A dry protective agent and a cell drying process for mesenchymal stem cells

Abstract

The application relates to the technical field of stem cells, and specifically discloses a drying protective agent and a cell drying process of mesenchymal stem cells. The drying protective agent provided by the application comprises the following components in the following contents: EGCG: 5-45 mg / 100 mL, hydroxytetrahydro pyrimidine: 400-520 mg / 100 mL, vitamin C: 350-500 mg / 100 mL, edaravone: 0.02-0.3 mg / 100 mL, quercitrin: 0.10-0.35 mg / 100 mL, aspartic acid: 180-280 mg / 100 mL, tetrahydro pyrimidine: 400-500 mg / 100 mL, trehalose: 10-60 g / 100 mL, dextran: 20-50 g / 100 mL, maltose: 25-40 g / 100 mL and 2,3,4,6-tetraacetyl-d-glucose: 30-60 mg / 100 mL, and the solvent is a phosphate buffer solution PBS; the application also provides a cell drying process, which comprises the following steps: cold-heat cycle treatment and drying; wherein the cold-heat cycle treatment adopts the aforementioned drying protective agent. The drying protective agent provided by the application can avoid damage of mesenchymal stem cells in the drying process, so that the survival rate of the mesenchymal stem cells after rehydration is improved.

Description

Technical Field

[0001] This application relates to the field of stem cell technology, specifically to a desiccant and a cell drying process for mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells found in various tissues and organs (such as bone marrow, adipose tissue, umbilical cord, skin, and dental pulp). They possess multipotent differentiation potential, capable of differentiating into adipocytes, osteoblasts, and chondrocytes, among others. Therefore, MSCs can be widely used in the treatment of various diseases and in tissue engineering research. Furthermore, MSCs are stable, easy to infuse, and readily transportable, making them of significant value in emerging medical fields.

[0003] In recent years, to extend the preservation time of mesenchymal stem cells (MSCs) at room temperature, researchers have typically freeze-dried MSCs under low-temperature, vacuum conditions to produce freeze-dried powder for storage. This allows MSCs to maintain their original biological activity for extended periods. However, the freeze-drying process has certain drawbacks. For example, it can easily damage cells, leading to decreased cell activity or even inactivation. Therefore, minimizing the damage to MSCs during freeze-drying and maintaining high cell viability is a pressing issue that needs to be addressed. Summary of the Invention

[0004] To reduce the damage to mesenchymal stem cells caused by freeze drying and improve their survival rate, this application provides a desiccant and a cell drying process for mesenchymal stem cells.

[0005] In a first aspect, this application provides a desiccant, employing the following technical solution:

[0006] A desiccant comprising the following components in the following amounts: EGCG: 5-45 mg / 100 mL, hydroxytetrahydropyrimidine: 300-520 mg / 100 mL, vitamin C: 350-500 mg / 100 mL, edaravone: 0.02-0.3 mg / 100 mL, quercetin: 0.10-0.35 mg / 100 mL, aspartic acid: 180-280 mg / 100 mL, tetrahydropyrimidine: 400-500 mg / 100 mL, trehalose: 10-60 g / 100 mL, dextran: 20-50 g / 100 mL, maltose: 25-40 g / 100 mL, and 2,3,4,6-tetraacetyl-d-glucose: 30-60 mg / 100 mL, in phosphate-buffered saline (PBS).

[0007] This application provides a desiccant that is added to mesenchymal stem cells. The active ingredients in the desiccant can be loaded into the cells, thereby playing a good stabilizing and protective role in the subsequent (freeze-drying) process of mesenchymal stem cells, effectively preventing morphological changes and damage to mesenchymal stem cells, and thus improving the survival rate of mesenchymal stem cells after freeze-drying.

[0008] In this application, EGCG (epigallocatechin gallate) is an effective active ingredient of tea polyphenols with antioxidant properties. Adding EGCG to the desiccant ensures that the heat-sensitive and easily oxidized components of mesenchymal stem cells are not damaged during the drying process, thereby effectively improving the survival rate of mesenchymal stem cells. Hydroxytetrahydropyrimidine is an excellent compatible solute that can be used to stabilize cells, especially during freeze-thaw cycles, providing excellent cell protection, reducing cell damage, and maintaining cell viability. Vitamin C maintains cell integrity and also has certain free radical scavenging and antioxidant effects. Edaravone can effectively scavenge free radicals in the system, inhibit oxidative damage to mesenchymal stem cells, and thus improve the survival rate of mesenchymal stem cells. Quercetin can scavenge various reactive oxygen free radicals and has excellent antioxidant effects. Aspartic acid is an α-amino acid that can act as a potassium phosphate (K+) sorbate. + Mg 2+ Ion carriers deliver electrolytes to the myocardium, thereby improving myocardial contractility and reducing oxygen consumption, thus protecting the myocardium during coronary circulatory disorders and hypoxia. This application found that adding aspartic acid to a desiccant effectively prevents protein denaturation and inactivation in mesenchymal stem cells, providing excellent protection. Trehalose, a non-reducing sugar composed of two glucose molecules linked by α,α,1,1-glycosidic bonds, can form a unique protective film on the cell surface under harsh conditions such as freezing and dehydration, effectively protecting protein molecules from denaturation and inactivation, thereby maintaining cell viability. Dextran improves the viscosity of cell suspensions and reduces cell aggregation; moreover, dextran adheres to the cell surface, providing coverage and protection. Maltose contains a large amount of carbohydrates, which can regulate and maintain cell viability. 2,3,4,6-Tetraacetyl-d-glucose is a glucose derivative with excellent water solubility, antioxidant properties and biocompatibility. In this application, 2,3,4,6-tetraacetyl-d-glucose is added to the desiccant, which can reduce the damage of heat-sensitive and easily oxidized components during cell drying and improve the survival rate of mesenchymal stem cells.

[0009] In one specific implementation, the content of aspartic acid can be 180 mg / 100 mL, 200 mg / 100 mL, 230 mg / 100 mL, 260 mg / 100 mL, or 280 mg / 100 mL.

[0010] In one specific implementation, the content of the 2,3,4,6-tetraacetyl-d-glucose can be 30 mg / 100 mL, 40 mg / 100 mL, 50 mg / 100 mL, or 60 mg / 100 mL.

[0011] Preferably, the desiccant comprises the following components in the following amounts: EGCG: 5-45 mg / 100 mL, hydroxytetrahydropyrimidine: 300-520 mg / 100 mL, vitamin C: 350-500 mg / 100 mL, edaravone: 0.02-0.3 mg / 100 mL, quercetin: 0.10-0.35 mg / 100 mL, aspartic acid: 200-260 mg / 100 mL, tetrahydropyrimidine: 400-500 mg / 100 mL, trehalose: 10-60 g / 100 mL, dextran: 20-50 g / 100 mL, maltose: 25-40 g / 100 mL, and 2,3,4,6-tetraacetyl-d-glucose: 40-60 mg / 100 mL, in phosphate-buffered saline (PBS).

[0012] More preferably, the desiccant comprises the following components in the following amounts: EGCG: 37 mg / 100 mL, hydroxytetrahydropyrimidine: 465 mg / 100 mL, vitamin C: 480 mg / 100 mL, edaravone: 0.20 mg / 100 mL, quercetin: 0.23 mg / 100 mL, aspartic acid: 230 mg / 100 mL, tetrahydropyrimidine: 465 mg / 100 mL, trehalose: 58 g / 100 mL, dextran: 32 g / 100 mL, maltose: 32 g / 100 mL, and 2,3,4,6-tetraacetyl-d-glucose: 50 mg / 100 mL, in phosphate-buffered saline (PBS).

[0013] Secondly, this application provides a cell drying process for mesenchymal stem cells.

[0014] A cell drying process for mesenchymal stem cells, wherein the cell drying process employs the aforementioned desiccant.

[0015] Preferably, the cell drying process includes the following steps: hot and cold cycling treatment and drying;

[0016] The hot and cold cycling process is as follows: a desiccant is added to the mesenchymal stem cells, and then hot and cold cycling is performed. The conditions for hot and cold cycling are: storage at 39±2℃ for 5-20 min, and then storage at 4±2℃ for 5-20 min. The above operation is repeated 2-8 times to obtain a cell suspension.

[0017] In this application, a desiccant is first added to mesenchymal stem cells, and then the components of the desiccant are fully loaded into the cells through a hot-cold cycle treatment, so that the desiccant can play a full role in the subsequent (freeze-drying) process.

[0018] In this application, the drying can be vacuum drying; the temperature of vacuum drying is 28-35℃; the vacuum drying procedure is as follows: first, maintain at 600-700Pa for 10-20 minutes, and then maintain at 100-120Pa for 55-80 minutes.

[0019] In one specific implementation, the vacuum drying temperature can be 28°C, 30°C, or 35°C.

[0020] In this application, the drying can also be vacuum freeze-drying, specifically: first, the cells after the cold and heat cycle treatment are frozen; then the frozen cells are freeze-dried once under the conditions of vacuum degree of 1-3 Pa and partition temperature of -(30-50)℃ for 15-24h; then the partition temperature is raised to 10-20℃ and maintained for 5-15h.

[0021] In this application, both vacuum drying and vacuum freeze drying can be used to dry mesenchymal stem cells. After rehydration, the cell survival rate of the stem cells obtained by the above drying process can reach more than 80%.

[0022] In this application, the cell freezing conditions are: pre-cooling at 2-6℃ for 10-20 min, and then freezing at -(75-85)℃ for 50-70 min.

[0023] Preferably, the temperature rise rate of the partition is 0.21-2.0℃ / min.

[0024] In one specific implementation, the temperature rise rate of the partition can be 1.5℃ / min.

[0025] Thirdly, this application provides a cell obtained by drying using the aforementioned cell drying process.

[0026] Fourthly, this application provides a rehydration method, comprising the following steps: adding a rehydration solution to mesenchymal stem cells until the cells are dissolved; wherein the rehydration solution is a PBS buffer and distilled water in a volume ratio of (2-4):1.

[0027] In one specific implementation, the rehydration solution is a PBS buffer and distilled water in a volume ratio of 3:1.

[0028] In summary, this application has the following beneficial effects:

[0029] 1. This application provides a desiccant made from EGCG, hydroxytetrahydropyrimidine, vitamin C, aspartic acid and 2,3,4,6-tetraacetyl-d-glucose, etc. This desiccant can play a good stabilizing and protective role in the process of mesenchymal stem cell (freeze-drying), effectively avoiding morphological changes and damage to mesenchymal stem cells, thereby effectively improving the survival rate of mesenchymal stem cells after freeze-drying.

[0030] 2. This application further controls the content of aspartic acid in the range of 200-260 mg / 100 mL and the content of 2,3,4,6-tetraacetyl-d-glucose in the range of 40-60 mg / 100 mL. The resulting desiccant can significantly reduce the impact of the drying process on mesenchymal stem cells and improve the survival rate of mesenchymal stem cells to over 90%.

[0031] 3. This application further controls the temperature of the partition during the drying process between 28-35℃, which can effectively prevent the mutation of mesenchymal stem cells and enable the survival rate of mesenchymal stem cells to reach more than 95% after drying and rehydration.

[0032] 4. In the freeze-drying process of mesenchymal stem cells, the mesenchymal stem cells are pre-cooled, which provides the cells with a certain buffer time, allowing residual water inside the cells to permeate out of the cells before freezing, further reducing the damage of ice crystals to the cells, thereby improving the cell survival rate. Attached Figure Description

[0033] Figure 1 This is a morphological image of the dried cells from Example 3 after rehydration under an optical microscope.

[0034] Figure 2 This is a morphological image of the dried cells of Comparative Example 11 after rehydration under an optical microscope.

[0035] Figure 3 This is a morphological image of the dried cells of Comparative Example 12 after rehydration under an optical microscope. Detailed Implementation

[0036] In a first aspect, this application provides a desiccant comprising the following components in the following amounts: EGCG: 5-45 mg / 100 mL, hydroxytetrahydropyrimidine: 300-520 mg / 100 mL, vitamin C: 350-500 mg / 100 mL, edaravone: 0.02-0.3 mg / 100 mL, quercetin: 0.10-0.35 mg / 100 mL, aspartic acid: 180-280 mg / 100 mL, tetrahydropyrimidine: 400-500 mg / 100 mL, trehalose: 10-60 g / 100 mL, dextran: 20-50 g / 100 mL, maltose: 25-40 g / 100 mL, and 2,3,4,6-tetraacetyl-d-glucose: 30-60 mg / 100 mL, in phosphate-buffered saline (PBS). Furthermore, the content of aspartic acid is 200-260 mg / 100 mL, and the content of 2,3,4,6-tetraacetyl-d-glucose is 40 mL-60 mg / 100 mL.

[0037] Secondly, this application provides a cell drying process for mesenchymal stem cells, comprising the following steps:

[0038] (1) Cold and heat cycling treatment: Add phosphate-buffered saline (PBS) solution to mesenchymal stem cells, then centrifuge at 300g for 5min and discard the supernatant; add PBS solution to the precipitate and mix well; add desiccant to the mesenchymal stem cells at a volume ratio of 1:5, gently mix and then perform cold and heat cycling under the following conditions: store at 39±2℃ for 5-20min, then store at 4±2℃ for 5-20min; repeat the above operation 2-8 times to obtain cell suspension.

[0039] (2) Drying: Dispense the cell suspension into vials and then perform vacuum drying or vacuum freeze drying.

[0040] (2-1) Vacuum drying: Raise the temperature of the freeze dryer partition to 28-35℃ and place the vial in the freeze dryer; then evacuate the freeze dryer to 600-700Pa and hold for 10-20min, then continue to evacuate to 100-120Pa and hold for 55-80min, then the freeze dryer is obtained.

[0041] (2-2) Vacuum freeze-drying: Pre-cool the vial at 2-6℃ for 10-20 min, and freeze it at -(75-85)℃ for 50-70 min to obtain frozen cells; then transfer the vial to a freeze dryer and freeze-dry it once under vacuum of 1-3 Pa and partition temperature of -(30-50)℃ for 15-24 h; finally, raise the partition temperature to 10-20℃ at a rate of 0.21-2.0℃ / min and maintain it for 5-15 h to obtain freeze-dried cells.

[0042] In a specific embodiment of this application, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells, model PCS-500-010, purchased from Beijing Bio-Innovation Technology Co., Ltd.; EGCG is purchased from Xi'an Weiao Biotechnology Co., Ltd.; the CAS number of hydroxytetrahydropyrimidine is 165542-15-4; the CAS number of vitamin C is 50-81-7; edaravone is purchased from Zhongshan Dixing Chemical Co., Ltd.; the CAS number of quercetin is 522-12-3; the CAS number of aspartic acid is 56-84-8; the CAS number of tetrahydropyrimidine is 96702-03-3; trehalose is purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; dextran is dextran 70, CAS number 58798-70-2; maltose is purchased from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd.; and the CAS number of 2,3,4,6-tetraacetyl-d-glucose is 10343-06-3, with a purity of 98%. All other raw materials, reagents, solvents, etc., can be obtained commercially.

[0043] The present application will be further described in detail below with reference to preparation examples, embodiments, performance testing tests and accompanying drawings.

[0044] Preparation Examples 1-5

[0045] Preparation Examples 1-5 each provide a desiccant; the difference between the above preparation examples is that the amount of aspartic acid added in the desiccant is shown in Table 1.

[0046] The above-mentioned desiccant is prepared by mixing the components of the desiccant and stirring at 400 r / min for 10 min to obtain the desiccant.

[0047] Table 1. Components and dosage of the desiccant in Preparation Examples 1-5

[0048]

[0049]

[0050] Preparation Examples 6-8

[0051] Preparation Examples 6-8 each provide a desiccant.

[0052] Preparation Examples 6-8 were prepared according to the method of Preparation Example 3. The difference between the above preparation examples and Preparation Example 3 is that the amount of 2,3,4,6-tetraacetyl-d-glucose added in the desiccant is shown in Table 2.

[0053] Table 2 shows the amount of 2,3,4,6-tetraacetyl-d-glucose added to the desiccant in Preparation Examples 3 and 6-8.

[0054] Example Amount of 2,3,4,6-tetraacetyl-d-glucose added (mg) 3 50 6 30 7 40 8 60

[0055] Comparative preparation examples 1-7

[0056] Comparative preparation examples 1-7 each provide a desiccant.

[0057] Comparative preparation examples 1-7 were prepared according to the method of preparation example 3. The difference between the above comparative preparation examples and preparation example 3 is that the components and amounts of the desiccant are as shown in Table 3.

[0058] Table 3 compares the components and amounts of the desiccant in Preparation Examples 1-7.

[0059]

[0060]

[0061] Comparative Preparation Example 8

[0062] Comparative preparation example 8 provides a desiccant.

[0063] The difference between Preparation Example 8 and Preparation Example 3 is that aspartic acid was replaced with acetylcysteine ​​(CAS No. 616-91-1).

[0064] Comparative preparation example 9

[0065] Comparative preparation example 9 provides a desiccant.

[0066] The difference between Preparation Example 9 and Preparation Example 3 is that aspartic acid was replaced with N-acetyl-L-glutamic acid (CAS No. 1188-37-0).

[0067] Comparative preparation example 10

[0068] Comparative preparation example 10 provides a desiccant.

[0069] The difference between Preparation Example 10 and Preparation Example 3 is that 2,3,4,6-tetraacetyl-d-glucose was replaced with N-acetyl-D-glucosamine (CAS No. 7512-17-6).

[0070] Comparative Preparation Example 11

[0071] Comparative preparation example 11 provides a desiccant.

[0072] The aforementioned desiccant comprises: component A and component B;

[0073] Component A includes EGCG 36mg / 100mL, hydroxytetrahydropyrimidine 465mg / 100mL, vitamin C 480mg / 100mL, edaravone 0.20mg / 100mL, quercetin 0.24mg / 100mL, acetylcysteine ​​475mg / 100mL, tetrahydropyrimidine 460mg / 100mL, and trehalose 58g / 100mL, in phosphate-buffered saline (PBS).

[0074] Component B includes EGCG 18mg / 100mL, hydroxytetrahydropyrimidine 465mg / 100mL, dextran 30g / 100mL, and maltose 30g / 100mL, in phosphate-buffered saline (PBS).

[0075] Comparative preparation example 12

[0076] Comparative preparation example 12 provides a desiccant.

[0077] The above-mentioned desiccant includes: trehalose 10g / 100mL, dextran 20g / 100mL, polyarginine 10mg / 100mL, mannitol 5.0g / 100mL, hydroxyethyl starch 5.0g / 100mL, poloxamer (P188) 5.0g / 100mL, glycerol 10g / 100mL, sucrose 5.0g / 100mL (0.25mol / L), polyvinylpyrrolidone 5.0g / 100mL, and the solvent is water for injection and serum-free basal culture medium (the basal culture medium is a mixture of DMEM and F12 in a 1:1 volume ratio).

[0078] Examples 1-8

[0079] Examples 1-8 each provide a cell drying process for mesenchymal stem cells.

[0080] The difference between the above embodiments is that the desiccant used in the cell drying process is derived from Preparation Examples 1-8.

[0081] The cell drying process for mesenchymal stem cells provided in Examples 1-8 includes the following steps:

[0082] (1) Cold and hot cycling treatment: Add 10 mL of phosphate-buffered saline (PBS) solution to 10 million mesenchymal stem cells, then centrifuge at 300 g for 5 min and discard the supernatant; add 0.1 mL of PBS solution to the precipitate and mix well; add 0.5 mL of desiccant to the above mesenchymal stem cells, gently mix and then perform cold and hot cycling under the following conditions: store at 39 °C for 10 min, then store at 4 °C for 10 min; repeat the above operation 6 times to obtain cell suspension.

[0083] (2) Drying: Dispense the cell suspension into vials; raise the temperature of the freeze dryer partition to 30°C and place the vials in the freeze dryer; then evacuate the freeze dryer to 665 Pa and hold for 15 min, then continue to evacuate to 106 Pa and hold for 65 min, and the dried cells are obtained.

[0084] Example 9

[0085] Example 9 provides a cell drying process for mesenchymal stem cells.

[0086] The difference between the above embodiment and embodiment 3 is that: step (2) freeze dryer partition temperature.

[0087] (2) Drying: Dispense the cell suspension into vials; raise the temperature of the freeze dryer partition to 28°C and place the vials in the freeze dryer; then evacuate the freeze dryer to 665 Pa and hold for 15 min, then continue to evacuate to 106 Pa and hold for 65 min, and the dried cells are obtained.

[0088] Example 10

[0089] Example 10 provides a cell drying process for mesenchymal stem cells.

[0090] The difference between the above embodiment and embodiment 3 is that: step (2) freeze dryer partition temperature.

[0091] (2) Drying: Dispense the cell suspension into vials; raise the temperature of the freeze dryer partition to 35°C and place the vials in the freeze dryer; then evacuate the freeze dryer to 665 Pa and hold for 15 min, then continue to evacuate to 106 Pa and hold for 65 min, and the dried cells are obtained.

[0092] Example 11

[0093] Example 11 provides a cell drying process for mesenchymal stem cells.

[0094] The difference between the above embodiment and embodiment 3 is that the vacuum degree of the lyophilizing agent in step (2) is different.

[0095] (2) Drying: Dispense the cell suspension into vials; raise the temperature of the freeze dryer partition to 30°C and place the vials in the freeze dryer; then evacuate the freeze dryer to 600 Pa and hold for 15 min, then continue to evacuate to 106 Pa and hold for 65 min, and the dried cells are obtained.

[0096] Example 12

[0097] Example 12 provides a cell drying process for mesenchymal stem cells.

[0098] The difference between the above embodiment and embodiment 3 is that step (2)

[0099] (2) Drying: The cell suspension was dispensed into vials; the vials were pre-cooled at 4°C for 15 min and then frozen at -80°C for 60 min to obtain frozen cells; the vials were then transferred to a freeze dryer and freeze-dried once under a vacuum of 2 Pa and a partition temperature of -45°C for 20 h; finally, the partition temperature was increased to 15°C at a rate of 1.5°C / min and maintained for 10 h to obtain freeze-dried cells.

[0100] Example 13

[0101] Example 13 provides a cell drying process for mesenchymal stem cells.

[0102] The difference between the above embodiment and embodiment 12 is that the vial in step (2) is not pre-cooled, but directly frozen.

[0103] (2) Drying: The cell suspension was dispensed into vials; the vials were directly frozen at -80℃ for 60 min to obtain frozen cells; the vials were then transferred to a freeze dryer and freeze-dried once under a vacuum of 2 Pa and a partition temperature of -45℃ for 20 h; finally, the partition temperature was increased to 15℃ at a rate of 1.5℃ / min and maintained for 10 h to obtain freeze-dried cells.

[0104] Comparative Examples 1-12

[0105] Comparative Examples 1-12 each provide a cell drying process for mesenchymal stem cells.

[0106] The difference between the above comparative examples and Example 3 is that the desiccant used in the cell drying process was derived from comparative preparation examples 1-12.

[0107] Comparative Example 13

[0108] Comparative Example 13 provides a cell drying process for mesenchymal stem cells.

[0109] The difference between the above comparative example and Example 3 is that the temperature of the freeze dryer partition in step (2) is different.

[0110] (2) Drying: Dispense the cell suspension into vials; raise the temperature of the freeze dryer partition to 25°C and place the vials in the freeze dryer; then evacuate the freeze dryer to 665 Pa and hold for 15 min, then continue to evacuate to 106 Pa and hold for 65 min, and the dried cells are obtained.

[0111] Comparative Example 14

[0112] Comparative Example 14 provides a cell drying process for mesenchymal stem cells.

[0113] The difference between the above comparative example and Example 3 is that the temperature of the freeze dryer partition in step (2) is different.

[0114] (2) Drying: Dispense the cell suspension into vials; raise the temperature of the freeze dryer partition to 40°C and place the vials in the freeze dryer; then evacuate the freeze dryer to 665 Pa and hold for 15 min, then continue to evacuate to 106 Pa and hold for 65 min, and the dried cells are obtained.

[0115] Performance testing

[0116] A rehydration solution was prepared by mixing PBS buffer and distilled water at a ratio of 3:1. Then, 1 mL of the rehydration solution was added to the vials containing dried cells from Examples 1-13 and Comparative Examples 1-14 at room temperature, and the vials were gently shaken until all the dried cells were dissolved. The morphology of the rehydrated cells was then observed under an optical microscope, and the cell viability (%) was directly read from the computer. The viability results are shown in Table 4.

[0117] The morphology of the dried cells obtained in Example 3 after rehydration under an optical microscope is as follows: Figure 1 As shown. The morphology of the dried cells obtained in Comparative Example 11 after rehydration under an optical microscope is as follows. Figure 2 As shown. The morphology of the dried cells obtained in Comparative Example 12 after rehydration under an optical microscope is as follows. Figure 3 As shown, living mesenchymal stem cells emit green fluorescence, while dead cells do not.

[0118] Table 4. Cell viability test results of dried cells after rehydration in Examples 1-13 and Comparative Examples 1-14.

[0119]

[0120]

[0121] The test results of Examples 1-5 show that the cell viability of the dried cells obtained in Examples 1-5 after rehydration is >80%, indicating that the aspartic acid content in Examples 1-5 of this application is controlled between 180-280 mg / 100 mL, and the resulting desiccant has a certain protective effect on mesenchymal stem cells. Further comparison revealed that the cell viability of the dried cells obtained in Examples 2-4 after rehydration is above 90%, indicating that the desiccant obtained in this application, by further controlling the aspartic acid content within the range of 200-260 mg / 100 mL, can significantly reduce the impact of the drying process on mesenchymal stem cells and improve their survival rate.

[0122] The test results of Examples 3 and 6-8 show that the dried cells from Examples 3 and 6-8 maintained a high survival rate after rehydration, with a cell survival rate >80%. Further investigation revealed that in Examples 3 and 9-10, when the content of 2,3,4,6-tetraacetyl-d-glucose was controlled between 40-60 mg / 100 mL, the cell survival rate was even higher, reaching over 90%. Therefore, it is demonstrated that by further controlling the content of 2,3,4,6-tetraacetyl-d-glucose within the range of 40-60 mg / 100 mL, the desiccant prepared in this application has a better protective effect on mesenchymal stem cells, resulting in a higher survival rate of mesenchymal stem cells after rehydration.

[0123] The test results of Examples 3, 9-10, and Comparative Examples 13-14 show that when the freeze dryer partition temperature was controlled at 25°C and 40°C in Comparative Examples 13 and 14, the cell viability rates of the dried cells after rehydration were 71.3% and 65.7%, respectively. In Examples 3 and 9-10, when the freeze dryer partition temperature was controlled between 28-35°C, the cell viability rate of the dried cells after rehydration was >95%. Therefore, it is demonstrated that in the cell drying process of this application, controlling the freeze dryer partition temperature between 28-35°C can effectively prevent mutations in mesenchymal stem cells, ensure that stem cells maintain normal morphology during the drying process, and thus allow mesenchymal stem cells to maintain high activity after drying and rehydration.

[0124] The test results of Examples 12-13 show that Example 12, which uses a process of pre-cooling, then freezing, and finally freeze-drying, achieved a 97.1% survival rate for rehydrated frozen stem cells. However, Example 13, which uses a process of freezing first and then freeze-drying, only achieved a 92.6% survival rate. Therefore, this indicates that in the freeze-drying process of mesenchymal stem cells, pre-cooling allows for a buffer time, enabling residual water to permeate out of the cells before freezing, reducing damage from ice crystals and improving cell survival.

[0125] Comparative Example 1 used a desiccant for mesenchymal stem cells that did not contain aspartic acid, and the cell viability after rehydration was only 58.5%. Comparative Example 2 used a desiccant with an aspartic acid content of 150 mg / 100 mL, and the cell viability after rehydration was 70.2%. Comparative Example 3 used a desiccant with an aspartic acid content of 300 mg / 100 mL, and the cell viability after rehydration was 75.4%. Therefore, it is indicated that adjusting the aspartic acid content to outside the range of 180-280 mg / 100 mL or omitting aspartic acid results in a desiccant that provides poor protection for mesenchymal stem cells and is unsuitable as a desiccant for mesenchymal stem cells.

[0126] Comparative Example 4 used a desiccant for mesenchymal stem cells that did not contain 2,3,4,6-tetraacetyl-d-glucose, and its cell viability after rehydration was only 69.3%. Comparative Example 5 used a desiccant with a 20 mg / 100 mL 2,3,4,6-tetraacetyl-d-glucose content, and its cell viability after rehydration was 73.2%. Comparative Example 6 used a desiccant with a 70 mg / 100 mL 2,3,4,6-tetraacetyl-d-glucose content, and its cell viability after rehydration was 76.8%. Therefore, it is shown that adjusting the 2,3,4,6-tetraacetyl-d-glucose content to 30-60 mg / 100 mL or omitting 2,3,4,6-tetraacetyl-d-glucose results in a desiccant that provides poor protection for mesenchymal stem cells and is unsuitable as a desiccant for mesenchymal stem cells.

[0127] The cell survival rate of the dried cells in Comparative Example 7 after rehydration was only 71.5%, indicating that the desiccant provided in Comparative Preparation Example 7 had a limited protective effect on mesenchymal stem cells.

[0128] Comparative Examples 8 and 9 used acetylcysteine ​​and N-acetyl-L-glutamic acid, respectively, to prepare desiccant solutions that replaced aspartic acid. The survival rates of the rehydrated dried cells in Comparative Examples 8 and 9 were 67.2% and 43.6%, respectively. Comparative Example 10 used N-acetyl-D-glucosamine to replace 2,3,4,6-tetraacetyl-d-glucose as a desiccant solution, and the survival rate of the rehydrated dried cells in Comparative Examples 10 was 64.7%. Comparative Examples 11 and 12 used desiccant solutions from related technologies to dehydrate mesenchymal stem cells, and the survival rates of the rehydrated dried cells in Comparative Examples 11 and 12 were 74.4% and 71.8%, respectively.

[0129] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A desiccant for mesenchymal stem cells, characterized in that, The mesenchymal stem cell desiccant comprises the following components in the following amounts: EGCG: 5-45 mg / 100 mL, hydroxytetrahydropyrimidine: 300-520 mg / 100 mL, vitamin C: 350-500 mg / 100 mL, edaravone: 0.02-0.3 mg / 100 mL, quercetin: 0.10-0.35 mg / 100 mL, aspartic acid: 200-260 mg / 100 mL, tetrahydropyrimidine: 400-500 mg / 100 mL, trehalose: 10-60 g / 100 mL, dextran: 20-50 g / 100 mL, maltose: 25-40 g / 100 mL, and 2,3,4,6-tetraacetyl-d-glucose: 40-60 mg / 100 mL, in phosphate-buffered saline (PBS).

2. The mesenchymal stem cell desiccant according to claim 1, characterized in that, The mesenchymal stem cell desiccant comprises the following components in the following amounts: EGCG: 37 mg / 100 mL, hydroxytetrahydropyrimidine: 465 mg / 100 mL, vitamin C: 480 mg / 100 mL, edaravone: 0.20 mg / 100 mL, quercetin: 0.23 mg / 100 mL, aspartic acid: 230 mg / 100 mL, tetrahydropyrimidine: 465 mg / 100 mL, trehalose: 58 g / 100 mL, dextran: 32 g / 100 mL, maltose: 32 g / 100 mL, and 2,3,4,6-tetraacetyl-d-glucose: 50 mg / 100 mL, in phosphate-buffered saline (PBS).

3. A cell drying process for mesenchymal stem cells, characterized in that, The cell drying process employs the mesenchymal stem cell drying protectant as described in any one of claims 1-2.

4. The cell drying process for mesenchymal stem cells according to claim 3, characterized in that, The cell drying process includes the following steps: hot and cold cycle treatment, and drying; The hot and cold cycling treatment is as follows: add the mesenchymal stem cell desiccant of any one of claims 1-3 to the mesenchymal stem cells, and then perform hot and cold cycling; the conditions for hot and cold cycling are: store at 39±2℃ for 5-20 min, and then store at 4±2℃ for 5-20 min; repeat the above operation 2-8 times to obtain a cell suspension.

5. The cell drying process for mesenchymal stem cells according to claim 4, characterized in that, The drying process is vacuum drying; the temperature of vacuum drying is 28-35℃; the vacuum drying procedure is as follows: first, maintain at 600-700Pa for 10-20 minutes, and then maintain at 100-120Pa for 55-80 minutes.

6. The cell drying process for mesenchymal stem cells according to claim 4, characterized in that, The drying process is vacuum freeze-drying, specifically: first, the cells after the cold and heat cycle treatment are frozen; then, the frozen cells are freeze-dried once under a vacuum of 1-3 Pa and a partition temperature of -30-50 °C for 15-24 hours; then, the partition temperature is raised to 10-20 °C and maintained for 5-15 hours. The cell freezing conditions are as follows: pre-cooling at 2-6℃ for 10-20 min, and then freezing at -(75-85)℃ for 50-70 min; The temperature rise rate of the partition is 0.21-2.0℃ / min.

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