran sodium chloride injection. The cryopreservation solution for dopaminergic neural progenitor cells also contains additives that can improve the recovery rate of dopaminergic neural progenitor cells.

[0007] Preferably, the dopaminergic neural progenitor cell cryopreservation solution consists of dextran sodium chloride injection, injection-grade HSA and injection-grade DMSO, as well as the additives that can improve the recovery rate of dopaminergic neural progenitor cells.

[0008] Preferably, the concentration of the additive that can improve the recovery rate of dopaminergic neural progenitor cells is 0.2-100 μg / ml.

[0009] More preferably, the concentration of the additive that can improve the recovery rate of dopaminergic neural progenitor cells is 0.2-60 μg / ml.

[0010] Preferably, the additive that can improve the recovery rate of dopaminergic neural progenitor cells is at least one of Y-27632, ascorbic acid, and vitamin E.

[0011] More preferably, the additive that can improve the recovery rate of dopaminergic neural progenitor cells is a combination of Y-27632 and ascorbic acid.

[0012] Preferably, the mass concentration of injectable grade DMSO in the dopaminergic neural progenitor cell cryopreservation solution is equal to or greater than 5% and less than 15%, and the mass concentration of injectable grade HSA is equal to or greater than 1% and less than 10%.

[0013] More preferably, the mass concentration of injectable grade DMSO in the dopaminergic neural progenitor cell cryopreservation solution is 7.5%-10%, and the mass concentration of injectable grade HSA is 1%-5%.

[0014] This invention provides a method for cryopreserving dopaminergic neural progenitor cells, comprising the following steps: cryopreserving dopaminergic neural progenitor cells using the cryopreservation solution provided by this invention.

[0015] Preferably, the cryopreservation density of the dopaminergic neural progenitor cells in the dopaminergic neural progenitor cell cryopreservation solution is 4 × 10⁻⁶. 6 -10×10 6 per mL.

[0016] Beneficial effects

[0017] The components of the dopaminergic neural progenitor cell cryopreservation solution of this invention work synergistically to significantly reduce cell damage during cryopreservation while ensuring high cell viability and significantly improving cell recovery rate after thawing, especially after 24 hours of culture. It also maintains cell purity and the potential for further maturation and differentiation. Furthermore, the dopaminergic neural progenitor cell cryopreservation solution of this invention has a simple composition, using only clinical-grade reagents, and is free of serum and animal-derived components, as well as macromolecules such as hydroxyethyl starch. After cryopreservation, only a small amount of washing is required for direct injection, thus significantly reducing cell loss during the washing process. This makes it more suitable for the storage of clinical-grade dopaminergic neural progenitor cells. Dopaminergic neural progenitor cells cryopreserved using the solution of this invention can be directly used for cell transplantation after thawing and can significantly improve the behavior of 6-OHDA-induced nude rats. Therefore, the cell cryopreservation solution of this invention provides an important tool for the preservation and clinical application of regenerative medicine drugs for the nervous system. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments. In the accompanying drawings:

[0019] Figure 1 A flowchart illustrating the cell cryopreservation process;

[0020] Figure 2 A flowchart illustrating the cell resuscitation process;

[0021] Figure 3 The effects of Y-27632, ascorbic acid, and / or vitamin E (VE) in the cryopreservation solution on the viability, 0-hour recovery, and 24-hour recovery of dopaminergic neural progenitor cells-A after cryopreservation.

[0022] Figure 4 The effects of DMSO and HSA concentrations in the cryopreservation solution on the viability and recovery rate of dopaminergic neural progenitor cells-A after cryopreservation at 0 and 24 hours were investigated.

[0023] Figure 5The effects of Y-27632, ascorbic acid and different concentrations of DMSO in the cryopreservation solution on the viability of dopaminergic neural progenitor cells B after cryopreservation and the recovery rate at 0 hours and 24 hours.

[0024] Figure 6 The cell growth status of dopaminergic neural progenitor cells-B after cryopreservation and thawing for 24 hours under different cryopreservation base solutions;

[0025] Figure 7 To investigate the effects of different cryopreservation base solutions on the viability of dopaminergic neural progenitor cells-B after cryopreservation and the recovery rate at 0 hours and 24 hours.

[0026] Figure 8 The test results of dopaminergic neural progenitor cells (A and B) at different differentiation stages under different cell cryopreservation densities;

[0027] Figure 9 The results of immunofluorescence staining of dopaminergic neural progenitor cells B after cryopreservation and thawing, followed by in vitro maturation and differentiation 14 days later.

[0028] Figure 10 Results of circling behavior test in nude Parkinson's model rats after injection of cryopreserved and thawed dopaminergic progenitor cells B.

[0029] Figure 11 Immunofluorescence staining results of brain slices from nude rats with Parkinson's disease model after injection of cryopreserved and thawed dopaminergic progenitor cells B. Detailed Implementation

[0030] The present invention will now be described in detail with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0031] The dopaminergic neural progenitor cell cryopreservation solution provided by this invention is free of serum and animal-derived components. The cryopreservation solution comprises a clinical-grade cryopreservation base solution, a non-permeable cryoprotectant, and a permeable cryoprotectant. The non-permeable cryoprotectant has a mass concentration of less than 10%, and the permeable cryoprotectant has a mass concentration of less than 15%. The non-permeable cryoprotectant is injectable grade HSA, and the permeable cryoprotectant is injectable grade DMSO. The clinical-grade cryopreservation base solution is dextran sodium chloride injection. The cryopreservation solution also contains additives that can improve the recovery rate of dopaminergic neural progenitor cells.

[0032] The dopaminergic neural progenitor cell cryopreservation solution provided by the present invention can be composed of dextran sodium chloride injection, injection-grade HSA and injection-grade DMSO, as well as the additives that can improve the recovery rate of dopaminergic neural progenitor cells.

[0033] The dextran sodium chloride injection, a cryopreservation base solution in this invention, is a pharmacopoeia-grade injection that can be directly applied clinically. When used as a cryopreservation base solution, it significantly increases the recovery rate of cryopreserved cells, especially the 24-hour recovery rate. The addition of HSA is necessary; HSA protects the cell membrane during cryopreservation and thawing, ensuring cell viability and increasing the cell recovery rate. DMSO in the cryopreservation solution of this invention can penetrate into the cells, increasing intracellular osmotic pressure, lowering the freezing point, and delaying the cryopreservation process. This allows some water to permeate out of the cells before freezing, reducing intracellular ice crystal formation during freezing and cooling, thereby protecting the cells, reducing cell freezing damage, and increasing the protective effect of the cryopreservation solution on cells. The additives described in this invention can significantly improve the cell recovery rate after thawing, especially the recovery rate after 24 hours of culture, while essentially maintaining the cell viability after thawing. The three components work synergistically to ensure high cell viability and recovery rate after thawing and maintain the cells' potential for further maturation and differentiation.

[0034] Dopaminergic neural progenitor cell cryopreservation solutions may be free of high-molecular-weight cryoprotectants such as hydroxyethyl starch and methylcellulose. This avoids the adverse effects on the human body caused by residual high-molecular-weight cryoprotectants after cell transplantation and subsequent washing.

[0035] In this invention, the concentration of the additive that can improve the recovery rate of dopaminergic neural progenitor cells is 0.2-100 μg / ml, preferably 0.2-60 μg / ml.

[0036] In this invention, the additive that can improve the recovery rate of dopaminergic neural progenitor cells is at least one of Y-27632 (4-[(1R)-1-aminoethyl]-N-(pyridin-4-yl)cyclohexane-1-carboxamide dihydrochloride), ascorbic acid, and vitamin E.

[0037] In this invention, the additive that can improve the recovery rate of dopaminergic neural progenitor cells is a combination of Y-27632 and ascorbic acid.

[0038] In this invention, Y-27632 and ascorbic acid in the cryopreservation solution can synergistically improve the recovery rate of dopaminergic neural progenitor cells, especially the 24-hour cell recovery rate. Poor cell condition after cryopreservation and thawing is detrimental to clinical applications. The 24-hour cell recovery rate provides a comprehensive understanding of the cell condition after cryopreservation and thawing, allowing for assessment of the cryopreservation effect of the solution.

[0039] In this invention, the mass concentration of injectable grade DMSO in the cryopreservation solution for dopaminergic neural progenitor cells is equal to or greater than 5% and less than 15%, and the mass concentration of injectable grade HSA is equal to or greater than 1% and less than 10%.

[0040] The dopaminergic neural progenitor cell cryopreservation solution of this invention contains 7.5%-10% DMSO and 1%-5% HSA. Within this concentration range, the cryopreservation solution can provide a higher cell recovery rate after cryopreservation and thawing, especially the 24-hour cell recovery rate.

[0041] The method for cryopreserving dopaminergic neural progenitor cells provided by the present invention includes the following steps: cryopreserving dopaminergic neural progenitor cells using the cryopreservation solution for dopaminergic neural progenitor cells provided by the present invention.

[0042] More specifically, it includes the following steps:

[0043] 1) Dopaminergic neural progenitor cells are mixed with the cryopreservation solution described in this invention. The specific steps are as follows: after cell digestion and collection, a certain amount of cell cryopreservation solution is slowly added while agitating the cell suspension to ensure thorough mixing. Cells are counted, and based on the counting results, the cell density is adjusted to the cryopreservation density using the cell cryopreservation solution.

[0044] 2) Cell cryopreservation. The cell mixture is aliquoted into cryovials according to standard quantities, then transferred to a Nalgene temperature-programmed chamber or a temperature-programmed freezer for cryopreservation, and finally transferred to liquid nitrogen.

[0045] The programmed cryopreservation of dopaminergic neural progenitor cells using the cryopreservation solution of the present invention includes the following steps: A. Equilibration at 4°C (after adjusting the density of dopaminergic neural progenitor cells in the cryopreservation solution, aliquot the cells into cryovials and equilibrate at 4°C); B. Cooling to -4°C at a cooling rate of 1°C / min; C. Cooling to -40°C at a cooling rate of 25°C / min; D. Heating to -12°C at a heating rate of 10°C / min; E. Cooling to -40°C at a cooling rate of 1°C / min; F. Cooling to -80°C at a cooling rate of 10°C / min.

[0046] In this invention, the cryopreservation density of dopaminergic neural progenitor cells in the dopaminergic neural progenitor cell cryopreservation solution can be 4 × 10⁻⁶. 6 -12×10 6 cells / mL (e.g., 5 × 10⁻⁶) 6 cells / mL, 6×10 6 cells / mL, 8×10 6 cells / mL, 10×10 6 (units / mL).

[0047] Furthermore, in this invention, the cryopreservation density of dopaminergic neural progenitor cells in the dopaminergic neural progenitor cell cryopreservation solution is 4-10 × 10⁻⁶. 6Cells / mL. Within this range, significantly higher cell recovery rates can be achieved, especially at 24 hours.

[0048] The preparation method of the dopaminergic neural progenitor cell cryopreservation solution of the present invention is described in detail below. In the preparation method, injectable grade HSA, injectable grade DMSO, and additives that can improve the recovery rate of dopaminergic neural progenitor cells are dissolved in dextran sodium chloride injection solution and mixed.

[0049] More specifically, for example, when the additive is ascorbic acid and / or vitamin E, the preparation method includes the following steps: 1) Dissolve ascorbic acid and / or vitamin E and HSA thoroughly in dextran sodium chloride injection solution, and filter sterilize using a filter screen; 2) Slowly add DMSO and mix thoroughly. When the additive is ascorbic acid and / or vitamin E and Y-27632, the preparation method includes the following steps: 1) Dissolve ascorbic acid and / or vitamin E and HSA thoroughly in dextran sodium chloride injection solution, and filter sterilize using a filter screen; 2) Add Y-27632 and slowly add DMSO, mixing thoroughly. The filter screen used is a 0.22 μM filter screen. During the preparation process, because the volume of DMSO added is large, it needs to be added slowly, and all components need to be thoroughly mixed. The prepared cryopreservation solution is stored at 4°C.

[0050] The dopaminergic neural progenitor cells described in this invention are resuscitated, washed with clinical-grade medical injection solution, centrifuged, and then resuspended to prepare a neural cell preparation. The medical injection solution includes dextran sodium chloride injection solution, compound electrolyte injection solution, etc.

[0051] Example

[0052] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] The sources of reagents and raw materials in the following embodiments are described below. It should be noted that the present invention does not limit the source of the reagents and materials used.

[0054] Blebbistatin was purchased from MCE, #HY-13441; ascorbic acid was purchased from Sigma, #A5960; Y-27632 was purchased from MCE, #HY-10071; DMSO was purchased from Origen. Biomedical, #CP-70; HSA was purchased from Chengdu Rongsheng, National Drug Approval Number S10940024; Dextran Sodium Chloride Injection was purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd., National Drug Approval Number H13022493; Compound Electrolyte Injection was purchased from Shanxi Zhendong Taisheng Pharmaceutical Co., Ltd.; Sodium Lactate Ringer's Injection was purchased from Otsuka Pharmaceutical Co., Ltd., National Drug Approval Number H12020009; Vitamin E was purchased from Sigma, #T3251; Trolox was purchased from MCE, HY-101445; Nude rats were purchased from Beijing Vital River, strain CR1: NIH-Foxnlnu; Midbrain dopaminergic neural progenitor cells were obtained by iPSC differentiation induced by Anhui Zhong Sheng Suyuan Biotechnology Co., Ltd. (CN202210329415.8).

[0055] Example 1: Effects of Y-27632, ascorbic acid, and / or vitamin E on the cryopreservation of dopaminergic neural progenitor cells-A

[0056] This example tested the effects of Blebbistatin, Y-27632, ascorbic acid, vitamin E, and Trolox on the cryopreservation of dopaminergic neural progenitor cells-A. The dopaminergic neural progenitor cells-A in this example were proliferative dopaminergic neural progenitor cells in the differentiation or expansion stage, expressing Lmx1a / Foxa2 / En1 / Otx2.

[0057] The preparation method of Formula 1-1 includes the following steps: HSA is fully dissolved in dextran sodium chloride injection solution and sterilized by filtration through a 0.22 μM filter; then DMSO is slowly added dropwise and thoroughly mixed, and stored at 4°C. The preparation methods of Formulas 1-2 to 1-9 include the following steps: Ascorbic acid, vitamin E, and / or Trolox are fully dissolved in dextran sodium chloride injection solution with HSA and sterilized by filtration through a 0.22 μM filter; and / or Blebbistatin or Y-27632 is added, and DMSO is slowly added dropwise and thoroughly mixed.

[0058] The specific preparation ratios of the cryopreservation solution for dopaminergic neural progenitor cells are shown in Table 1. The concentrations of HSA and DMSO are expressed as volume percentages, the concentrations of Blebbistatin, Y-27632, vitamin E, and Trolox are expressed in μM, and the concentration of ascorbic acid is expressed in μg / ml. Conversions between μM and μg / ml are possible.

[0059] Table 1. Distribution ratio of cryopreservation solutions with different compositions

[0060]

[0061] Cell cryopreservation steps are as follows Figure 1 As shown, cultured dopaminergic neural progenitor cells were digested and collected. After cell counting, cell cryopreservation solution was added to adjust the cell cryopreservation density to 6 × 10⁻⁶ cells / year. 6 After dispensing the cells at / ml, transfer them to cryovials, place the cryovials in a programmed cooling box, and gradually cool them to 80°C before storing them in liquid nitrogen.

[0062] Cell resuscitation steps are as follows Figure 2 As shown, remove the cryovial from the frozen cells and place it in a 37.0°C water bath, shaking it continuously to rapidly thaw the frozen cells.

[0063] After resuscitation, cell counts were performed to calculate the 0-hour cell viability and 0-hour cell recovery rate. The 0-hour cell viability was calculated as: 0-hour cell viability = number of viable cells detected after resuscitation / total number of cells detected after resuscitation; and the 0-hour cell recovery rate was calculated as: 0-hour cell viability = number of viable cells detected after resuscitation / total number of cells before cryopreservation. After 24 hours of incubation, the resuscitated cells were digested and collected. Cell counting yielded the 24-hour adhesion rate as: 24-hour cell adhesion rate = number of viable cells actually adhering to the culture medium after 24 hours of incubation / number of cells inoculated. The 24-hour cell recovery rate was calculated as: 24-hour cell adhesion rate × 0-hour cell recovery rate.

[0064] Cell viability was assessed after cryopreserved cells were thawed. 50 μL of the thawed cell suspension was added to 450 μL of DPBS (Dubor's phosphate buffer), and cell viability was measured using Vi-cell assay. Figure 3 A) and the number of living cells.

[0065] Determination of cell recovery rate at 0 h after cryopreservation. 0 h cell recovery rate = viable cell count / 6 × 10⁻⁶ 6 ( Figure 3 B). Based on the count results, administer 2 × 10⁻⁶ doses. 6 Dopaminergic neural progenitor cells were cultured in one well of a 6-well plate coated with VTN (human vitrin). After 24 hours, the cell status was observed under a microscope, and the cells were digested and counted using TrypLE. The 24-hour adhesion rate was calculated based on the counting results: (number of adherent viable cells / 2 × 10⁻⁶) 6 .

[0066] Cell recovery rate 24 hours after cryopreservation = 24-hour adhesion rate × 0-hour recovery rate Figure 3 C).

[0067] like Figure 3As shown in A-3C, comparing the experimental results of formulations 1-3 to 1-5 and 1-7 to 1-9 with those of formulation 1-1 reveals that adding any one of Y-27632 (a rock inhibitor), ascorbic acid, and vitamin E (both antioxidants), or a combination thereof, significantly improves cell recovery rate, especially the 24-hour cell recovery rate, while maintaining high cell viability. The combination of Y-27632 and ascorbic acid is particularly effective in improving the 24-hour cell recovery rate after cryopreservation. However, comparing formulation 1-2 with formulation 1-1 shows that although both Blebbistatin and Y-27632 are rock inhibitors, Blebbistatin does not improve cell recovery rate like Y-27632, including both 0-hour and 24-hour cell recovery rates. In formulation 1-6, adding Trolox (a vitamin E derivative) together with Y-27632 only significantly improves the 0-hour cell recovery rate, but not the 24-hour cell recovery rate.

[0068] Example 2: Effects of HSA and DMSO concentrations on cryopreservation of dopaminergic neural progenitor cells-A

[0069] The specific preparation ratios of the cryopreservation solution for dopaminergic neural progenitor cells are shown in Table 2, and the specific preparation method is the same as in Example 1.

[0070] Table 2. Composition ratios of cryopreservation solutions with different HSA and DMSO concentrations

[0071]

[0072] The cell cryopreservation density is 6 × 10⁶ 6 / ml, 1ml / tube, cell cryopreservation steps are the same as in Example 1. Figure 1 As shown. The cell resuscitation steps are the same as in Example 1. Figure 2 As shown. 50 μL of the revived cell suspension was added to 450 μL of DPBS, and cell viability was detected using Vi-cell. Figure 4 A) and viable cell count. 0h cell recovery rate = viable cell count / 6 × 10 6 ( Figure 4 B). Based on the count results, administer 2 × 10⁻⁶ doses. 6 Dopaminergic progenitor cells-A were cultured in one well of a VTN-coated 6-well plate for 24 hours. Cell status was observed under a microscope, and cells were digested and counted using TrypLE. The 24-hour adhesion rate was calculated based on the count results: (number of adherent viable cells / 2 × 10⁻⁶). 6 24-hour cell recovery rate = 24-hour cell adhesion rate × 0-hour recovery rate Figure 4 C).

[0073] like Figure 4As shown in A-4C, formulations 2-4, 2-8, and 2-12, which do not contain HSA, exhibit significantly lower cell recovery rates, especially at 24 hours, compared to formulations containing HSA. Therefore, the addition of HSA is necessary and can increase the recovery rate of cryopreserved cells. Furthermore, formulations 2-1 to 2-3, 2-5 to 2-7, and 2-9 to 2-11 all demonstrate high cell viability, 0-hour cell recovery rate, and 24-hour cell recovery rate.

[0074] Example 3: Verification of the cryopreservation effect of dopaminergic neural progenitor cells-B in cryopreservation solution

[0075] Dopaminergic progenitor cells-B are dopaminergic neurons in the early maturation stage. They are obtained by continuing the maturation and differentiation of dopaminergic progenitor cells-A for 3-7 days. They express early-specific markers of dopaminergic neurons such as Nurr1 / Sox6 and have decreased cell proliferation. They are often used as a cell source for intracranial transplantation.

[0076] The specific preparation ratios of the cryopreservation solution for dopaminergic neural progenitor cells are shown in Table 3, and the specific preparation method is the same as in Example 1.

[0077] Table 3. Partition ratio of cryopreservation solution for dopaminergic neural progenitor cells

[0078]

[0079]

[0080] The cell cryopreservation density is 6 × 10⁶ 6 / ml, 1ml / tube, cell cryopreservation steps are the same as in Example 1. Figure 1 As shown. The cell resuscitation steps are the same as in Example 1. Figure 2 As shown. 50 μL of the revived cell suspension was added to 450 μL of DPBS, and cell viability was detected using Vi-cell. Figure 5 A) and viable cell count. 0h cell recovery rate = viable cell count / 6 × 10 6 ( Figure 5 B). Based on the count results, administer 2 × 10⁻⁶ doses. 6 Dopaminergic progenitor cells-B were cultured in one well of a VTN-coated 6-well plate for 24 hours. Cell status was observed under a microscope, and cells were digested and counted using TrypLE. The 24-hour adhesion rate was calculated based on the count results: (number of adherent viable cells / 2 × 10⁻⁶). 6 24-hour cell recovery rate = 24-hour cell adhesion rate × 0-hour recovery rate Figure 5 C).

[0081] like Figure 5As shown in A-5C, adding at least one of Y-27632 and ascorbic acid to the cryopreservation solutions of formulations 3-2 to 3-10 has a cryopreservation protective effect on dopaminergic neural progenitor cells B. The concentration range of DMSO is 7.5%-10%, especially at 10%, the 24-hour cell recovery rate of cryopreserved cells is the highest and the effect is the best. In particular, the simultaneous addition of Y-27632 and ascorbic acid can synergistically increase the 24-hour recovery rate of cryopreserved cells.

[0082] Example 4: Optimization of the basal solution for cryopreservation of dopaminergic neural progenitor cells

[0083] Different pharmacopoeia-grade injection solutions were selected as base solutions to test the cryopreservation effect. The specific preparation ratios of the cell cryopreservation solutions are shown in Table 4, and the specific preparation methods are the same as in Example 1.

[0084] Table 4. Partition ratio of cell cryopreservation solutions containing different cryopreservation base solutions

[0085]

[0086] The cell cryopreservation density is 6 × 10⁶ 6 / ml, 1ml / tube, cell cryopreservation steps are the same as in Example 1. Figure 1 As shown. The cell resuscitation steps are the same as in Example 1. Figure 2 As shown. 50 μL of the revived cell suspension was added to 450 μL of DPBS, and cell viability was detected using Vi-cell. Figure 7 A) and viable cell count. 0h cell recovery rate = viable cell count / 6 × 10 6 ( Figure 7 B). Based on the count results, administer 2 × 10⁻⁶ doses. 6 Dopaminergic neural progenitor cells were cultured in one well of a VTN-coated 6-well plate for 24 hours. Afterward, they were observed and photographed under a microscope, digested with TrypLE, and counted. The 24-hour adhesion rate was calculated based on the count results: (number of adherent viable cells / 2 × 10⁻⁶). 6 24-hour cell recovery rate = 24-hour cell adhesion rate × 0-hour recovery rate Figure 7 C).

[0087] like Figure 6 As shown, microscopic observation of cell adhesion after 24 hours of culture revealed that formulation 4-3, using dextran sodium chloride injection as the cryopreservation base solution, significantly increased the 24-hour cell adhesion rate after cryopreservation compared to formulations 4-1 and 4-2. Furthermore, the recovery rate calculations showed that, compared to compound electrolyte injection and sodium lactate Ringer's solution, dextran sodium chloride injection as the cryopreservation base solution significantly increased the cell recovery rate, especially the 24-hour recovery rate.

[0088] Example 5: Cryopreservation density test of dopaminergic neural progenitor cells

[0089] Cell cryopreservation density affects the cryopreservation effect. This example further tested the effect of different cell cryopreservation densities on the cryopreservation of dopaminergic neural progenitor cells-A and dopaminergic neural progenitor cells-B. The cell cryopreservation density was selected as 4 × 10⁻⁶. 6 / ml, 6×10 6 / ml, 8×10 6 / ml, 10×10 6 / ml, 12×10 6 / ml.

[0090] Dopaminergic neural progenitor cells A and B were aliquoted into cryovials containing the cryopreservation solution described in this invention at the test density described above, 1 ml / cryovial. The cryovials were placed in a programmed cooling box and cooled to -80°C. The next day, the cryovials were transferred to liquid nitrogen. For cell thawing, cells were transported to the intercellular space using dry ice, and the cryovials were then rapidly thawed in a 37°C water bath until the ice crystals disappeared. 50 μL of the cell suspension was added to 450 μL of DPBS, and cell viability was detected using Vi-cell assay. Figure 8 A) and viable cell count. 0h cell recovery rate = viable cell count / initial viable cell count ( Figure 8 B). Based on the count results, administer 2 × 10⁻⁶ doses. 6 Dopaminergic neural progenitor cells were cultured in one well of a VTN-coated 6-well plate for 24 hours. Cell status was observed under a microscope, and cells were digested and counted using TrypLE. The 24-hour adhesion rate and 24-hour recovery rate were calculated based on the counting results. Figure 8 C).

[0091] like Figure 8 As shown in A-8C, the optimal cryopreservation density for dopaminergic neural progenitor cells is 4 × 10⁻⁶. 6 -10×10 6 / ml.

[0092] Example 6: In vitro maturation and differentiation of dopaminergic neural progenitor cells B after cryopreservation and thawing

[0093] Dopaminergic neural progenitor cells-B, cryopreserved and revived using cryopreservation solutions formulated 1-7 in Table 1 of Example 1, were then subjected to maturation differentiation using a 3D differentiation approach. The cells were differentiated at a ratio of 1×10⁻⁶. 6 / ml was suspended and cultured in Poly-HEMA-coated T25 flask; after 14 days of further culture in mature culture medium (Anhui Zhong Sheng Su Yuan Biotechnology Co., Ltd.), immunofluorescence detection was performed.

[0094] The staining procedure for 3D cultured dopaminergic neural progenitor cells-B is as follows:

[0095] (1) Fix EB with 4% PFA for 1 day, dehydrate with 30% sucrose for 3-4 days, and quick-freeze to section with a thickness of 30μm;

[0096] (2) Place the EB section onto the anti-detachment film, air dry, and then stain to ensure that the section adheres tightly to the anti-detachment film.

[0097] (3) Wash once with DPBS, then permeabilize with 0.2% Triton X-100 for 10 minutes;

[0098] (4) Block with 10% donkey serum at room temperature for 1 hour;

[0099] (5) Wash once with DPBS, add primary antibody Mouse anti-TH (1:500), incubate at room temperature for 30 minutes, then transfer to 4°C and incubate overnight;

[0100] (6) On the second day, transfer the incubated slices to room temperature and let them stand for 30 minutes;

[0101] (7) Wash once with DPBS, add secondary antibody Donkey anti mouse A488 (1:500), and incubate at room temperature in the dark for 30 minutes;

[0102] (8) Wash 3 times with DPBS, add DAPI (1:1000), and incubate at room temperature in the dark for 10 minutes;

[0103] (9) Wash twice with DPBS and once with ddH2O;

[0104] (10) The stained dopaminergic progenitor cells B were observed and photographed under an immunofluorescence microscope.

[0105] The results are as follows Figure 9 As shown, midbrain dopaminergic neurons (TH+) exhibit green fluorescence, while their nuclei show blue fluorescence. Therefore, dopaminergic neural progenitor cells-B, cryopreserved and thawed using the aforementioned cryopreservation solution, can continue to mature and differentiate into mature dopaminergic neurons with typical characteristics.

[0106] Example 7: In vivo differentiation and functional verification of dopaminergic neural progenitor cells-B after cryopreservation and thawing

[0107] Establishment of a nude rat Parkinson's disease model: Nude rats were anesthetized by isoflurane inhalation (2.5%). The skin of the head was prepared, disinfected with alcohol, and the skin was cut to expose the skull. The anterior fontanelle of the skull was located and used as the center to determine the origin. The brain localization instrument was used according to the coordinates: X = +1.92 mm (rightward shift), Y = -2.00 mm (anterior shift), Z = -8.5 mm (depth). The MFB position was marked: a small hole (0.6 mm in diameter) was carefully made at the marked location using a small animal bone drill. After slowly inserting the needle and adjusting the depth, the needle was left in place for 2 minutes. 4 μl of 6-OHDA compound solution was injected, and the injection time was 10 minutes. After the injection, the needle was left in place for 5 minutes, then slowly withdrawn for 5 minutes. Finally, the small hole was sealed with bone wax.

[0108] Dopaminergic neural progenitor cells-B were revived using cryopreservation solutions formulated 1-7 in Table 1 of Example 1. After revival, the cells were washed once with compound electrolyte injection solution, centrifuged, and resuspended in compound electrolyte solution to a concentration of 1×10⁻⁶. 5 / μl (cell resuscitation steps are the same as in Example 1) Figure 2 As shown), according to 4×10 5 The injection was administered only to the striatum of nude rats with a Parkinson's disease model (6-OHDA injection). Two months after administration, circling behavior tests were performed on rats in the treatment group (modeling + cell injection) and the untreated group (modeling only). The results showed that after cell therapy, the behavior of the Parkinson's disease model rats was significantly improved, with the number of circles decreasing to about 6 circles / min (≤7 circles / min was considered an improvement). The behavior of the untreated Parkinson's disease model rats showed virtually no improvement. Figure 10 ).

[0109] Following behavioral tests, nude rats underwent perfusion, brain harvesting, and brain slices (30 μm). Brain slices from larger graft areas were stained with immunofluorescence. Primary antibodies were Rabbit anti-TH (1:500) and Mouse anti-human nuclei (1:1000); secondary antibodies were Donkey anti-rabbit A594 (1:1000) and Donkey anti-mouse A488 (1:500). Immunofluorescence microscopy was used for observation and photography. Figure 11 As shown, midbrain dopaminergic neurons (TH+ cells) exhibit red fluorescence, while human cell nuclei (hNuc+ cells) exhibit green fluorescence.

[0110] In summary, dopaminergic progenitor cells B, after being cryopreserved and thawed using the cryopreservation solution described in this invention, can continue to mature and differentiate into mature dopaminergic neurons when transplanted into nude Parkinson's disease model rats, and can exert physiological functions in vivo, thereby improving the behavior of the model rats.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A cryopreservation solution for dopaminergic neural progenitor cells, free of serum and animal-derived components, wherein the cryopreservation solution comprises a clinical-grade cryopreservation base solution, a non-permeable cryoprotectant, and a permeable cryoprotectant, wherein the mass concentration of the non-permeable cryoprotectant is less than 10% and the mass concentration of the permeable cryoprotectant is less than 15%, the non-permeable cryoprotectant is injectable grade HSA, the permeable cryoprotectant is injectable grade DMSO, the clinical-grade cryopreservation base solution is dextran sodium chloride injection, and the cryopreservation solution for dopaminergic neural progenitor cells further comprises an additive capable of improving the recovery rate of dopaminergic neural progenitor cells, wherein the additive capable of improving the recovery rate of dopaminergic neural progenitor cells is at least one selected from Y-27632, ascorbic acid, and vitamin E.

2. The dopaminergic neural precursor cell cryo- solution according to claim 1, characterized in that, The dopaminergic neural progenitor cell cryopreservation solution consists of dextran sodium chloride injection, injection-grade HSA and injection-grade DMSO, as well as the additives that can improve the recovery rate of dopaminergic neural progenitor cells.

3. The dopaminergic neural precursor cell cryo- solution according to claim 1 or 2, characterized in that, The concentration of the additive that can improve the recovery rate of dopaminergic neural progenitor cells is 0.2-100 μg / ml.

4. The dopaminergic neural precursor cell cryo- solution according to claim 3, characterized in that, The concentration of the additive that can improve the recovery rate of dopaminergic neural progenitor cells is 0.2-60 μg / ml.

5. The dopaminergic neural precursor cell cryo- solution according to claim 1 or 2, characterized in that, The additive that can improve the recovery rate of dopaminergic neural progenitor cells is a combination of Y-27632 and ascorbic acid.

6. The dopaminergic neural precursor cell cryo- solution according to claim 1 or 2, characterized in that, The dopaminergic neural progenitor cell cryopreservation solution contains an injection-grade DMSO concentration of 5% or greater and less than 15%, and an injection-grade HSA concentration of 1% or greater and less than 10%.

7. The dopaminergic neural precursor cell cryo- solution according to claim 6, characterized in that, The dopaminergic neural progenitor cell cryopreservation solution contains 7.5%-10% DMSO and 1%-5% HSA.

8. A method for cryopreservation of dopaminergic neural precursor cells, characterized in that, The procedure includes the following steps: cryopreserving dopaminergic neural progenitor cells using the cryopreservation solution for dopaminergic neural progenitor cells as described in any one of claims 1-7.

9. The cryopreservation method of claim 8, wherein, The freezing density of the dopaminergic neural precursor cells in the dopaminergic neural precursor cell freezing solution is 4 x 10 6 -10 x 10 6 cells / mL.

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