Dopaminergic neural precursor cell cryoprotectant without dmsO and use thereof

Abstract

This invention belongs to the field of biological cell cryopreservation technology, specifically relating to a DMSO-free cryopreservation protectant for dopaminergic neural progenitor cells and its application. The cryopreservation cell agent of this invention uses propylene glycol as the core permeability protectant, combined with human serum albumin, methylcellulose, ascorbic acid derivatives, ROCK inhibitors, and optional neurotrophic factors, achieving highly efficient cryopreservation protection while completely eliminating DMSO. The protectant of this invention has a recovery rate of ≥80% and a live cell recovery rate of ≥80%, which is superior to mainstream commercial cryopreservation solutions containing DMSO. Furthermore, it has no cytotoxicity, differentiation interference, or clinical infusion risks. Its composition is clearly defined, complies with GMP standards, and is suitable for the cryopreservation and production of clinical-grade cell therapy products for neurodegenerative diseases such as Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of biological cell cryopreservation technology, specifically relating to a DMSO-free cryopreservation protectant for dopaminergic neural progenitor cells and its application. Background Technology

[0002] Dopaminergic progenitor cells (iDANPCs) are key seed cells for cell therapy of neurodegenerative diseases such as Parkinson's disease. Long-term, stable cryopreservation (freezing) is a prerequisite for ensuring the quality of cell therapy products and achieving clinical application. Currently, dimethyl sulfoxide (DMSO) is the most widely used cryoprotectant for cell cryopreservation. DMSO is a permeable cryoprotectant that can penetrate cell membranes, lower the freezing point, and reduce intracellular ice crystal formation, thereby protecting cell structure. Commercial cryopreservation solutions (such as CryoStor® CS10, PSC Cryopreservation Medium, etc.) typically contain 5%-10% DMSO, supplemented with components such as human serum albumin (HSA).

[0003] Existing research also explores DMSO-free cryopreservation solutions. For example, Drummond et al. (2020, Front. Cell Dev. Biol.) systematically compared the protective effects of various commercial cryopreservation solutions on human midbrain dopaminergic neural progenitor cells. The study found that a DMSO-free commercial cryopreservation solution, "Cellvation," showed significantly lower cell viability and recovery rate after 24 hours of thawing compared to the best-performing DMSO-containing cryopreservation solution, "PSC," but was superior to some other DMSO-containing products (such as Synth-a-Freeze).

[0004] Despite its widespread use, DMSO has significant inherent drawbacks: (1) Cytotoxicity: DMSO is toxic to cells at room temperature, has a short operation window, and if it is not completely removed after thawing, it may affect the subsequent function, survival and transplantation safety of cells.

[0005] (2) Risk of differentiation interference: DMSO may alter the epigenetic state or signaling pathways of cells. Especially in therapeutic cells such as iDANPC that need to maintain a specific precursor identity, residual DMSO poses a risk of inducing unintended differentiation or affecting their in vivo integration function, which is unacceptable for clinical applications.

[0006] (3) Clinical concerns: Infusion of cell products containing DMSO may cause adverse reactions in patients, such as allergies, hemolysis, and cardiovascular events. Regulatory agencies have strict limits on the residual amount of DMSO in clinical-grade cell products, which increases the complexity of the manufacturing process and the cost of quality control.

[0007] While existing DMSO-free cryopreservation technologies (such as Cellvation) avoid the toxicity of DMSO, their cryopreservation and recovery efficiencies (viability and recovery rate) are generally lower than those of optimal DMSO-containing formulations, making it difficult to meet the stringent requirements of clinical-grade iDANPC cell products for high viability, high recovery rate, and functional integrity. Therefore, developing a highly efficient, safe, and DMSO-free dedicated iDANPC cryoprotectant is a critical technological bottleneck that the industry urgently needs to overcome. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a DMSO-free cryopreservation protectant for dopaminergic neural progenitor cells, so as to completely eliminate DMSO-related toxicity, differentiation interference and clinical infusion risks, and achieve a cryopreservation recovery rate of ≥80% and a live cell recovery rate of ≥80%, while stably maintaining the expression of iDANPC specific markers and neural differentiation potential, thus ensuring the function of cell therapy.

[0009] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a cryopreservation protectant for dopaminergic neural progenitor cells that does not contain DMSO, comprising: propylene glycol, human serum albumin, methylcellulose, ascorbic acid or a stable derivative thereof, and a ROCK signaling pathway inhibitor. The propylene glycol has a volume fraction of 5-15%, the human serum albumin has a mass fraction of 1-5%, the methylcellulose has a mass fraction of 0.5-2%, the ROCK signaling pathway inhibitor has a dilution factor of 100-200 times, and the ascorbic acid or its stable derivative has a concentration of 100-300 μM.

[0010] The DMSO-free cryopreservative for dopaminergic neural progenitor cells provided by this invention is composed of propylene glycol, human serum albumin, methylcellulose, ascorbic acid or its stable derivatives, and a ROCK signaling pathway inhibitor. This overcomes the bottleneck of traditional DMSO cryopreservatives' toxicity to neural progenitor cells and high incidence of apoptosis after resuscitation. It has crucial supporting significance for the industrialization and clinical translation of neural cell bank construction and cell transplantation therapy for neurodegenerative diseases such as Parkinson's disease. Propylene glycol, as a low-toxicity, permeable cryopreservative, replaces DMSO, reducing ice crystal damage and cytotoxicity, while human serum albumin provides stability. The cell membrane structure is maintained and colloidal osmotic pressure is preserved. Methylcellulose optimizes the viscosity of the cryopreservation system and further reduces physical damage. Ascorbic acid and its derivatives effectively neutralize oxidative stress throughout the freeze-thaw process and protect intracellular biomolecules. ROCK signaling pathway inhibitors specifically inhibit apoptosis caused by cytoskeleton rearrangement in the early stage of resuscitation, significantly improving adherent survival rate. The components work synergistically at the optimal concentrations to greatly improve the resuscitation survival rate and live cell recovery rate of dopaminergic neural progenitor cells, balancing safety and functionality, and providing a more suitable and safer solution for the long-term stable cryopreservation of sensitive neural progenitor cells.

[0011] Preferably, the volume fraction of the propylene glycol is 7.5%.

[0012] Preferably, the mass fraction of the human serum albumin is 5%, and the mass fraction of the methylcellulose is 0.5%.

[0013] Preferably, the ascorbic acid stable derivative is magnesium ascorbate phosphate with a concentration of 300 μM.

[0014] Preferably, the ROCK signaling pathway inhibitor is RevitaCell supplement diluted 100 times.

[0015] This invention achieves more efficient and safer DMSO-free cryopreservation of dopaminergic neural progenitor cells through more precise optimization and systematic formulation of the types and concentrations of each component: 7.5% propylene glycol (PG) is selected as the core permeability protectant, ensuring sufficient intracellular water replacement and reducing ice crystal damage while avoiding the cytotoxicity caused by high concentrations of alcohol; it is combined with 5% high-concentration human serum albumin (HAS) to form a synergistic protective system with propylene glycol, significantly improving cell membrane stability and intracellular buffering capacity; 0.5% methylcellulose (MC) is used to maintain the viscosity of the cryopreservation system, further reducing ice crystal formation and mechanical damage; 300μM magnesium ascorbate phosphate (MAP) is introduced as a stabilizing antioxidant to specifically eliminate reactive oxygen species generated during cryopreservation and thawing, reducing oxidative stress damage to dopaminergic neural progenitor cells; and simultaneously, RevitaCell supplement at a 1:100 volume ratio is added to effectively inhibit apoptosis and adherent cell death induced by cryopreservation and thawing, achieving specific protection for induced dopaminergic neural progenitor cells. The above-mentioned optimal ratio is not a simple superposition of the components, but a synergistic effect through multiple dimensions and multiple targets. Under the premise of completely replacing DMSO, it maximizes the protection effect and cell safety, and significantly improves the survival rate, viability and functional integrity of cryopreserved dopaminergic neural progenitor cells.

[0016] Preferably, it further comprises brain-derived neurotrophic factor, wherein the brain-derived neurotrophic factor is BDNF and the concentration is 1-10 ng / mL.

[0017] BDNF, as a key neurotrophic factor, can provide timely survival signal support to damaged cells in the very early stages of cell cryopreservation and thawing, mitigating the initiation of apoptosis caused by low-temperature stress and cell membrane damage. It works synergistically with the basic cryopreservation components such as propylene glycol, human serum albumin, and methylcellulose, as well as ROCK signaling pathway inhibitors in the cryopreservation agent, to further enhance cell survival rate and structural integrity throughout the cryopreservation-thawing process. Simultaneously, controlling the BDNF concentration within the trace range of 1-10 ng / mL ensures its effective neuroprotective and survival-promoting effects while avoiding abnormal cell differentiation or metabolic disorders caused by excessively high concentrations, thus balancing protective efficacy with maintenance of cell stemness.

[0018] Preferably, it further includes a basal culture medium, wherein the basal culture medium is DMEM / F-12.

[0019] More preferably, the cryoprotectant consists of the following components: 7.5% (v / v) propylene glycol, 5% (w / v) human serum albumin, 0.5% (w / v) methylcellulose, 300 μM magnesium ascorbate phosphate, 1:100 (v / v) RevitaCell supplement, 1 ng / mL brain-derived neurotrophic factor, and the balance being basal culture medium.

[0020] Secondly, the present invention provides a method for cryopreserving dopaminergic neural progenitor cells, wherein the cryopreservation agent is used for cell cryopreservation.

[0021] Thirdly, the present invention provides the application of the cryopreservation protectant in the preparation of cell therapy drugs for treating Parkinson's disease.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) Extremely high safety: The cryoprotectant contains no DMSO, eliminating DMSO-related cytotoxicity, potential differentiation induction risks and clinical infusion side effects, significantly improving the safety of the product in clinical translation and making it more compliant with regulatory requirements; (2) Excellent protective efficacy: The preferred formulation of this invention has achieved or surpassed the performance of commercial cryopreservation solution (CryoStor CS10) containing 10% DMSO and another commercial DMSO-free cryopreservation solution (Cellvation) in terms of key indicators such as cell viability (>80%) and live cell recovery rate (>80%), thus solving the industry problem of low efficiency of DMSO-free formulations; (3) Good functional maintenance: The formulation of this invention has clear and mild ingredients and contains specific protective ingredients for nerve cells, which can better maintain the cell activity, identity marker expression and subsequent differentiation potential of iDANPC after cryopreservation and thawing, thus ensuring the "quality" of the therapeutic cells. (4) Clear ingredients and high degree of standardization: All ingredients are traceable and meet the requirements of Good Manufacturing Practice (GMP) for pharmaceuticals, which facilitates quality control and large-scale production and is suitable for the production of clinical-grade cell therapy products. Detailed Implementation

[0023] The following detailed embodiments illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0024] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0025] Examples 1-4 Examples 1-4 provide a DMSO-free cryoprotectant for dopaminergic neural progenitor cells, the formulation of which is shown in Table 1. The preparation method of the cryoprotectant is as follows: (1) Under ice bath conditions, take DMEM / F-12 (HEPES) basal medium; (2) Add the remaining components to the culture medium, stir and mix evenly, filter with a 0.22μm filter membrane for sterilization, and store at 4℃ in the dark to obtain the cryoprotectant.

[0026] Table 1. Formulation of the cryopreservation cell agents described in Examples 1-4 Note: 1:100 or 1:200 in RevitaCell supplements means that RevitaCell supplements are diluted 100 times or 200 times in basal culture medium.

[0027] Comparative Examples 1-11 Comparative Examples 1-11 provide a DMSO-free cryoprotectant for dopaminergic neural progenitor cells, the formulation of which is shown in Table 2. The preparation method of the cryoprotectant is the same as that in the examples, the only difference being the formulation.

[0028] Table 2 Formulation table of the cryopreservation cell agents described in Comparative Examples 1-11 Note: In RevitaCell supplements, 1:50, 1:100, and 1:250 refer to diluting RevitaCell supplements in basal culture medium by 50, 100, and 250 times, respectively.

[0029] Comparative Example 7 aims to explore the effect of replacing methylcellulose with trehalose, while Comparative Examples 9-10 are used to verify the effect of the concentration of each component exceeding the preferred range of the present invention.

[0030] Example 1 This efficacy example uses the cryopreservation cell agents described in Examples 1-4 and Comparative Examples 1-11 as test samples to test the cryopreservation and thawing effect on dopaminergic neural progenitor cells (iDANPC). Two positive controls were also set up: Control Group 1: CryoStorCS10 containing 10% DMSO + 5% HSA + RevitaCell (1:100); Control Group 2: Commercially available PSC cryopreservation solution containing DMSO + RevitaCell (1:100). The specific testing methods are as follows: iDANPC was digested into single-cell suspensions with a viability ≥90%. After centrifugation, the cells were resuspended in different cryoprotectants prepared according to the experimental design and aliquoted into cryovials. Cells were cryopreserved at -80°C using a programmed cooling box and rapidly thawed at 37°C after 24 hours. The thawed cells were washed with pre-chilled culture medium containing antioxidants using a 1:3 two-stage dilution method, centrifuged, and resuspended. Viability and cell count were measured using an NC-200 automated cell counter, and the viable cell recovery rate was calculated as (total number of thawed viable cells / total number of viable cells before cryopreservation × 100%). Each sample was tested three times. Specific test results are shown in Tables 3-5.

[0031] Table 3. Results of the first experimental thaw count using iDANPC cryoprotectant. Note: The cell viability before cryopreservation was 95.6%, the cryopreservation density was 1E7 / ml, and the volume was 0.5ml / tube.

[0032] Table 4. Results of the second iDANPC cryopreservation experiment and thaw counts. Note: The cell viability before cryopreservation was 90.00%, the cryopreservation density was 0.75E7 / ml, and the volume was 0.375ml / tube.

[0033] Table 5 Results of the third iDANPC cryoprotectant experiment's recovery count. Note: The cell viability before cryopreservation was 90.00%, the cryopreservation density was 0.75E7 / ml, and the volume was 0.375ml / tube.

[0034] Tables 3-5 show that the DMSO-free cryoprotectant developed in this invention, with propylene glycol, high-concentration human serum albumin, methylcellulose, magnesium ascorbate phosphate, and ROCK inhibitor as its core functional components, can achieve efficient and stable cryoprotection of iDANPC without the addition of DMSO. The cryoprotectant formulations in Examples 1-2 are particularly advantageous; after three repeated experiments, the cell recovery survival rate was consistently maintained above 80%, demonstrating excellent protective efficacy and batch stability.

[0035] In contrast, in each comparative group, regardless of whether one or more of the five core functional components were missing, the dosage of each component deviated from the limits of this invention, or materials such as ethylene glycol, trehalose, and KnockOut SR serum substitutes were used to replace the core active components in equal amounts, the cell survival rate after cryopreservation was significantly lower than that in the embodiments of this invention. This indicates that the synergistic combination, specific dosage ratio, and selection of specific components of the five core components—propylene glycol, high-concentration human serum albumin, methylcellulose, magnesium ascorbate phosphate, and ROCK inhibitor—are key to ensuring efficient cryopreservation of iDANPC and maintaining a high survival rate under the DMSO-free system. The absence of a single component, deviation in dosage, or replacement of core raw materials will significantly weaken the cryopreservation protection effect.

[0036] Furthermore, compared to positive control group 1 (CryoStor CS10 containing 10% DMSO), the preferred formulation of this invention showed a significantly higher recovery viability (CS10 control viability was approximately 60-80%), while completely avoiding DMSO. Compared to positive control group 2 (the optimal DMSO-containing product "PSC"), the preferred formulation of this invention showed a higher recovery viability than PSC (PSC control viability was 80.65% and 71.10%, respectively), demonstrating better batch-to-batch stability of recovery viability while completely avoiding DMSO. Compared to the data reported by Drummond et al. (2020, Front. Cell Dev. Biol.), the recovery viability of the preferred formulation of this invention is far higher than that of their reported DMSO-free commercial cryopreservation solution Cellvation, and is comparable to or even better than the optimal DMSO-containing product PSC.

[0037] Example 2 This effect example uses the cryopreserved cell agents described in Example 1 and Comparative Example 11 as test samples to detect the upregulation of stress genes in dopaminergic neural progenitor cells (iDANPC) after thawing. The specific test results are shown in Table 6.

[0038] Table 6. Data on upregulation of stress genes after cryopreserved cell thawing Table 6 shows that after treatment with the MAP-containing cryoprotectant of this invention (Example 1), the CT value of the PRODH gene in the revived cells was significantly higher than that in Comparative Example 11, and closer to the level of fresh cells. This indicates that in the cryopreservation system of this invention, which combines the ROCK inhibitor with propylene glycol, human serum albumin, and methylcellulose, the addition of MAP can significantly inhibit the abnormally high expression of stress genes in revived cells, effectively alleviating oxidative stress and cellular stress damage induced by cryopreservation-revival. Furthermore, although the immediate revived viability of Comparative Example 11 (lacking MAP) was acceptable, the expression of the stress gene PRODH in its revived cells was significantly upregulated, indicating that the cells were in a state of high oxidative stress, which may seriously affect their subsequent survival and function. This again demonstrates that MAP is crucial for ensuring the health of cells after revival.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A DMSO-free cryopreservation agent for dopaminergic neural progenitor cells, characterized in that, Contains: propylene glycol, human serum albumin, methylcellulose, ascorbic acid or its stable derivatives, and ROCK signaling pathway inhibitors; The propylene glycol has a volume fraction of 5-15%, the human serum albumin has a mass fraction of 1-5%, the methylcellulose has a mass fraction of 0.5-2%, the ROCK signaling pathway inhibitor has a dilution factor of 100-200 times, and the ascorbic acid or its stable derivative has a concentration of 100-300 μM.

2. The cryoprotectant as described in claim 1, characterized in that, The volume fraction of the propylene glycol is 7.5%.

3. The cryoprotectant as described in claim 1, characterized in that, The human serum albumin has a mass fraction of 5%, and the methylcellulose has a mass fraction of 0.5%.

4. The cryoprotectant as described in claim 1, characterized in that, The ascorbic acid stable derivative is magnesium ascorbate phosphate with a concentration of 300 μM.

5. The cryoprotectant as described in claim 1, characterized in that, The ROCK signaling pathway inhibitor is RevitaCell supplement, diluted 100 times.

6. The cryoprotectant as described in claim 1, characterized in that, It also contains brain-derived neurotrophic factor, wherein the concentration of brain-derived neurotrophic factor is 1-10 ng / mL.

7. The cryoprotectant as described in claim 1, characterized in that, It also contains a basal culture medium, which is DMEM / F-12.

8. A method for cryopreserving dopaminergic neural progenitor cells, characterized in that, Cell cryopreservation is performed using the cryoprotectant described in any one of claims 1-7.

9. The use of the cryoprotectant as described in any one of claims 1-7 in the preparation of a cell therapy drug for treating Parkinson's disease.

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