Serum-free cryopreservation solution for cells and use thereof

Abstract

This application discloses a serum-free cryopreservation solution for cells and its applications. The serum-free cryopreservation solution comprises: 1-2% recombinant human albumin, 5-10 μM (-)-Blebbistatin, 50-100 μM antioxidant, 5-10% permeable cryoprotectant, and a basic culture medium free of phenol red indicator. This serum-free cryopreservation solution is suitable for the cryopreservation of various cell types, particularly muscle cells, especially human induced pluripotent stem cell (iPSC) differentiated cardiomyocytes. It can significantly improve the survival rate, structural integrity, and effective preservation of the physiological functions of cardiomyocytes after resuscitation. It is applicable to basic scientific research, disease modeling, drug screening, and clinical treatment related to cardiomyocytes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a serum-free cryopreservation solution for cells and its applications. Background Technology

[0002] In the biomedical field, especially in the research and treatment of heart diseases, human cardiomyocytes play a crucial role. In recent years, cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have become the preferred tool for mimicking cardiac function, establishing disease models, drug screening, and cell therapy. However, the unique properties of CMs—including their complex sarcomere structure and subtle electrophysiological characteristics—make their handling during cryopreservation and thawing a significant challenge. Traditional cryopreservation techniques often rely on formulations such as fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO), but these have shown significant limitations and drawbacks in practical applications.

[0003] 1. The Standardization Challenges of Serum Addition: Many existing CM cryopreservation solutions rely on fetal bovine serum (FBS) as a non-permeable preservative, such as the commonly used 90% FBS + 10% DMSO formulation. However, the significant batch-to-batch variation of FBS leads to variability and unpredictability in cryopreservation effects, severely hindering experimental standardization and result reproducibility. The presence of FBS also introduces the risk of pathogen contamination, posing an obstacle to the pursuit of high-quality, safe clinical-grade cell therapy. Due to the complex composition of FBS, containing various unknown growth factors, this could unintendedly affect the function of CMs, thereby interfering with pharmacological studies and the efficacy of transplantation therapy.

[0004] 2. Poor compatibility with mature cardiomyocytes: Existing cryopreservation solutions are mainly suitable for cardiomyocytes in the early differentiation stage (≤20 days), but they are not protective enough for highly mature cardiomyocytes (greater than 60 days). The survival rate after thawing is often less than 50%, which greatly limits their application in long-term research on heart disease and high-level functional analysis.

[0005] 3. Insufficient maintenance of electrophysiological function: Even after successful thawing, cardiomyocytes often experience a significant decline in contractility and electrophysiological function after cryopreservation. This is mainly due to the damage to the cell membrane and internal structures caused by ice crystal formation and sudden changes in osmotic pressure during cryopreservation. This loss of function is fatal for drug screening and cardiac regenerative medicine, as it directly affects the cell's physiological responses and therapeutic potential.

[0006] In summary, while current cardiomyocyte cryopreservation technology has made some progress, it still fails to effectively address issues such as high survival rates of cardiomyocytes (CMs), maintenance of electrophysiological function, and compatibility with mature cells. Therefore, developing a novel cryopreservation solution and technology that can significantly improve the cryopreservation and recovery rate of mature CMs without the use of frozen bone shavings (FBS) while effectively maintaining their electrophysiological function has become an urgent need in this field. Summary of the Invention

[0007] The present invention aims to provide a serum-free cryopreservation solution for cells and its application, in order to solve the technical problems of low survival rate (usually <50%) and alteration of normal electrophysiological function caused by cryopreservation and resuscitation of cardiomyocytes in the prior art.

[0008] To achieve the above objectives, according to one aspect of the present invention, a serum-free cryopreservation solution for cells is provided. This serum-free cryopreservation solution comprises: 1-2% recombinant human albumin, 5-10 μM (-)-Blebbistatin, 50-100 μM antioxidant, 5-10% permeable cryoprotectant, and a basic culture medium free of phenol red indicator.

[0009] Furthermore, the recombinant human albumin is plant-derived recombinant human albumin; preferably, the recombinant human serum albumin is rice-derived recombinant human albumin.

[0010] Furthermore, the antioxidant is selected from one or more of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, β-mercaptoethanol, and N-acetylcysteine.

[0011] Furthermore, the penetrating cryoprotectant is DMSO.

[0012] Furthermore, the basic culture medium was selected from 1640 medium or DME / F-12 medium without phenol red indicator.

[0013] Furthermore, the content of recombinant human albumin is 2%; and / or the content of (-)-Blebbistatin is 10 μM; and / or the content of antioxidant is 100 μM; and / or the content of permeable cryoprotectant is 8%.

[0014] Furthermore, the serum-free cryopreservation solution includes 2% plant-derived recombinant human albumin, 10 μM (-)-Blebbistatin, 100 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate, 8% DMSO, and 1640 medium without phenol red indicator.

[0015] Further, the cells include one or more of muscle cells, iPS cells, neural stem cells, and tumor cells; optionally, the muscle cells are cardiomyocytes, and more preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for more than 20 days; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for 20-30 days.

[0016] According to another aspect of the present invention, an application of the above-described serum-free cryopreservation solution for cells in the cryopreservation of muscle cells is provided; optionally, the muscle cells include one or more of cardiomyocytes, iPS cells, neural stem cells and tumor cells; optionally, the muscle cells are cardiomyocytes, further optionally, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for more than 20 days; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for 20-30 days.

[0017] According to another aspect of the present invention, a method for cryopreservation and thawing of muscle cardiomyocytes is provided. The method includes the following steps: cryopreserving muscle cells using any of the serum-free cryopreservation solutions for cells described above; during the thawing process, adding liquid to the cryopreserved muscle cells slowly, optionally adding liquid drop by drop at a rate of 1 drop every 3-5 seconds, and gently shaking to mix during the addition; optionally, the liquid is a muscle cell culture medium and / or a cell suspension of thawed cryopreserved muscle cells.

[0018] The serum-free cryopreservation solution of this invention is suitable for the cryopreservation of various cell types, especially muscle cells, particularly human induced pluripotent stem cell (iPSC) differentiated cardiomyocytes. It can significantly improve the survival rate, structural integrity, and effective preservation of the physiological functions of cardiomyocytes after resuscitation. It is applicable to basic scientific research, disease modeling, drug screening, and clinical treatment related to cardiomyocytes. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1 Phase contrast micrographs (200X) of human cardiomyocytes before and after cryopreservation (30 days) in Example 1 are shown;

[0021] Figure 2 Immunofluorescence staining images of human cardiomyocyte markers α-Actinin and TNNT2 before and after cryopreservation (30 days) in Example 1 are shown;

[0022] Figure 3The myocardial field potentials before and after cryopreservation in Example 1 are shown; and

[0023] Figure 4 The number of beats of human cardiomyocytes (30 days) before and after cryopreservation in Example 1 is shown. Detailed Implementation

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0025] In view of the technical problems of low survival rate (usually <50%) and alteration of normal electrophysiological function caused by cryopreservation and thawing of cardiomyocytes in the prior art, this application proposes the following technical solution.

[0026] According to a typical embodiment of this application, a serum-free cryopreservation solution for muscle cells is provided. This serum-free cryopreservation solution comprises, by volume percentage: 1-2% recombinant human albumin, 5-10 μM (-)-Blebbistatin, 50-100 μM antioxidant, 5-10% permeable cryoprotectant, and a basic culture medium without phenol red indicator.

[0027] Among them, recombinant human albumin is preferably plant-derived, containing no animal-derived components, thus avoiding the problems associated with animal-derived components such as serum. Recombinant human albumin can effectively protect cell structures from damage. Its core function is a multi-layered synergistic protective network: 1. During freezing: It protects the delicate structures of cardiomyocytes, such as the cell membrane and internal myofibrils and mitochondria, from damage by ice crystals and drastic changes in osmotic pressure by physically altering ice crystal morphology and providing colloid osmotic pressure support; 2. Throughout the process: It resists oxidative stress by chelating metal ions, protecting cardiomyocyte organelles sensitive to oxidative damage; 3. In terms of structure and function: It provides physical support by mimicking the extracellular matrix, maintaining the normal morphology and polarity of cardiomyocytes, and protects the stability of intracellular proteins through its "molecular chaperone"-like activity.

[0028] Therefore, adding recombinant human serum albumin to the cryopreservation solution is equivalent to dressing cardiomyocytes in a high-level protective suit that integrates "buffer clothing," "antioxidant," and "life support system," thereby achieving efficient preservation of cardiomyocytes and their complex three-dimensional structure.

[0029] Preferably, the recombinant human serum albumin is rice-derived recombinant human albumin. In one embodiment of this application, rice-derived recombinant human albumin produced by Wuhan Heyuan Biotechnology Co., Ltd. was used.

[0030] (-)-Blebbistatin primarily improves cell survival and physiological integrity during cardiomyocyte cryopreservation by inhibiting myosin II ATPase activity, thereby regulating cytoskeleton contraction and energy metabolism. Specifically, it inhibits myosin II-mediated contractile damage, maintaining cytoskeleton stability; reduces ATP depletion and apoptosis signal activation, protecting mitochondrial function; and regulates membrane nanotube formation, promoting intercellular repair substance exchange after thawing.

[0031] According to a typical embodiment of this application, the antioxidant is selected from one or more of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, β-mercaptoethanol, and N-acetylcysteine. Among them, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate overcomes the inherent defects of ordinary vitamin C through molecular structure modification, achieving comprehensive superiority in stability and functional diversity. By modifying the unstable 2-position hydroxyl group into a phosphate ester bond and forming a magnesium salt, L-ascorbic acid 2-phosphate sesquimagnesium salt achieves the following objectives: 1. Significantly improves chemical stability, making it less susceptible to oxidation; 2. Transforms strong acidity into near-neutral, reducing irritation; 3. Retains biological activity, releasing native vitamin C through enzymatic hydrolysis in vivo.

[0032] It provides better antioxidant protection during cryopreservation. By protecting mitochondria, it better ensures the energy supply for ion channels.

[0033] According to a typical embodiment of this application, the permeable cryoprotectant is DMSO. Specifically, 8% DMSO can reduce ice crystal formation, stabilize cell membranes, and provide antioxidant effects.

[0034] According to a typical embodiment of this application, the basic culture medium is selected from 1640 medium without phenol red indicator or DME / F-12 medium. 1640 medium can provide nutrient support, act as a solvent for DMSO, and maintain osmotic pressure and pH stability.

[0035] In this application, the above-mentioned components of the serum-free cryopreservation solution work together and coordinate with each other to create a pioneering "electrophysiological-contraction dual protection" mechanism: - (-)-Blebbistatin inhibits contraction, reduces mechanical damage, and maintains the myosin heavy chain structure; - L-ascorbic acid 2-phosphate sesquimagnesium hydrate has antioxidant properties, protects mitochondria, and ensures the energy supply of ion channels.

[0036] In some preferred embodiments of this application, the content of recombinant human serum albumin is 2%; and / or the content of (-)-Blebbistatin is 10 μM; and / or the content of antioxidant is 100 μM; and / or the content of permeable cryoprotectant is 8%. Serum-free cryopreservation solutions with these specific component contents have better performance.

[0037] In particular, in one embodiment of this application, the serum-free cryopreservation solution comprises 2% plant-derived recombinant human serum albumin, 10 μM (-)-Blebbistatin, 100 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate, 8% DMSO, and 1640 culture medium free of calcium ions and phenol red indicator.

[0038] The serum-free cryopreservation solution of this application can be used to cryopreserve various cells, especially muscle cells, including one or more of cardiomyocytes, iPS cells, neural stem cells and tumor cells; optionally, the muscle cells are cardiomyocytes, more preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for more than 20 days; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for 20-30 days.

[0039] According to a typical embodiment of this application, the above-described serum-free cryopreservation solution for cells is provided for the cryopreservation of muscle cells; optionally, the muscle cells include one or more of cardiomyocytes, iPS cells, neural stem cells, and tumor cells; optionally, the muscle cells are cardiomyocytes, and more preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for more than 20 days; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for 20-30 days.

[0040] According to a typical embodiment of this application, a method for cryopreservation and thawing of muscle cells is provided. The method includes the following steps: cryopreserving muscle cells using the serum-free cryopreservation solution described above; during thawing, adding liquid to the cryopreserved muscle cells slowly, optionally adding the liquid dropwise at a rate of 1 drop every 3-5 seconds, gently shaking to mix during the addition; optionally, the liquid is a muscle cell culture medium and / or a cell suspension of thawed cryopreserved muscle cells.

[0041] If long-term preservation of cardiomyocytes is required, they can be cryopreserved. In one embodiment of this application, the cardiomyocyte cryopreservation procedure includes:

[0042] 1. Equilibrate PBS solution and CardioEasy at room temperature. ® Human cardiomyocyte digestive fluid I / II. CardioEasy is then aspirated. ® Human cardiomyocyte maintenance medium (cardiomyocytes were cultured using CardioEasy® human cardiomyocyte maintenance medium), and washed once with PBS solution.

[0043] 2. Add CardioEasy ® Human cardiomyocyte digestion solution I, ensuring it completely covers the bottom of the dish / bottle.

[0044] Incubate at 37℃ for 5–10 minutes. Under a microscope, a certain gap is found between the cells, indicating that the first step of digestion has been achieved.

[0045] 4. Immediately inhale CardioEasy ® Human cardiomyocyte digestion solution I, added CardioEasy ® Human cardiomyocyte digestion solution II, ensuring it completely covers the bottom of the dish / bottle.

[0046] Incubate at 5.37℃ for 15-20 minutes. Under a microscope, the cells were found to be completely rounded, and most of the cells floated up after being tapped, indicating that the second step of digestion had been achieved.

[0047] 6. Use a pipette to fan-shaped blows on the bottom of the culture dish / flask to detach the cardiomyocytes attached to the bottom of the dish / flask. Transfer the cells to a 15mL centrifuge tube, then add an appropriate amount of PBS solution to rinse the bottom of the dish / flask. Collect the remaining cells and add them to a 15mL centrifuge tube.

[0048] Note: The blowing and tapping should be gentle to avoid damaging the myocardial cells.

[0049] Centrifuge at 7.200g for 5 min.

[0050] 8. Discard the supernatant and wash once with 5-10 mL of PBS solution.

[0051] Centrifuge at 9.200g for 5 min.

[0052] 10. Discard the supernatant, resuspend the cells in cryopreservation solution, and gently pipette to mix.

[0053] 11. Cryopreserve 1 mL per tube according to the required amount for resuscitation. Do not use a programmed cooling box; simply place the cells directly into a -80°C freezer. After incubating overnight at -80°C, transfer the cryopreserved cells to liquid nitrogen for storage.

[0054] In one embodiment of this application, myocardial cell resuscitation includes:

[0055] 1. Room temperature equilibrium CardioEasy ® Human cardiomyocytes were resuscitated and inoculated with complete culture medium, and the coated CardioEasy cells were removed. ® In culture dishes / flasks containing human cardiomyocytes, remove the coating solution and add an appropriate amount of CardioEasy. ® Human cardiomyocytes were resuscitated and inoculated with complete culture medium, and placed in a 37°C CO2 cell culture incubator.

[0056] Thaw the cells at 2.37℃, shake rapidly to melt the cells until only a small ice crystal remains (avoid contact between the cryovial cap and the water surface), quickly remove them and disinfect the outer surface of the cryovial with 75% alcohol, then transfer them to a sterile operating table.

[0057] 3. Carefully transfer the cell suspension to a new 15 mL centrifuge tube using a 1 mL pipette, avoiding vigorous aspiration or blow-drip.

[0058] 4. Use 1mL CardioEasy ® The cryovials were rinsed with human cardiomyocyte maintenance culture medium to collect the remaining cells. Then, the cells were added dropwise to a 15 mL centrifuge tube containing the cell suspension at a slow rate of one drop every 5 seconds, while gently shaking to mix continuously.

[0059] 5. Maintaining the same dropping rate of 1 drop every 5 seconds, add 4 mL of CardioEasy dropwise. ® Human cardiomyocyte maintenance culture medium was added dropwise with gentle shaking to mix thoroughly.

[0060] Centrifuge at 6.200g for 5 min.

[0061] 7. Discard the supernatant and use CardioEasy ® Human cardiomyocytes were resuscitated, inoculated into complete culture medium, resuspended, gently pipetted to mix, and then inoculated into prepared culture dishes / flasks. The cells were then evenly distributed by horizontal cross-vibration.

[0062] Note: Cardiac cell conduction of action potential requires the formation of intercellular connections. The reference seeding density that enables the formation of intercellular connections is shown in Table 1 below.

[0063] Table 1

[0064]

[0065] Cells were cultured at 37°C in a CO2 incubator for 48 hours and then continued to be cultured using CardioEasy. ® Human cardiomyocyte maintenance culture medium.

[0066] The beneficial effects of this application will be further illustrated below with reference to the embodiments.

[0067] Example 1

[0068] Cryopreservation of cardiomyocytes

[0069] Serum-free cryopreservation solution for muscle cells, by volume percentage, includes: 2% recombinant human serum albumin, 10 μM (-)-Blebbistatin, 100 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate, 8% DMSO, and phenol red-free basal medium: 1640 medium.

[0070] Cardiomyocytes: Cardiomyocytes differentiated from iPSCs.

[0071] 1. Equilibrate PBS solution and CardioEasy at room temperature. ® Human cardiomyocyte digestion solution I / II, stored at 4°C.

[0072] 2. Absorb CardioEasy ® Human cardiomyocyte maintenance culture medium was prepared and washed once with PBS solution.

[0073] 3. Add CardioEasy ® Human cardiomyocyte digestion solution I, ensuring it completely covers the bottom of the dish / bottle.

[0074] 4. Incubate at 37℃ for 5-10 minutes. Under a microscope, a certain gap is found between the cells, indicating that the first step of digestion has been achieved.

[0075] 5. Immediately inhale CardioEasy ® Human cardiomyocyte digestion solution I, added CardioEasy ® Human cardiomyocyte digestion solution II, ensuring it completely covers the bottom of the dish / bottle.

[0076] Incubate at 6.37℃ for 15-20 minutes. Under a microscope, the cells were found to be completely rounded, and most of the cells floated up after being tapped, indicating that the second step of digestion had been achieved.

[0077] 7. Use a pipette to fan-shaped blows on the bottom of the culture dish / flask to detach the cardiomyocytes attached to the bottom of the dish / flask. Transfer the cells to a 15mL centrifuge tube, then add an appropriate amount of PBS solution to rinse the bottom of the dish / flask. Collect the remaining cells and add them to a 15mL centrifuge tube.

[0078] Note: The blowing and tapping should be gentle to avoid damaging the myocardial cells.

[0079] Centrifuge at 8.200g for 5 min.

[0080] 9. Discard the supernatant and wash once with 5-10 mL of PBS solution.

[0081] Centrifuge at 10.200g for 5 min.

[0082] 11. Discard the supernatant, resuspend the cells in cryopreservation solution, and gently pipette to mix.

[0083] 12. Cryopreserve 1 mL per tube according to the required amount for resuscitation. Do not use a programmed cooling box; simply place the cells directly into a -80°C freezer. After incubating overnight at -80°C, transfer the cryopreserved cells to liquid nitrogen for storage.

[0084] After being frozen for several days, the frozen cells were thawed, including the following steps:

[0085] 1. Room temperature equilibrium CardioEasy ® Human cardiomyocytes were resuscitated and inoculated with complete culture medium, and the coated CardioEasy cells were removed. ® In culture dishes / flasks containing human cardiomyocytes, remove the coating solution and add an appropriate amount of CardioEasy. ® Human cardiomyocytes were resuscitated and inoculated with complete culture medium, and placed in a 37°C CO2 cell culture incubator.

[0086] Thaw the cells at 2.37℃, shake rapidly to melt the cells until only a small ice crystal remains (avoid contact between the cryovial cap and the water surface), quickly remove them and disinfect the outer surface of the cryovial with 75% alcohol, then transfer them to a sterile operating table.

[0087] 3. Carefully transfer the cell suspension to a new 15 mL centrifuge tube using a 1 mL pipette, avoiding vigorous aspiration or blow-drip.

[0088] 4. Use 1mL CardioEasy ® The cryovials were rinsed with human cardiomyocyte maintenance culture medium to collect the remaining cells. Then, the cells were added dropwise to a 15 mL centrifuge tube containing the cell suspension at a slow rate of one drop every 5 seconds, while gently shaking to mix continuously.

[0089] 5. Maintaining the same dropping rate of 1 drop every 5 seconds, add 4 mL of CardioEasy dropwise. ® Human cardiomyocyte maintenance culture medium was added dropwise with gentle shaking to mix thoroughly.

[0090] Centrifuge at 6.200g for 5 min.

[0091] 7. Discard the supernatant and use CardioEasy ® Human cardiomyocytes were resuscitated, inoculated into complete culture medium, resuspended, gently pipetted to mix, and then inoculated into prepared culture dishes / flasks. The cells were then evenly distributed by horizontal cross-vibration.

[0092] Note: Cardiac cell conduction of action potential requires the formation of intercellular connections. The reference seeding density that enables the formation of intercellular connections is shown in Table 1.

[0093] Cells were cultured at 37°C in a CO2 incubator for 48 hours and then continued to be cultured using CardioEasy. ® Human cardiomyocyte maintenance culture medium.

[0094] Experimental data

[0095] Table 2. Comparison of myocardial resuscitation rates in humans after cryopreservation for different periods:

[0096]

[0097] Note: Data is derived from the results of testing iPSC-differentiated cardiomyocytes (30 days) that were cryopreserved in liquid nitrogen for 6 months (n=6).

[0098] Table 3. Comparison of recovery rates of human cardiomyocytes (30 days) after different liquid nitrogen cryopreservation times:

[0099]

[0100] Note: Data are from the results of testing iPSC-differentiated cardiomyocytes (30 days) after liquid nitrogen cryopreservation for one year, three years, and five years (n=6).

[0101] Figure 1 Phase contrast micrographs (200X) of human cardiomyocytes before and after cryopreservation (30 days) show no significant morphological differences (left: before cryopreservation; right: after cryopreservation).

[0102] Figure 2 Immunofluorescence staining images (200X) of human cardiomyocyte markers α-Actinin and TNNT2 before and after cryopreservation (30 days) are shown, indicating no significant difference in sarcomere structure.

[0103] Figure 3 and Figure 4 The electrophysiological data of human cardiomyocytes (30 days) before and after cryopreservation are shown. Figure 3 There was no significant difference in myocardial field potential before and after cryopreservation (left: before cryopreservation; right: after cryopreservation). Figure 4 The results showed no significant difference in the number of heartbeats of human cardiomyocytes (30 days) before and after cryopreservation (left: before cryopreservation; right: after cryopreservation).

[0104] Tables 4 and 5 show the field potential time history corrected values ​​(FPDc, detected using microelectrode array technology) of human cardiomyocytes (30 days) before and after cryopreservation. The mean values ​​before and after cryopreservation, SD and %cv values ​​are similar and have no significant difference.

[0105] Table 4 Before freezing

[0106]

[0107] Table 5 After freezing

[0108]

[0109] Note: Data were obtained from iPSC-differentiated cardiomyocytes that were cryopreserved in liquid nitrogen for 6 months after differentiation (n=6).

[0110] The length of FPDc, in milliseconds.

[0111] The average values ​​before and after freezing, SD and %cv values ​​are quite similar.

[0112] Example 2

[0113] Based on Example 1, in order to study the effect of a change in a single component on the recovery rate of cardiomyocytes after cryopreservation while keeping other components of the cryopreservation solution unchanged, multiple experiments were conducted, and the results are as follows:

[0114] Table 6

[0115]

[0116] Note: Data is derived from the results of testing iPSC-differentiated cardiomyocytes (30 days) that were cryopreserved in liquid nitrogen for 6 months (n=6).

[0117] Example 3

[0118] Using the myocardial cryopreservation solution and experimental methods of Example 1, the recovery rates after cryopreservation of other cell types are as follows:

[0119] Table 7

[0120]

[0121] Note: Data is derived from the results of cell testing after 6 months of cryopreservation in liquid nitrogen (n=6).

[0122] Example 4

[0123] Using the experimental method of Example 1, human cardiomyocytes were thawed after cryopreservation using commercially available cryopreservation solution. The results are as follows:

[0124] Table 8

[0125]

[0126] Note: Data is derived from the results of testing iPSC-differentiated cardiomyocytes (30 days) that were cryopreserved in liquid nitrogen for 6 months (n=6).

[0127] Example 5

[0128] Using the experimental method of Example 1, different resuscitation methods were employed to resuscitate cryopreserved human cardiomyocytes. The results are as follows:

[0129] Table 9

[0130]

[0131] Note: Data is derived from the results of testing iPSC-differentiated cardiomyocytes (30 days) that were cryopreserved in liquid nitrogen for 6 months (n=6).

[0132] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0133] 1. The cryopreservation solution of this application uses plant-derived recombinant human albumin instead of animal-derived albumin and serum, which ensures the cryopreservation effect while avoiding the harm caused by the use of animal-derived components.

[0134] 2. The cryopreservation solution of this application contains effective components for protecting cardiomyocytes (recombinant human albumin, (-)-Blebbistatin, and -L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate synergistic effect), which can significantly improve the recovery rate of myocardium and achieve good results for cardiomyocytes older than 60 days.

[0135] 3. The cryopreservation solution of this application can effectively preserve the contractile and electrophysiological functions of the myocardium.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A serum-free cryopreservation solution for cells, characterized in that, include: The formula consists of 1-2% recombinant human albumin, 5-10 μM (-)-Blebbistatin, 50-100 μM antioxidant, 5-10% permeable cryoprotectant, and a basic culture medium without phenol red indicator.

2. The serum-free cryopreservation solution according to claim 1, characterized in that, The recombinant human albumin is plant-derived recombinant human albumin; preferably, the recombinant human serum albumin is rice-derived recombinant human albumin.

3. The serum-free cryopreservation solution according to claim 1, characterized in that, The antioxidant is selected from one or more of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, β-mercaptoethanol, and N-acetylcysteine.

4. The serum-free cryopreservation solution according to claim 1, characterized in that, The permeable cryoprotectant is DMSO.

5. The serum-free cryopreservation solution according to claim 1, characterized in that, The basic culture medium is selected from 1640 medium or DME / F-12 medium without phenol red indicator.

6. The serum-free cryopreservation solution according to any one of claims 1 to 5, characterized in that, The content of the recombinant human albumin is 2%; and / or The content of (-)-Blebbistatin is 10 μM; and / or The antioxidant content is 100 μM; and / or The content of the permeable cryoprotectant is 8%.

7. The serum-free cryopreservation solution according to claim 1, characterized in that, The serum-free cryopreservation solution comprises 2% plant-derived recombinant human albumin, 10 μM (-)-Blebbistatin, 100 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate, 8% DMSO, and 1640 medium without phenol red indicator.

8. The serum-free cryopreservation solution according to claim 1, characterized in that, The cells include one or more of muscle cells, iPS cells, neural stem cells, and tumor cells; Optionally, the muscle cells are cardiomyocytes; more preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for more than 20 days; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for 20-30 days.

9. The application of the serum-free cryopreservation solution for cells as described in any one of claims 1 to 8 in the cryopreservation of muscle cells; optionally, the cells include one or more of muscle cells, iPS cells, neural stem cells, and tumor cells; optionally, the muscle cells are cardiomyocytes; Optionally, the muscle cells are cardiomyocytes; more preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for more than 20 days; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells differentiated for 20-30 days.

10. A method for cryopreservation and thawing of muscle and cardiomyocytes, characterized in that, Includes the following steps: Muscle cells were cryopreserved using the serum-free cryopreservation solution for cells as described in any one of claims 1 to 8. When adding liquid to the frozen muscle cells during the revival process, the speed should be slow. Alternatively, the liquid can be added drop by drop at a rate of 1 drop every 3-5 seconds, and the mixture should be gently shaken during the addition. Optionally, the liquid is a muscle cell culture medium, and / or a cell suspension of the frozen muscle cells after thawing.

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