Cryopreservation liquid containing L-carnitine and application of cryopreservation liquid in stem cell cryopreservation

Abstract

The invention discloses a cryopreservation solution containing L-carnitine and application of the cryopreservation solution in stem cell cryopreservation, and particularly, the cryopreservation solution contains L-carnitine; the content of the L-carnitine in the freezing medium is 0.02 mol / L to 0.5 mol / L. The cryopreservation liquid can also comprise low-temperature protective agents such as polyhydric alcohol, sugar, sugar alcohol, DMSO (Dimethylsulfoxide), DMSO derivatives and the like. The invention provides an effective and safe low-temperature protective agent and an application method thereof for the cell freezing medium at the present stage.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to cryopreservation solutions containing L-carnitine and their application in stem cell cryopreservation. Background Technology

[0002] Deep cryopreservation is currently the only method that can maintain cell function and activity for extended periods. Its principle is to reduce intracellular biochemical reactions to extremely low levels using cryogenics, thus enabling storage for decades or even centuries. During deep cryopreservation, cells are primarily affected by two factors: intracellular ice damage caused by water crystallization and osmotic damage caused by increased solute concentration due to water crystallization in the solution. Ideal cryopreservation involves using cryoprotectants to inhibit extracellular water crystallization while simultaneously preventing intracellular ice formation.

[0003] Mesenchymal stem cells (MSCs) have become an important resource in the field of cell therapy due to their unique self-replication characteristics, immunomodulatory capabilities, and multi-lineage differentiation potential. MSCs can be obtained from dental pulp, bone marrow, adipose tissue, and umbilical cord tissue through specific isolation methods. Among these, umbilical cord-derived MSCs are one of the most important cell resources due to their abundant availability, non-invasive sample acquisition, and strong anti-inflammatory, immunomodulatory, and tissue repair capabilities.

[0004] Current cryopreservation solutions for mesenchymal stem cells may contain osmotic cryoprotectants such as Me2SO4, glycerol, ethylene glycol, and propylene glycol, supplemented with non-osmotic cryoprotectants such as glucose, trehalose, and dextran to control ice crystal formation inside and outside the cells and reduce osmotic damage. However, the corresponding cryopreservation results are still unsatisfactory. Therefore, there is an urgent need for a cryopreservation solution formulation with stronger protective capabilities to provide more stable and higher-quality cryopreservation results.

[0005] L-carnitine (LC) is an amino acid-like compound widely distributed in animal livers. LC promotes fatty acid degradation and lowers blood lipids; it is naturally non-toxic and biocompatible. This invention incorporates LC into the cryopreservation solution for mesenchymal stem cells to achieve better cryopreservation results. Summary of the Invention

[0006] To address the current problems in cryopreservation technology for mesenchymal stem cells, this invention applies L-carnitine to the cryopreservation solution for human mesenchymal stem cells to improve their cryopreservation effect. This invention provides a natural and highly effective protectant and its formulation for mesenchymal stem cells.

[0007] To solve the above-mentioned technical problems, the first aspect of the present invention provides a cryopreservation solution containing L-carnitine.

[0008] In some embodiments, the L-carnitine content in the cryopreservation solution is 0.02-0.5 mol / L.

[0009] In some embodiments, the L-carnitine content in the cryopreservation solution is 0.05-0.5 mol / L.

[0010] In some embodiments, the content of L-carnitine in the cryopreservation solution is 0.1-0.5 mol / L.

[0011] In some embodiments, the content of L-carnitine is 0.04-0.3 mol / L.

[0012] In some embodiments, the cryopreservation solution also contains a cryoprotectant.

[0013] In some embodiments, the cryoprotectant comprises one or more of polyols, sugars and sugar alcohols, and DMSO and its derivatives.

[0014] In some embodiments, the cryopreservation solution further comprises one or more of plasma proteins, culture medium, and buffer solutions.

[0015] In some embodiments, the polyol comprises one or more of ethylene glycol, glycerol, and propylene glycol.

[0016] In some embodiments, the sugar and sugar alcohol comprise one or more of glucose, trehalose, and dextran.

[0017] In this invention, dimethyl sulfoxide is equivalent to DMSO and Me2SO. In this invention, glycerol is glycerol.

[0018] In some embodiments, the plasma protein is albumin.

[0019] In some embodiments, the volume content of the cryoprotectant in the cryopreservation solution is 2-20%.

[0020] In some embodiments, the volume content of the cryoprotectant in the cryopreservation solution is 3-10%.

[0021] In some embodiments, the volume content of plasma proteins in the cryopreservation solution is 1-10%.

[0022] In some embodiments, the volume content of plasma proteins in the cryopreservation solution is 1.5-7.5%.

[0023] In some embodiments, the plasma protein is selected from albumin.

[0024] In some embodiments, the content of L-carnitine is 0.05-0.2 mol / L.

[0025] In some embodiments, the content of L-carnitine is 0.1-0.2 mol / L.

[0026] In some embodiments, the cryoprotectant comprises one or more of DMSO, ethylene glycol, 1,2-propylene glycol, and trehalose.

[0027] In some embodiments, the plasma protein is selected from human serum albumin.

[0028] In some embodiments, the cryopreservation solution is:

[0029] 2-20% Me2SO, 0.02-0.5 mol / L L-carnitine and 1-10% human serum albumin;

[0030] 2-20% ethylene glycol, 0.02-0.5 mol / L L-carnitine, and 1-10% human serum albumin;

[0031] 2-20% 1,2-propylene glycol, 0.02-0.5 mol / L L-carnitine, and 1-10% human serum albumin; or,

[0032] 2-20% glycerol, 0.02-0.5 mol / L L-carnitine and 1-10% human serum albumin.

[0033] In some embodiments, the cryopreservation solution is:

[0034] 4-10%, for example, 8% Me2SO, 0.03-0.5 mol / L L-carnitine, and 2-8% human serum albumin;

[0035] 4-10% ethylene glycol, 0.03-0.5 mol / L L-carnitine, and 2-8% human serum albumin;

[0036] 4-10% 1,2-propylene glycol, 0.03-0.5 mol / L L-carnitine, and 2-8% human serum albumin; or,

[0037] 4-10% glycerol, 0.03-0.5 mol / L L-carnitine and 2-8% human serum albumin.

[0038] In some embodiments, the cryopreservation solution is:

[0039] 4-10% Me2SO, 0.05-0.3 mol / L L-carnitine and 2-6% human serum albumin;

[0040] 4-10% ethylene glycol, 0.05-0.3 mol / L L-carnitine, and 2-6% human serum albumin; or,

[0041] 4-10% 1,2-propylene glycol, 0.05-0.3 mol / L L-carnitine, and 2-6% human serum albumin.

[0042] In this invention, the base solution for the cryopreservation solution can be physiological saline, culture medium, or buffer solution.

[0043] To solve the above-mentioned technical problems, a second aspect of the present invention provides a method for preparing a cryopreservation solution as described in the first aspect of the present invention, the method comprising preparing the components required for the cryopreservation solution and mixing the required components to obtain the cryopreservation solution.

[0044] A third aspect of this invention provides the application of L-carnitine in the preparation of stem cell cryopreservation solutions.

[0045] To address the aforementioned technical problems, the fourth aspect of this invention provides the application of L-carnitine or the cryopreservation solution as described in the first aspect of this invention in the cryopreservation of stem cells.

[0046] In some embodiments, the stem cells are mesenchymal stem cells.

[0047] In some specific embodiments, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells. The cryopreservation solution of the present invention also has similar effects on mesenchymal stem cells from other sources. The cryopreservation solution of the present invention is not limited to human umbilical cord mesenchymal stem cells.

[0048] To address the aforementioned technical problems, a fifth aspect of the present invention provides a method for cryopreservation of stem cells, the method comprising the following steps:

[0049] The stem cells are mixed with L-carnitine or a cryopreservation solution as described in the first aspect of the invention and then cooled to a biological sample storage temperature, for example, -70 to -90°C.

[0050] In some embodiments, the stem cells are mesenchymal stem cells.

[0051] In some embodiments, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0052] In some embodiments, the cooling rate is 0.5-10 °C / min.

[0053] In some embodiments, the volume / number ratio of the cryopreservation solution to the stem cells is 1 mL:(1×10⁻⁶) / mL. 6 -5×10 7 ) each, for example, 1 mL: (3 × 10 6 )indivual.

[0054] In some embodiments, after the cryopreservation solution is mixed with the stem cells, the method further includes a low-temperature equilibration step before cooling.

[0055] In some implementations, the low temperature is 0-20°C.

[0056] In some implementations, the low temperature is 0-10°C.

[0057] In some implementations, the low temperature is 0-4°C.

[0058] In some implementations, the balancing process takes 5-120 minutes.

[0059] In some implementations, the balancing process takes 10-50 minutes.

[0060] In some implementations, the method further includes: resuscitation and washing.

[0061] In some embodiments, the resuscitation temperature is 35-39°C, and the washing uses a buffer solution or culture medium, such as PBS solution.

[0062] In some implementations, the method of using L-carnitine in the cryopreservation of mesenchymal stem cells includes the following steps:

[0063] Human umbilical cord mesenchymal stem cells were isolated from umbilical cord tissue via adherent culture; complete culture medium was added, and the human umbilical cord mesenchymal stem cells were passaged; the human umbilical cord mesenchymal stem cells were digested and collected; the cells were resuspended in cryopreservation solution; the cell suspension was transferred to cryovials and placed in a programmed cooling box for cryopreservation at -80°C; the cryovials were placed in a 37°C constant temperature water bath to revive the cells; the cells were washed with PBS solution and resuspended in complete culture medium.

[0064] In some embodiments, the culture steps of the human umbilical cord tissue are as follows: fresh umbilical cord is cut into 5-10 mm tissue particles with surgical scissors, placed in a culture flask, and cultured using complete culture medium; when the primary cells that have crawled out of the tissue reach 90% confluence, TrypLE Express enzyme is added, and the cells are digested in a 37°C incubator for 3 minutes; the digested cells are centrifuged at 1000 rpm / min for 8 minutes, the supernatant is discarded, and the human umbilical cord mesenchymal stem cells are resuspended in complete culture medium;

[0065] The complete culture medium consists of: 94% (v / v) mesenchymal stem cell basal medium, 5% (v / v) ADV, and 1% penicillin-streptomycin mixture.

[0066] In some embodiments, the culture step of the human umbilical cord mesenchymal stem cells is as follows: culturing human umbilical cord mesenchymal stem cells using complete culture medium at a density of 10,000 cells / cm³. 2 .

[0067] In some embodiments, the step of resuspending cells in the cryopreservation solution includes: preparing a cryopreservation solution containing L-carnitine; taking 3.3 × 10⁻⁶ cells... 6 Centrifuge each cell at 1000 rpm for 8 minutes and discard the supernatant; resuspend the cells in 1.1 ml of cryopreservation solution containing L-carnitine.

[0068] In some embodiments, the steps for freezing cells are as follows: take 1 ml of a mixed suspension of cells and cryopreservation solution, load it into a cryopreservation tube; place the cryopreservation tube in a programmed cooling box, and freeze the cells at -80°C.

[0069] In some embodiments, the step of reviving the cells is as follows: the cryovial is removed from the -80°C freezer and placed in a 37°C constant temperature water bath for rewarming for 150 seconds.

[0070] In some embodiments, the steps of washing and counting cells are as follows: wash cells with 9 ml PBS, centrifuge at 1000 rpm / min for 8 minutes, and discard the supernatant; resuspend human umbilical cord mesenchymal stem cells in 1 ml of complete culture medium, mix well, take 23 μl of cell suspension, mix with 23 μl of AO / PI dye, and perform counting and viability detection.

[0071] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0072] The reagents and raw materials used in this invention are all commercially available.

[0073] The positive and progressive effects of this invention are as follows:

[0074] This invention provides an effective and safe cryoprotectant and its application method for mesenchymal stem cell cryopreservation solutions at present, which can provide better cryopreservation survival rate and recovery rate than existing technologies. Attached Figure Description

[0075] Figure 1 A comparison of L-carnitine and traditional non-permeable cryoprotectants.

[0076] Figure 2 The combined effect of a low concentration of L-carnitine and a conventional non-permeable cryoprotectant.

[0077] Figure 3 The combined effect is due to a high concentration of L-carnitine and a conventional non-permeable cryoprotectant.

[0078] Figure 4 To demonstrate the effect of adding L-carnitine on improving the base cryopreservation solution.

[0079] Figure 5 To screen the optimal concentration of the permeability protectant under the condition of combining L-carnitine.

[0080] Figure 6 To further test the combined effects of different types of osmotic protectants and L-carnitine under the optimal concentration conditions of the osmotic protectant.

[0081] Figure 7 To compare the freeze survival rate and recovery rate of the preferred embodiment with those of the comparative embodiment under other cooling rate conditions (programmed temperature control of 2℃ / min or 5℃ / min). Detailed Implementation

[0082] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0083] Example 1

[0084] This embodiment compares L-carnitine with traditional non-permeable cryoprotectants to demonstrate that L-carnitine can be used in the cryopreservation of human umbilical cord mesenchymal stem cells.

[0085] The complete culture medium consisted of 94% (v / v) mesenchymal stem cell basal medium (Dakorway), 5% (v / v) ADV (EliteCell), and 1% penicillin-streptomycin mixture.

[0086] Part 1: Cell Digestion and Collection

[0087] 1) Cut the fresh umbilical cord into 5-10 mm tissue particles using surgical scissors and culture them using complete culture medium;

[0088] 2) When the primary cells that have crawled out of the tissue reach 90% confluence, digest them with TrypLE Express enzyme in a 37°C incubator for 3 minutes;

[0089] 3) Centrifuge the digested cells at 1000 rpm / min for 8 minutes, discard the supernatant, and resuspend the human umbilical cord mesenchymal stem cells in complete culture medium;

[0090] 4) Human umbilical cord mesenchymal stem cells were cultured using complete culture medium at a density of 10,000 cells / cm³. 2 ;

[0091] 5) When the cells reach 90% confluence, digest them with TrypLE Express enzyme in a 37°C incubator for 3 minutes;

[0092] 6) Centrifuge the digested cells at 1000 rpm / min for 8 minutes, discard the supernatant, and resuspend the human umbilical cord mesenchymal stem cells in complete culture medium.

[0093] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0094] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 1;

[0095] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0096] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0097] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0098] 5) Place the cryovials into the programmed cooling box and transfer them to a -80°C freezer for cell cryopreservation. The programmed cooling box buffers the rate of sample cooling, allowing the cryovials to cool to -80°C at a rate of approximately 1°C / minute.

[0099] Part Three: Cell Resuscitation and Cell Viability Testing

[0100] 1) Remove the cryovials from the -80℃ freezer and place them in a 37℃ constant temperature water bath for 150 seconds to rewarm.

[0101] 2) Wash the cells with 9 ml of PBS and centrifuge at 1000 rpm / min for 8 minutes, then discard the supernatant;

[0102] 3) Resuspend human umbilical cord mesenchymal stem cells in 1 ml of complete culture medium, mix well, take 23 μl of cell suspension, mix with 23 μl of AO / PI for counting and viability detection.

[0103] Table 1. Composition of cryopreservation solution, cryopreservation survival rate, and recovery rate in Implementation 1

[0104]

[0105]

[0106] Experimental results show that ( Figure 1 In mesenchymal stem cells, L-carnitine can achieve a certain cryopreservation effect at a low concentration, and has a better cryopreservation effect than conventional non-permeable cryoprotectants; the conventional non-permeable cryoprotectants include monosaccharides (glucose), disaccharides (trehalose) and polysaccharides (dextran).

[0107] Example 2

[0108] This embodiment combines L-carnitine with a low concentration (0.1–0.2 mol / L) of a conventional non-permeable cryoprotectant to demonstrate that L-carnitine can be used for the cryopreservation of human umbilical cord mesenchymal stem cells; the conventional non-permeable cryoprotectant is represented by trehalose.

[0109] Part 1: Cell Digestion and Collection

[0110] The specific implementation steps in this section are the same as in Example 1.

[0111] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0112] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 2;

[0113] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0114] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0115] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0116] 5) Place the cryovials into the programmed cooling box and freeze the cells at -80°C.

[0117] Part Three: Cell Resuscitation and Cell Viability Testing

[0118] The specific implementation steps in this section are the same as in Example 1.

[0119] Table 2. Composition of cryopreservation solution, survival rate, and recovery rate in Implementation 2

[0120]

[0121] Experimental results show that ( Figure 2 Compared to using it alone, conventional non-permeable cryoprotectants can be combined with low concentrations (0.1–0.2 mol / L) of L-carnitine to achieve better cryopreservation results; the conventional non-permeable cryoprotectant is trehalose.

[0122] Example 3

[0123] This embodiment combines L-carnitine with a high concentration (0.3 mol / L) of conventional non-permeable cryoprotectant to demonstrate that L-carnitine can be used for cryopreservation of human umbilical cord mesenchymal stem cells; the conventional non-permeable cryoprotectant is represented by trehalose.

[0124] Part 1: Cell Digestion and Collection

[0125] The specific implementation steps in this section are the same as in Example 1.

[0126] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0127] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 2;

[0128] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0129] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0130] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0131] 5) Place the cryovials into the programmed cooling box and freeze the cells at -80°C.

[0132] Part Three: Cell Resuscitation and Cell Viability Testing

[0133] The specific implementation steps in this section are the same as in Example 1.

[0134] Table 3. Composition of cryopreservation solution, cryopreservation survival rate, and recovery rate in Implementation 3

[0135]

[0136] Experimental results show that ( Figure 3 Compared to using it alone, conventional non-permeable cryoprotectants can be combined with a higher concentration of L-carnitine (0.3 mol / L) to achieve better cryopreservation results; the conventional non-permeable cryoprotectant is trehalose.

[0137] Example 4

[0138] This embodiment combines L-carnitine with conventional cryopreservation solution to demonstrate that L-carnitine can be used in the cryopreservation of human umbilical cord mesenchymal stem cells; the conventional cryopreservation solution is a combination of a permeable cryoprotectant (Me2SO) and a non-permeable cryoprotectant (trehalose).

[0139] Part 1: Cell Digestion and Collection

[0140] The specific implementation steps in this section are the same as in Example 1.

[0141] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0142] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 4;

[0143] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0144] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0145] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0146] 5) Place the cryovials in a programmed cooling box and freeze the cells at -80°C. Part Three: Cell Recovery and Cell Viability Assay

[0147] The specific implementation steps in this section are the same as in Example 1.

[0148] Table 4. Composition of cryopreservation solution, cryopreservation survival rate, and recovery rate in Implementation 4

[0149]

[0150] Experimental results show that ( Figure 4 Adding L-carnitine can improve the cryopreservation effect of conventional cryopreservation solutions; the conventional cryopreservation solution is a combination of a permeable cryoprotectant (Me2SO) and a non-permeable cryoprotectant (trehalose).

[0151] Example 5

[0152] This embodiment combines L-carnitine with a permeable cryoprotectant, demonstrating that L-carnitine can be used in combination with a permeable cryoprotectant to obtain a cryopreservation solution with good freezing effect; the permeable cryoprotectant is ethylene glycol, and different concentrations are set to optimize the concentration of the permeable cryoprotectant.

[0153] Part 1: Cell Digestion and Collection

[0154] The specific implementation steps in this section are the same as in Example 1.

[0155] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0156] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 5;

[0157] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0158] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0159] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0160] 5) Place the cryovials into the programmed cooling box and freeze the cells at -80°C.

[0161] Part Three: Cell Resuscitation and Cell Viability Testing

[0162] The specific implementation steps in this section are the same as in Example 1.

[0163] Table 5. Composition of cryopreservation solution, cryopreservation survival rate, and recovery rate in Implementation 5.

[0164]

[0165] Experimental results show that ( Figure 5 L-carnitine can be combined with ethylene glycol to form cryopreservation solutions with high cell viability and high recovery rates. Specifically, an 8% concentration of ethylene glycol combined with L-carnitine resulted in the highest cell viability and recovery rates after cryopreservation. Therefore, under the conditions of using these two in combination, an 8% concentration of ethylene glycol is preferred. Subsequently, the application of L-carnitine has been expanded from ethylene glycol to other common permeability protectants, including dimethyl sulfoxide, 1,2-propylene glycol, and glycerol.

[0166] Example 6

[0167] This embodiment combines L-carnitine with a permeable cryoprotectant to demonstrate that L-carnitine can be used in combination with a permeable cryoprotectant to obtain a cryopreservation solution with good cryopreservation effect. In order to verify the cryopreservation effect of the cryopreservation solution obtained by the present invention based on L-carnitine, an existing Me2SO-free cryopreservation solution (CN111602652A) is introduced as a comparative example in this embodiment. The permeable cryoprotectant includes Me2SO, ethylene glycol, 1,2-propylene glycol and glycerol.

[0168] Part 1: Cell Digestion and Collection

[0169] The specific implementation steps in this section are the same as in Example 1.

[0170] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0171] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 5;

[0172] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0173] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0174] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0175] 5) Place the cryovials into the programmed cooling box and freeze the cells at -80°C.

[0176] Part Three: Cell Resuscitation and Cell Viability Testing

[0177] The specific implementation steps in this section are the same as in Example 1.

[0178] Table 6. Composition of cryopreservation solution, cryopreservation survival rate, and recovery rate in Implementation 6

[0179]

[0180]

[0181] Experimental results show that ( Figure 6 The cryopreservation solution containing L-carnitine showed higher cryopreservation survival and recovery rates than the comparative example. Specifically, the combination of Me2SO, ethylene glycol, and propylene glycol with L-carnitine significantly reduced the damage to mesenchymal stem cells at low temperatures, resulting in better cryopreservation compared to the comparative example. The cryopreservation survival rate of Examples 6-3 was significantly better than that of the comparative example, while the recovery rate was comparable.

[0182] Example 7

[0183] This embodiment broadens the range of cooling rates, verifying that the cryopreservation solution obtained by the present invention based on L-carnitine can achieve better cryopreservation results than the comparative example under other cooling rates or cooling methods (using a programmed cooling device). The comparative example is consistent with the comparative example of Example 6, and the cryopreservation solution compositions of Examples 7-1 and 7-2 correspond to those of Examples 6-1 and 6-2, respectively; the permeable cryoprotectant includes Me2SO4, ethylene glycol, 1,2-propylene glycol, and glycerol.

[0184] Part 1: Cell Digestion and Collection

[0185] The specific implementation steps in this section are the same as in Example 1.

[0186] Part Two: Preparation of Cryopreservation Solutions and Cell Cryopreservation

[0187] 1) Prepare the required cell cryopreservation solution according to the cryopreservation solution formula shown in Table 5;

[0188] 2) Take 3.3 × 10 6 Centrifuge each cell at 1000 rpm / min for 8 minutes and discard the supernatant;

[0189] 3) Resuspend the cells using 1.1 ml of cryopreservation solution and cryopreservation solution with added L-carnitine;

[0190] 4) Take 1 ml of cell suspension and load it into a cryovial;

[0191] 5) Place the cryovials in a programmed freezing apparatus and freeze the cells at freezing rates of 2℃ / min and 5℃ / min respectively. Transfer them to liquid nitrogen when the temperature drops below -85℃.

[0192] Part Three: Cell Resuscitation and Cell Viability Testing

[0193] The specific implementation steps in this section are the same as in Example 1.

[0194] Table 7. Composition of cryopreservation solution, cryopreservation survival rate, and recovery rate in Implementation 7.

[0195]

[0196]

[0197] As shown in Table 7 and Figure 7 As shown, the experimental results indicate that under other cooling rates, namely, using a programmed cooling system at 2℃ / min and 5℃ / min, the cryopreservation solution containing L-carnitine still achieved significantly higher viability and recovery rates than the control group. The combination of Me₂SO₄ and ethylene glycol with L-carnitine can greatly reduce cell damage at low temperatures; therefore, even under other cooling rates, the cryopreservation effects of Examples 7-1 and 7-2 were significantly better than the control group.

[0198] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cryopreservation solution, characterized in that, The cryopreservation solution contains L-carnitine; Preferably, the content of L-carnitine in the cryopreservation solution is 0.02-0.5 mol / L.

2. The cryopreservation solution as described in claim 1, characterized in that, The cryopreservation solution further comprises a cryoprotectant, which includes one or more of polyols, sugars and sugar alcohols, and DMSO and its derivatives; and / or, The cryopreservation solution further comprises one or more of plasma proteins, culture medium, and buffer solutions; and / or, The content of L-carnitine is 0.04-0.3 mol / L.

3. The cryopreservation solution as described in claim 2, characterized in that, The cryopreservation solution contains 2-20% by volume of cryoprotectant; preferably 3-10%; and / or, The volume content of plasma proteins in the cryopreservation solution is 1-10%; preferably 1.5-7.5%; and / or, The plasma protein is selected from albumin.

4. The cryopreservation solution as described in claim 2 or 3, characterized in that, The cryoprotectant comprises one or more of DMSO, ethylene glycol, 1,2-propylene glycol, and trehalose; and / or, The plasma protein was selected from human serum albumin.

5. A method for preparing the cryopreservation solution according to any one of claims 1-4, characterized in that, The method includes preparing the components required for the cryopreservation solution and mixing the required components to obtain the cryopreservation solution.

6. Application of L-carnitine in the preparation of stem cell cryopreservation solution.

7. The use of L-carnitine or the cryopreservation solution as described in any one of claims 1-4 in the cryopreservation of stem cells; Preferably, the stem cells are mesenchymal stem cells.

8. A method for cryopreservation of stem cells, characterized in that, The method includes the following steps: The stem cells were mixed with L-carnitine or the cryopreservation solution as described in any one of claims 1-4 and then cooled to the biological sample storage temperature, for example -70 to -90°C. Preferably, the stem cells are mesenchymal stem cells.

9. The method as described in claim 8, characterized in that, The cooling rate is 0.5-10℃ / min; and / or, the volume / number ratio of the cryopreservation solution to the stem cells is 1mL:(1×10⁻⁶) / min. 6 -5×10 7 ) each, for example, 1 mL: (3 × 10 6 )indivual.

10. The method as described in claim 8 or 9, characterized in that, After the cryopreservation solution is mixed with the stem cells and before cooling, the method further includes a low-temperature equilibration step. Preferably, the low temperature is 0-20°C, for example, 0-10°C; and / or, The balancing period is 5-120 minutes.

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