Cell freezing medium and application thereof

Abstract

The invention relates to a cell cryopreservation solution which is suitable for cryopreservation of animal cells. The compound electrolyte comprises a compound electrolyte solution, a human serum albumin solution and dimethyl sulfoxide (DMSO). The cell cryopreservation liquid has good biocompatibility, the survival rate and functionality of cells under the low-temperature condition are remarkably improved, and the cell cryopreservation liquid is particularly suitable for cryopreservation and recovery of immune cells and stem cells (such as natural killer cells, T cells and mesenchymal stem cells). The cryopreservation liquid not only can be used for in-vitro cryopreservation of cells, but also can be directly applied to a clinical tested object together with the cryopreserved cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a cell cryopreservation solution. Background Technology

[0002] Cell cryopreservation technology plays a vital role in the biomedical field, especially in cell therapy and regenerative medicine. With the development of technologies such as cell therapy, stem cell transplantation, and immunotherapy, long-term cell preservation has become a crucial step in scientific research and clinical applications. The main purpose of cell cryopreservation is to minimize cellular metabolic activity under ultra-low temperature conditions to maintain cell viability and function, ensuring that cells remain clinically viable even after long-term storage.

[0003] Currently, widely used cell cryopreservation media typically contain cryoprotectants containing dimethyl sulfoxide (DMSO). DMSO is a commonly used permeable cryoprotectant that effectively prevents damage to cell membranes from ice crystals formed during freezing. However, the cytotoxicity of DMSO and its effects on certain sensitive cell types (such as immune cells and stem cells) remain a concern. Furthermore, existing cryopreservation media formulations often fail to meet the specific needs of different cell types, exhibiting deficiencies in aspects such as cell viability after freezing, cell functionality, and cell activity after thawing.

[0004] In the development of cell cryopreservation technology, although various cryopreservation media have been developed for cell cryopreservation, many technical bottlenecks still exist in improving cryopreservation efficiency, reducing cell damage, and adapting to the cryopreservation needs of different types of cells. In particular, for highly sensitive cell types such as immune cells and stem cells, existing cell cryopreservation solutions have limited effectiveness in maintaining cell viability, reducing cell damage during freezing, and improving cell functionality after thawing.

[0005] Compound electrolyte injection and human serum albumin, as basic components of cell cryopreservation solutions, possess good biocompatibility and stability. Compound electrolyte injection maintains osmotic pressure balance in cells, while human serum albumin provides a stable growth environment and reduces mechanical damage during cryopreservation. However, how to rationally combine these components to meet the freezing requirements of different cell types and minimize adverse reactions during the freezing process remains a significant challenge in the field. Summary of the Invention

[0006] Based on this, the present invention proposes an optimized culture medium composition for cell cryopreservation, aiming to overcome the shortcomings of the prior art, especially for the cryopreservation needs of sensitive cells such as immune cells and stem cells, and to provide a cryopreservation solution formulation that can significantly improve cell viability and functionality, so as to better meet the needs of cell therapy and other biomedical applications.

[0007] The first aspect of this invention provides a cell cryopreservation solution and its application, particularly a cryopreservation solution suitable for the cryopreservation of animal cells such as immune cells and stem cells. This solution effectively improves cell survival rates during low-temperature storage and is widely applicable to long-term cell preservation and clinical applications. The cell cryopreservation solution of this invention comprises: a compound electrolyte solution, a human serum albumin solution, and dimethyl sulfoxide (DMSO); the volume percentage content of each component is as follows: compound electrolyte solution 20%–65%, human serum albumin solution 20%–70%, and dimethyl sulfoxide (DMSO) 4%–8%.

[0008] Other aspects of the present invention also provide kits comprising the components of the cryopreservation solution, immune cell and stem cell preparations comprising the cryopreservation solution, and their use in the prevention and / or treatment of cancer, infectious diseases, degenerative diseases, and immune disorders.

[0009] Compared with the prior art, the beneficial technical effects of the present invention are as follows: When the cryopreservation solution of the present invention is applied to the cryopreservation of sensitive cells such as immune cells and stem cells, it can significantly improve the cell survival rate and the proliferation capacity after thawing, and greatly reduce the cost. It is particularly suitable for the cryopreservation of immune cells or stem cells produced on a large scale. Attached Figure Description

[0010] Figure 1 Cell survival rate after resuscitation.

[0011] Figure 2 Cell fold expansion after resuscitation. Detailed Implementation

[0012] The compound electrolyte solution described in this invention, commonly referred to as compound electrolyte injection, has the following main components: 5.26g sodium chloride, 5.02g sodium gluconate, 3.68g sodium acetate, 0.37g potassium chloride, and 0.30g magnesium chloride per 1000ml.

[0013] The compound electrolyte solution of the present invention has a content of 20% to 65% by volume percentage, preferably 20% to 50%; in some embodiments, the content is 45%; in some embodiments, the content is 25%; and in some embodiments, the content is 22.5%.

[0014] The compound electrolyte solution serves to stabilize cell osmotic pressure, maintain cell morphology, and act as a diluent during cell infusion.

[0015] The human serum albumin solution described in this invention is a 20% human serum albumin solution, for example, a commercially available 200 g / L or 10 g / 50 mL human serum albumin solution. In this invention, the human serum albumin is used as a non-permeable cryoprotectant, reducing the content of free water in the solution and decreasing the formation of ice crystals; simultaneously, due to its large molecular weight, it reduces the electrolyte concentration in the solution, thereby mitigating solute damage and significantly improving cell viability.

[0016] The human serum albumin solution of the present invention has a volume percentage content of 20% to 70%, preferably 30% to 70%; in some embodiments, the content is 30%; in some embodiments, the content is 40%; in some embodiments, the content is 50%; in some embodiments, the content is 60%; and in some embodiments, the content is 70%.

[0017] The DMSO described in this invention is clinical-grade or pharmaceutical-grade DMSO. Cells cryopreserved using the cryopreservation solution of this invention can be directly infused into test subjects after being thawed to room temperature. In this invention, the clinical-grade DMSO is used as a permeable cryoprotectant, which can lower the freezing point of cells, reduce the formation of ice crystals during cryopreservation, and thus reduce damage to cells.

[0018] The DMSO content of the present invention is 4%-8% by volume percentage; preferably, it is 5%-7.5%; in some embodiments, it is 5%; in some embodiments, it is 7.5%.

[0019] In one specific embodiment, the volume percentage content of each component is as follows: 45% compound electrolyte solution, 50% human serum albumin, and 5% dimethyl sulfoxide (DMSO).

[0020] In one specific embodiment, the volume percentage content of each component is as follows: 25% compound electrolyte solution, 70% human serum albumin, and 5% dimethyl sulfoxide (DMSO).

[0021] In one specific embodiment, the volume percentage content of each component is as follows: compound electrolyte injection 22.5%, human serum albumin 70%, and dimethyl sulfoxide (DMSO) 7.5%.

[0022] The present invention does not have any special limitation on the source of the composition of the above solution; conventional commercially available products known to those skilled in the art can be used.

[0023] The cryopreservation solution can be used for the cryopreservation of various animal cells, especially sensitive cells such as immune cells and stem cells (such as mesenchymal stem cells, bone marrow stem cells, human neural stem cells and oral mucosal lamina propria stem cells).

[0024] Immune cells can be one or more of the following: T cells, B cells, NK cells, macrophages, dendritic cells, etc. In some embodiments, the immune cells are NK cells.

[0025] Immune cells and stem cells can be wild-type immune cells or stem cells, or engineered immune cells or stem cells, such as CAR-T, CAR-NK, TCR-T, etc.

[0026] The cryopreservation solution of this invention is suitable for cryopreservation of cells within a wide range of cell concentrations, with a cryopreservation density of 1E7-1E8 cells / mL, preferably 3E7-5E7 cells / mL. By optimizing the component ratios of the cryopreservation solution, this invention significantly improves cell viability and functionality during cryopreservation.

[0027] To facilitate use, this invention also provides a kit comprising individually packaged compound electrolyte solution, human serum albumin solution, dimethyl sulfoxide (DMSO), and instructions containing the proportions of the three components of the cryopreservation solution. This kit not only meets the clinical needs of cell therapy but also supports basic cell research and drug development.

[0028] In some embodiments, the present invention provides an immune cell preparation comprising the above-described cryopreservation solution and immune cells.

[0029] In some embodiments, the present invention provides a stem cell preparation comprising: the above-described cryopreservation solution and stem cells.

[0030] In some embodiments, the present invention provides the use of the aforementioned immune cell preparations or stem cell preparations in the prevention and / or treatment of cancer, infectious diseases, degenerative diseases, and immune disorders; wherein the cancers include solid tumors and hematologic malignancies; and the immune disorders are selected from: graft-versus-host disease, transplant rejection, autoimmune diseases, chronic inflammatory diseases, inflammatory pain, neuropathic pain, and chronic obstructive pulmonary disease (COPD).

[0031] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0032] Example

[0033] 1. Preliminary optimization of NK cell cryopreservation and cryopreservation solution formulation

[0034] NK cell culture: CD3-positive T cells were removed by PBMC sorting, and the cell density was adjusted to 1.5E6 / mL. Inactivated feeder cells were added at a 1:1 ratio and mixed, then incubated at 37°C in a 5% CO2 incubator. Culture medium was added (0.8-1E6 Cell / mL) according to the color of the cell suspension or the cell count. Cell counting was performed on days 5-7, and culture medium was added to maintain an NK cell concentration of 1E6 Cell / mL, with 5% plasma added. Observation or counting was performed daily on days 8-10, with 1% plasma added, and culture medium added to maintain a cell concentration slightly below 1E6 Cell / mL. After 10-20 days of expansion, the cells were washed and cryopreserved.

[0035] Preparation process of cryopreservation solution:

[0036] Eight stock samples of cryopreservation solutions were prepared (Table 1). Human serum albumin (200 g / L) was purchased from Grifols Therapeutics LLC; human AB serum was purchased from Beijing Novobio; dextran 40 (100 g / L) and glucose (50 g / L) were purchased from Ecosai Biotechnology Co., Ltd.; DMSO was purchased from OriGen; PBS was purchased from Corning; and compound electrolytes were purchased from Shanghai Baxter Medical Supplies Co., Ltd. The percentages of each component in the example table are % (v / v). The above reagents were mixed to the required concentrations to prepare cryopreservation solutions of the desired working concentrations for later use.

[0037] Table 1 Formulations of different cryopreservation solutions

[0038]

[0039]

[0040] The cells were cryopreserved using eight different cryopreservation solutions as shown in Table 1, with the same cell density of 2.7E7 cells / mL in each group. The cryopreserved cells were then thawed and cultured in NK medium. The results showed that at 48 hours post-thaw, cryopreservation solutions No. 6, 7, and 8 exhibited the highest cell viability, indicating that they better protected the cells from cryopreservation-induced damage. Furthermore, at 2 and 4 days post-thaw, cryopreservation solutions No. 6, 7, and 8 showed the highest cell count and cell viability, indicating that they better maintained the proliferative potential of the cryopreserved cells after thawing.

[0041] Table 2. Survival rate (%) after thawing for different cryopreservation solution formulations

[0042]

[0043] Table 3. NK cell expansion folds after thawing with different cryopreservation solution formulations

[0044]

[0045]

[0046] 2. NK cell cryopreservation and cryopreservation solution formulations have been further optimized.

[0047] NK cell culture: CD3-positive T cells were removed by PBMC sorting, and the cell density was adjusted to 1.5E6 / mL. Inactivated feeder cells were added at a 1:1 ratio and mixed, then incubated at 37°C in a 5% CO2 incubator. Culture medium was added (0.8-1E6 Cell / mL) according to the color of the cell suspension or the cell count. Cell counting was performed on days 5-7, and culture medium was added to maintain an NK cell concentration of 1E6 Cell / mL, with 5% plasma added. Observation or counting was performed daily on days 8-10, with 1% plasma added, and culture medium added to maintain a cell concentration slightly below 1E6 Cell / mL. After 10-20 days of expansion, the cells were washed and cryopreserved.

[0048] Preparation process of cryopreservation solution:

[0049] Fourteen stock samples of cryopreservation solutions were prepared (Table 4). Human serum albumin (200 g / L) was purchased from Grifols Therapeutics LLC (HSA); DMSO was purchased from OriGen; and compound electrolytes were purchased from Shanghai Baxter Medical Supplies Co., Ltd. The percentages of each component in the example table are % (v / v). The above reagents were mixed to the required concentrations to prepare cryopreservation solutions of the desired working concentrations for later use.

[0050] Table 4 Formulations of different cryopreservation solutions

[0051]

[0052]

[0053] The cells were cryopreserved using 14 different cryopreservation solutions as shown in Table 4, with the same cell density of 3E7 cells / mL in each group. The cryopreserved cells were then thawed and cultured in NK medium. Results showed that on day 3 after thaw, cryopreservation solution #1 exhibited the highest cell count and viability. On days 3 and 4 after thaw, the groups with the best viability were, in descending order: cryopreservation solutions #1, #2, #5, #7, and #11; the cryopreservation solutions with the most significant cell proliferation were, in descending order: cryopreservation solutions #1, #3, #4, and #7. Among these, with human serum albumin remaining constant, the 5% DMSO group was generally superior to the 7.5% DMSO group. The last two groups, 3% and 10% DMSO concentrations, showed the worst proliferation and viability. Results are shown in Tables 5 and 6. Figure 1 , Figure 2 .

[0054] Table 5. Survival rates after thawing for different cryopreservation solution formulations

[0055]

[0056]

[0057] Table 6. NK cell amplification folds after thawing using different cryopreservation solution formulations.

[0058] cryopreservation solution number D0 D3 D4 D6 1 1 2.86 7.82 18.34 2 1 2.23 6.30 17.50 3 1 2.38 6.95 19.06 4 1 2.38 6.64 18.49 5 1 2.13 6.22 19.45 6 1 2.15 6.24 18.83 7 1 2.65 6.72 16.97 8 1 2.21 6.36 18.60 9 1 1.94 5.74 16.12 10 1 1.99 6.22 14.39 11 1 1.90 5.93 14.77 12 1 1.85 4.87 16.11 13 1 1.42 3.10 6.35 14 1 1.05 2.19 6.48

[0059] 3. Production scale (WAVE reactor) for NK cell culture and cryopreservation

[0060] To meet the needs of large-scale NK cell culture and cryopreservation, and to explore closed systems for future production, this embodiment tested NK cells expanded under large-scale wave reactor (WAVE) culture conditions, and then cryopreserved after harvest using Flex Pro, thereby testing the effect of cryopreservation solution on NK cell cryopreservation.

[0061] NK cell culture: CD3-positive T cells were removed by PBMC sorting, and inactivated feeder cells were added and mixed before being cultured in a 37°C, 5% CO2 incubator. Culture medium (0.8-1.0 × 1E6 Cell / mL) was added based on the color of the cell suspension or the cell count. Cell counts were performed on days 5-7, and culture medium was added to maintain an NK cell concentration of 1E6 Cell / mL. Platelet lysate was added at 5%. When the cell count reached 1E9, the cells were transferred to a wave reactor and expanded for 3-8 days to a volume of 5L. Cells were harvested by manual centrifugation or by direct washing with Flex Pro, followed by cryopreservation.

[0062] Preparation process of cryopreservation solution:

[0063] The components of the cryopreservation solution are shown in Table 7. Human serum albumin (200 g / L) was purchased from Grifols Therapeutics LLC (HSA); DMSO was purchased from OriGen; and compound electrolytes were purchased from Shanghai Baxter Medical Supplies Co., Ltd. The percentages of each component in the example table are % (v / v). Mix the above reagents to the required concentration to prepare the cryopreservation solution at the desired working concentration for later use.

[0064] Table 7. Frozen solution formulation

[0065]

[0066] The NK cells described above were cryopreserved using the cryopreservation solutions shown in Table 5, with cryopreserved cell densities of 3.3E7 and 1E8 / mL, respectively. The cryopreserved cells were then thawed and cultured in NK medium. Results showed that on days 3, 5, 7, and 10 after thaw, the cell viability recovered under all cryopreservation conditions and maintained continuous expansion capacity. The differences in cell viability after thaw were small among different cryopreservation densities, indicating that the cryopreservation solutions could meet the cryopreservation requirements for large-scale NK cell expansion. The results are shown in Tables 8 and 9.

[0067] Table 8. Viability of cells harvested and cryopreserved under different conditions after thawing

[0068]

[0069] Table 9. Fold-up of cells harvested and cryopreserved under different conditions after thawing.

[0070]

[0071] 3. Cryopreservation of CAR-T cells

[0072] CD3 cells were obtained by PBMC sorting and resuspended in X VIVO-15 medium containing IL2 and serum. The cells were then added to wells coated with CD3 and CD28 antibodies and incubated at 37°C in a 5% CO2 incubator. After 24 hours, lentivirus was added at a specific MOI. After transduction for 24 hours, the medium was replaced with fresh medium. Cell density (0.8-1.0 × E6 Cell / mL) was adjusted by adding more medium based on the color of the cell suspension or cell count. After 7-10 days of expansion, the cells were washed and cryopreserved.

[0073] Preparation process of cryopreservation solution:

[0074] Prepare cryopreservation solutions according to the formulations in Table 10, and prepare stock samples of three cryopreservation solutions. Human serum albumin (200 g / L) was purchased from Grifols Therapeutics LLC (HSA); DMSO was purchased from OriGen; compound electrolytes and physiological saline were purchased from Shanghai Baxter Medical Supplies Co., Ltd. CS10 was purchased from STEMCELL; the percentages of various components in the examples table are % (v / v). Mix the above reagents to the required concentrations to prepare cryopreservation solutions of the required working concentrations for later use.

[0075] Table 10 Formulations of Different Cryopreservation Solutions

[0076]

[0077] The CAR-T cells were cryopreserved using the three different cryopreservation solutions shown in Table 7. The cell density was the same in all groups, 5E7 cells / mL. The cryopreserved cells were then thawed, and the results showed little difference between the groups (Table 11).

[0078] Currently, there are many cryopreservation solutions using CS10-related components in clinical CAR-T therapy, which are relatively expensive. The viability of the No. 1 cryopreservation solution prepared in this experiment is similar to that of the cryopreservation solution containing CS10 components, but the cost of the No. 1 cryopreservation solution is significantly lower than that of the cryopreservation solution containing CS10 components, which is beneficial to reducing the cost for CAR-T cell therapy companies.

[0079] Table 11 Viability of CAR-T cells after thawing with different cryopreservation solutions

[0080] cryopreservation solution number 1 2 3 Survival rate 82.64％ 81％ 85.43％

[0081] 4. Cryopreservation of the tumor cell line K562

[0082] The cryopreserved human chronic myeloid leukemia cell line K562 was revived and cultured in 1640 medium containing 10% FBS. The cell density (0.8-1.0×E6 Cell / mL) was adjusted by adding culture medium according to the color of the cell suspension or the number of cells. After passage for 4-7 days, the cells were collected, washed, and cryopreserved.

[0083] Preparation process of cryopreservation solution:

[0084] Prepare cryopreservation solutions according to the formulations in Table 12, and prepare stock samples of three cryopreservation solutions. Human serum albumin (200 g / L) was purchased from Grifols Therapeutics LLC (HSA); DMSO was purchased from OriGen; compound electrolytes and physiological saline were purchased from Shanghai Baxter Medical Supplies Co., Ltd. CS10 was purchased from STEMCELL; the percentages of various components in the examples table are % (v / v). Mix the above reagents to the required concentrations to prepare cryopreservation solutions of the required working concentrations for later use.

[0085] Table 12 Formulations of different cryopreservation solutions

[0086]

[0087] The K562 cells were cryopreserved using the three different cryopreservation solutions shown in Table 12. The cell density was the same in all groups, at 4E7 cells / mL. The cryopreserved cells were then thawed, and the results showed little difference between the groups (Table 13). However, the cost of cryopreservation solution No. 1 was significantly lower than that of cryopreservation solution containing CS10, and it can replace CS10 cryopreservation solution in use.

[0088] Table 13 Viability of K562 cells after thawing with different cryopreservation solutions

[0089] cryopreservation solution number 1 2 3 Survival rate 89.68％ 87.9％ 88.53％

[0090] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A cell cryopreservation solution, characterized in that, The compound electrolyte solution, the human serum albumin solution and the dimethyl sulfoxide (DMSO) have volume percentage contents of 20-65%, 20-70% and 4-8%, respectively. The volume percentage content of the compound electrolyte solution is 20-50%.

2. The cryo-protectant solution of claim 1, wherein, The volume percentage content of the human serum albumin is 30-70%.

3. The cryo-protectant solution of claim 1 or 2, wherein, The volume percentage content of the dimethyl sulfoxide (DMSO) is 5-7.5%.

4. The cryo-protectant solution of any one of claims 1 to 3, wherein, The volume percentage contents of the compound electrolyte solution, the human serum albumin and the dimethyl sulfoxide (DMSO) are 45%, 50% and 5%, respectively; alternatively, the volume percentage contents of the compound electrolyte solution, the human serum albumin and the dimethyl sulfoxide (DMSO) are 25%, 70% and 5%, respectively; alternatively, the volume percentage contents of the compound electrolyte solution, the human serum albumin and the dimethyl sulfoxide (DMSO) are 22.5%, 70% and 7.5%, respectively.

5. The cryo-protectant solution of any one of claims 1 to 4, wherein, The cell is an animal cell; preferably, the animal cell is an immune cell. Preferably, the immune cell is a natural killer cell or a T cell; preferably, the animal cell is a stem cell, preferably, the stem cell comprises a bone marrow stem cell, a mesenchymal stem cell, a human neural stem cell or a buccal mucosa lamina propria stem cell.

6. The cryo-protectant solution of any one of claims 1 to 5, wherein, The freezing density of the cell is 3E7-5E7 cells / mL.

7. The cryopreservation solution of any one of claims 1-6, wherein, 8. A kit comprising a compound electrolyte solution, a human serum albumin solution and dimethyl sulfoxide (DMSO) packaged separately and a package insert containing information on the three-component proportioning of the cryopreservation solution according to any one of claims 1-7. The cryopreservation solution and the immune cell according to any one of claims 1-7.

9. An immune cell preparation comprising: The cryopreservation solution and the stem cell according to any one of claims 1-7.

10. A stem cell preparation comprising: ​

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