Serum-free, animal-source-component-free and DMSO-free cell freezing medium as well as preparation method and application thereof

Abstract

The invention relates to a serum-free, animal-source-component-free and DMSO-free cell freezing medium and a preparation method and application thereof, and relates to the technical field of cell and biological engineering, and the serum-free, animal-source-component-free and DMSO-free cell freezing medium comprises the following components (according to the concentration range): 10-50mM of lacturonic acid; 50 to 200 mM of raffinose; 2-8% (w / v) of hydroxyethyl starch; 1 to 5 mM of monopotassium phosphate; 0.5 to 2 mM of magnesium sulfate; 1 to 5 mM of glutathione; allopurinol: 0.1 to 1 mM; 0.5 to 2 mM of adenosine; insulin: 0.1 to 1 [mu] g / mL; 50 to 150 mM of trehalose; proline: 20 to 100 mM; 10-20% (v / v) of glycerol; and a pH buffer. The application does not contain serum, animal-derived substances and DMSO, and avoids the problems of toxicity, immunogenicity and ethics.

Description

Technical Field

[0001] The present application relates to the field of cell and bioengineering technology, and in particular to a serum-free, animal-derived component-free, DMSO-free cell freezing solution, and a preparation method and application thereof. Background Art

[0002] Cell freezing technology is a key link in the biomedicine field and is used to preserve cell activity and function for a long time. Traditional freezing fluids usually rely on serum (such as fetal bovine serum), animal-derived components (such as albumin) and dimethyl sulfoxide (DMSO) as core protective agents. However, these components have significant defects. Serum and animal components may carry pathogens (such as viruses, mycoplasma), causing immune rejection or quality fluctuations between batches, and do not meet the ethical and safety requirements of clinical applications. DMSO is cytotoxic and may cause abnormal cell function or differentiation changes. Additional washing steps are required after thawing, which increases the complexity of the operation. Existing serum-free formulas mostly rely on high molecular polymers or single sugars, which have limited protective effects on complex cell types (such as stem cells and primary cells), and lack antioxidant and energy metabolism support.

[0003] Therefore, there is an urgent need to develop a safe, efficient and completely serum-free, animal component-free and DMSO-free freezing solution to meet the high standards required in fields such as regenerative medicine, cell therapy and biopharmaceuticals. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a serum-free, animal-derived component-free, DMSO-free cell freezing solution and its preparation method and application, which solves the technical problem.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A serum-free, animal-derived component-free, DMSO-free cell freezing solution comprising the following components (in concentration range): lactobionic acid: 10-50 mM; raffinose: 50-200 mM; hydroxyethyl starch: 2-8% (w / v); potassium dihydrogen phosphate: 1-5 mM; magnesium sulfate: 0.5-2 mM; glutathione: 1-5 mM; allopurinol: 0.1-1 mM; adenosine: 0.5-2 mM; insulin: 0.1-1 μg / mL; trehalose: 50-150 mM; proline: 20-100 mM; glycerol: 10-20% (v / v).

[0007] Furthermore, a pH buffer is included to adjust the pH value to 7.2-7.4.

[0008] The method for preparing a serum-free, animal-derived component-free, and DMSO-free cell freezing solution as described above comprises:

[0009] S1: Take 50-80% of the final volume of PBS buffer (pH 7.2-7.4) and dissolve potassium dihydrogen phosphate and magnesium sulfate in sequence;

[0010] S2: Maintain a water bath at 25-37°C and add lactobionic acid, raffinose, trehalose, and proline in sequence;

[0011] S3: Add hydroxyethyl starch and stir in a 50-60°C water bath for 1-2 hours until completely dissolved;

[0012] S4: After the solution is cooled to below 25°C, glycerol, glutathione, allopurinol and adenosine are added in sequence;

[0013] S5: Finally, insulin was added and PBS buffer was added to the final volume;

[0014] S6: Sterilize by positive pressure filtration through a 0.22 μm PVDF filter membrane, and store at 2-8°C after aliquoting.

[0015] Furthermore, in said S1:

[0016] The amount of potassium dihydrogen phosphate added is 1-5mM, and the amount of magnesium sulfate added is 0.5-2mM;

[0017] Maintain magnetic stirring (200-400 rpm) for 20-30 minutes while dissolving.

[0018] Furthermore, in said S2:

[0019] Each sugar was added according to a concentration gradient: lactobionic acid (10-50 mM) was added first and stirred for 10 min;

[0020] Raffinose (50-200 mM) was then added, and finally trehalose (50-150 mM) and proline (20-100 mM) were added.

[0021] Furthermore, in S3:

[0022] The amount of hydroxyethyl starch added is 2-8% (w / v);

[0023] The pH value was maintained in the range of 7.2-7.4 during the dissolution process.

[0024] Furthermore, in said S4:

[0025] The interval between adding each component is 5-10 minutes;

[0026] Glutathione should be prepared and used immediately, and added under light-protected conditions.

[0027] Furthermore, in said S5:

[0028] Insulin was added as follows: first diluted with PBS buffer to a working concentration of 0.1-1 μg / mL;

[0029] Then slowly add it dropwise to the solution for no less than 5 minutes.

[0030] Furthermore, in said S6:

[0031] The filtration pressure is controlled at 0.1-0.3MPa;

[0032] After subpackaging, seal and store in nitrogen atmosphere.

[0033] The serum-free, animal-derived component-free, DMSO-free cell freezing solution described above is used to freeze mammalian cells, including stem cells, primary cells, and immune cells. The cells are suspended in the freezing solution and programmed to cool to below -80°C for long-term storage.

[0034] In summary, the present application includes at least one of the following serum-free, animal-derived component-free, and DMSO-free cell freezing solutions, as well as its preparation method and application beneficial technical effects:

[0035] Eliminate risky ingredients: does not contain serum, animal-derived substances and DMSO, avoiding toxicity, immunogenicity and ethical issues.

[0036] A multi-mechanism synergistic protection system: Lactobionic acid and raffinose create an osmotic pressure gradient, balancing water migration inside and outside the cell and preventing ice crystal damage. Hydroxyethyl starch replaces traditional polymers, inhibiting ice crystal formation through vitrification. Potassium dihydrogen phosphate and magnesium sulfate maintain intracellular ion homeostasis and prevent pH fluctuations. Glutathione and allopurinol synergistically scavenge free radicals and reduce oxidative stress damage. Adenosine provides a precursor for ATP synthesis, and insulin promotes cellular metabolic activity. Trehalose and proline stabilize cell membranes and protein structures through a water-displacement effect.

[0037] Broad-spectrum applicability: Applicable to a variety of cell types (such as mesenchymal stem cells, immune cells, and primary cells), with a survival rate of ≥90% after cryopreservation and no significant decrease in cell function (such as proliferation and differentiation). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This application mainly provides a comparison of the resuscitation rate of cryopreserved mesenchymal stem cells;

[0039] Figure 2 This application mainly provides a comparison of the proliferation of cryopreserved mesenchymal stem cells after inoculation and thawing at various time points between the experimental group and the control group;

[0040] Figure 3This application mainly provides a comparison of the resuscitation rate of PBMCs frozen at different densities in the freezing solution of the experimental group and the control group;

[0041] Figure 4 This application mainly provides a comparison of the resuscitation rate in the cryopreservation solution of the experimental group and the control group. DETAILED DESCRIPTION

[0042] The technical solutions of the present invention are described in detail below in conjunction with specific embodiments and their accompanying drawings. The embodiments described herein are specific embodiments of the present invention and are used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary and should not be understood as limiting the embodiments of the present invention and the scope of protection of the present invention. In addition to the embodiments described herein, those skilled in the art can also adopt other obvious technical solutions based on the contents disclosed in the claims of this application and its specification, including technical solutions that adopt any obvious replacements and modifications to the embodiments described herein.

[0043] The chemical reagents used in the examples and comparative examples of the present invention are all commercially available products without further purification or treatment.

[0044] The following is combined with Figure 1-4 This application is described in further detail.

[0045] A serum-free, animal-derived component-free, DMSO-free cell freezing solution comprises the following components (in concentration range): lactobionic acid: 10-50 mM; raffinose: 50-200 mM; hydroxyethyl starch: 2-8% (w / v); potassium dihydrogen phosphate: 1-5 mM; magnesium sulfate: 0.5-2 mM; glutathione: 1-5 mM; allopurinol: 0.1-1 mM; adenosine: 0.5-2 mM; insulin: 0.1-1 μg / mL; trehalose: 50-150 mM; proline: 20-100 mM; glycerol: 10-20% (v / v); and contains a pH buffer, the pH value of which is adjusted to 7.2-7.4.

[0046] Example 1

[0047] This example provides a method for preparing a serum-free, animal-derived component-free, and DMSO-free cell freezing solution. The specific preparation method includes:

[0048] S1: Dissolve 1 mM potassium dihydrogen phosphate and 0.5 mM magnesium sulfate in 50-80% of the final volume of PBS buffer (pH 7.2-7.4) in this solution, stirring magnetically (200-400 rpm) for 20-30 minutes.

[0049] S2: Maintain a 25-37°C water bath, add 10 mM lactobionic acid, stir for 10 minutes, then add 50 mM raffinose, 50 mM trehalose, and 20 mM proline;

[0050] S3: Add 2% (w / v) hydroxyethyl starch and stir in a 50-60°C water bath for 1-2 hours until completely dissolved. Maintain the pH value within the range of 7.2-7.4 during the dissolution process.

[0051] S4: After the solution is cooled to below 25°C, 10% (v / v) glycerol, 1 mM glutathione, 0.1 mM allopurinol, and 0.1 mM adenosine are added in sequence; each component is added 5-10 minutes apart; glutathione is prepared immediately before use and added in the dark.

[0052] S5: First dilute insulin with PBS buffer to a working concentration of 0.1 μg / mL; then slowly add it dropwise to the solution for at least 5 minutes, and add PBS buffer to the final volume;

[0053] S6: Sterilize by positive pressure filtration through a 0.22 μm PVDF filter membrane with the filtration pressure controlled at 0.3 MPa. After aliquoting, seal with nitrogen and store at 2-8°C.

[0054] Example 2

[0055] This example provides a method for preparing a serum-free, animal-derived component-free, and DMSO-free cell freezing solution. The specific preparation method includes:

[0056] S1: Dissolve 5 mM potassium dihydrogen phosphate and 2 mM magnesium sulfate in 50-80% of the final volume of PBS buffer (pH 7.2-7.4) in this solution, stirring magnetically (200-400 rpm) for 20-30 minutes.

[0057] S2: Maintain a 25-37°C water bath, add 50 mM lactobionic acid, stir for 10 minutes, then add 200 mM raffinose, 150 mM trehalose, and 100 mM proline;

[0058] S3: Add 8% (w / v) hydroxyethyl starch and stir in a 50-60°C water bath for 1-2 hours until completely dissolved. Maintain the pH value within the range of 7.2-7.4 during the dissolution process.

[0059] S4: After the solution is cooled to below 25°C, add 20% (v / v) glycerol, 5 mM glutathione, 1 mM allopurinol, and 0.5 mM adenosine in sequence; add each component 5-10 minutes apart; prepare glutathione immediately before use and add in the dark.

[0060] S5: First dilute insulin with PBS buffer to a working concentration of 0.5 μg / mL; then slowly add it dropwise to the solution for at least 5 minutes, and add PBS buffer to the final volume;

[0061] S6: Sterilize by positive pressure filtration through a 0.22 μm PVDF filter membrane with the filtration pressure controlled at 0.3 MPa. After aliquoting, seal with nitrogen and store at 2-8°C.

[0062] Example 3

[0063] This example provides a method for preparing a serum-free, animal-derived component-free, and DMSO-free cell freezing solution. The specific preparation method includes:

[0064] S1: Dissolve 3 mM potassium dihydrogen phosphate and 1 mM magnesium sulfate in 50-80% of the final volume of PBS buffer (pH 7.2-7.4) in this solution, stirring magnetically (200-400 rpm) for 20-30 minutes.

[0065] S2: Maintain a 25-37°C water bath, add 30 mM lactobionic acid, stir for 10 minutes, then add 100 mM raffinose, 100 mM trehalose, and 50 mM proline;

[0066] S3: Add 5% (w / v) hydroxyethyl starch and stir in a 50-60°C water bath for 1-2 hours until completely dissolved. Maintain the pH value within the range of 7.2-7.4 during the dissolution process.

[0067] S4: After the solution is cooled to below 25°C, 15% (v / v) glycerol, 3 mM glutathione, 0.5 mM allopurinol, and 1 mM adenosine are added in sequence; each component is added 5-10 minutes apart; glutathione is prepared immediately before use and added in the dark.

[0068] S5: First dilute insulin with PBS buffer to a working concentration of 0.5 μg / mL; then slowly add it dropwise to the solution for at least 5 minutes, and add PBS buffer to the final volume;

[0069] S6: Sterilize by positive pressure filtration through a 0.22 μm PVDF filter membrane with the filtration pressure controlled at 0.3 MPa. After aliquoting, seal with nitrogen and store at 2-8°C.

[0070] Example 4

[0071] Evaluation of cryopreservation effects of human mesenchymal stem cells

[0072] S1: Rinse the fresh umbilical cord repeatedly with D-Hanks solution 3-5 times to remove surface blood. Cut the umbilical cord longitudinally, dissect the two arteries and one vein, remove the Wharton's jelly, and mince the Wharton's jelly until it becomes a paste.

[0073] S2: Add 0.1% (m / v) collagenase IV and digest at 37°C for 2-4 hours with shaking. Add an equal volume of complete medium to terminate the digestion and filter through a 200-mesh filter to remove undigested tissue.

[0074] S3: The filtrate was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded.

[0075] S4: Resuspend the cells in complete medium (DMEM + 10% FBS), inoculate into culture flasks, and culture at 37°C, 5% CO2. When the cell confluence reaches 80%-90%, subculture.

[0076] S5: After continuous passage to passage 5, harvest the mesenchymal stem cells. Resuspend the cells in the freezing solution prepared in Example 3 at a density of 1×10^7 / mL (experimental group) and aliquot into cryovials. Use conventional freezing solution (90% FBS + 10% DMSO) as a control. Cooling program: 4°C for 30 minutes → -20°C for 2 hours → -80°C overnight → storage in liquid nitrogen.

[0077] S6: Three tubes of cells were recovered in each of the experimental and control groups, thawed quickly in a 37°C water bath, centrifuged to remove the freezing solution, and resuspended in complete culture medium. Trypan blue staining was used to detect cell viability in both groups. Figure 1 The cell viability of the experimental group was (93±2)%, and that of the control group was (92±3)%. There was no statistical difference between the two groups (p>0.05).

[0078] S7: The two groups of recovered cells were resuspended in complete medium and inoculated into six-well cell culture plates at 37°C and 5% CO2. The proliferation of the two groups of cells was detected by CCK-8 method at 0, 2, 4, 6, and 8 days after inoculation. Figure 2 : There was no significant difference in the proliferation rate of revived mesenchymal stem cells between the experimental group and the control group at each time point after inoculation.

[0079] Example 5

[0080] Evaluation of cryopreservation effects of human peripheral blood mononuclear cells (PBMC)

[0081] S1: Prepare PBMCs: Take fresh anticoagulated human whole blood and dilute it with PBS at a 1:1 ratio. Add an appropriate amount of Ficoll separation buffer (GE Biosciences) to a sterile centrifuge tube and spread the diluted blood sample evenly on top of the separation buffer. Centrifuge at 800g for 30 minutes at room temperature. After centrifugation, discard the plasma layer and carefully aspirate the PBMC layer (i.e., the buffy coat layer) and transfer it to a 15 mL centrifuge tube. Centrifuge at 400g for 30 minutes at room temperature and discard the supernatant.

[0082] S2: Cryopreservation: Resuspend cells at varying densities (1×10⁷ / mL, 2×10⁷ / mL, 4×10⁷ / mL) in the cryopreservation solution prepared in Example 1 (experimental group) and dispense into cryovials. Use conventional cryopreservation solution (90% FBS + 10% DMSO) as a control. Cooling cycle: 4°C for 30 minutes → -20°C for 2 hours → -80°C overnight → storage in liquid nitrogen.

[0083] S3: Three tubes of cells were recovered in each of the experimental and control groups, thawed quickly in a 37°C water bath, centrifuged to remove the freezing solution, and resuspended in complete culture medium. Trypan blue staining was used to detect cell viability in both groups. Figure 2 : There was no significant difference in the resuscitation rate of PBMCs frozen at different densities in the freezing medium of the experimental group compared with that of the control group.

[0084] S4: Three tubes of cells were recovered in each of the experimental and control groups, thawed quickly in a 37°C water bath, centrifuged to remove the freezing solution, and resuspended in complete culture medium. Trypan blue staining was used to detect cell viability in both groups. Results are shown in the table. Figure 3 : The resuscitation rate of PBMCs frozen at different densities in the freezing medium of the experimental group had no significant difference compared with that of the control group

[0085] Example 6

[0086] Evaluation of cryopreservation efficacy of human T cells, natural killer (NK) cells, and monocytes

[0087] S1: Isolate human PBMC cells according to the method of Example 5

[0088] S2: T cells, NK cells, and monocytes were negatively sorted from PBMCs using the human T cell negative sorting kit (Miltenyi Biotec), NK cell negative sorting kit (Miltenyi Biotec), and monocyte negative sorting kit (Miltenyi Biotec) according to the manufacturer's instructions.

[0089] S3: Cryopreservation: Resuspend the cells in the cryopreservation solution prepared in Example 1 at a density of 1×10^7 / mL (experimental group) and dispense into cryopreservation tubes. Use conventional cryopreservation solution (90% FBS + 10% DMSO) as a control. Cooling program: 4°C for 30 minutes → -20°C for 2 hours → -80°C overnight → liquid nitrogen storage

[0090] S4: Three tubes of cells were recovered in each of the experimental and control groups, thawed quickly in a 37°C water bath, centrifuged to remove the freezing solution, and resuspended in complete culture medium. Trypan blue staining was used to detect cell viability in both groups. Results are shown in the table. Figure 4 There was no significant difference in the resuscitation rate of T cells, NK cells and monocytes in the cryopreservation fluid of the experimental group compared with that of the control group.

[0091] The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A serum-free, animal-derived component-free, DMSO-free cell freezing solution, characterized in that: Contains the following components (in concentration range): Lactobionic acid: 10-50 mM; Raffinose: 50-200 mM; Hydroxyethyl starch: 2-8% (w / v); Potassium dihydrogen phosphate: 1-5 mM; Magnesium sulfate: 0.5-2 mM; Glutathione: 1-5 mM; Allopurinol: 0.1-1 mM; Adenosine: 0.5-2 mM; Insulin: 0.1-1 μg / mL; Trehalose: 50-150 mM; Proline: 20-100 mM; Glycerol: 10-20% (v / v).

2. The serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 1, characterized in that: The product further comprises a pH buffer to adjust the pH value to 7.2-7.

4.

3. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 1-2, characterized in that: The preparation method comprises: S1: Take 50-80% of the final volume of PBS buffer (pH 7.2-7.4) and dissolve potassium dihydrogen phosphate and magnesium sulfate in sequence; S2: Maintain a water bath at 25-37°C and add lactobionic acid, raffinose, trehalose, and proline in sequence; S3: Add hydroxyethyl starch and stir in a 50-60°C water bath for 1-2 hours until completely dissolved; S4: After the solution is cooled to below 25°C, glycerol, glutathione, allopurinol and adenosine are added in sequence; S5: Finally, insulin was added and PBS buffer was added to the final volume; S6: Sterilize by positive pressure filtration through a 0.22 μm PVDF filter membrane, and store at 2-8°C after aliquoting.

4. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 3, characterized in that: In S1: The amount of potassium dihydrogen phosphate added is 1-5mM, and the amount of magnesium sulfate added is 0.5-2mM; Maintain magnetic stirring (200-400 rpm) for 20-30 minutes while dissolving.

5. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 3, characterized in that: In S2: Each sugar was added according to a concentration gradient: lactobionic acid (10-50 mM) was added first and stirred for 10 min; Raffinose (50-200 mM) was then added, and finally trehalose (50-150 mM) and proline (20-100 mM) were added.

6. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 3, characterized in that: In the S3: The amount of hydroxyethyl starch added is 2-8% (w / v); The pH value was maintained in the range of 7.2-7.4 during the dissolution process.

7. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 3, characterized in that: In said S4: The interval between adding each component is 5-10 minutes; Glutathione should be prepared and used immediately, and added under light-protected conditions.

8. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 3, characterized in that: In said S5: Insulin was added as follows: first diluted with PBS buffer to a working concentration of 0.1-1 μg / mL; Then slowly add it dropwise to the solution for no less than 5 minutes.

9. The method for preparing a serum-free, animal-derived component-free, DMSO-free cell freezing solution according to claim 3, characterized in that: In said S6: The filtration pressure is controlled at 0.1-0.3MPa; After subpackaging, seal and store in nitrogen atmosphere.

10. Use of the serum-free, animal-derived component-free, DMSO-free cell freezing solution according to any one of claims 1-2 in freezing mammalian cells, including stem cells, primary cells, and immune cells, by suspending the cells in the freezing solution and programmed cooling to below -80°C for long-term storage.

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