CN114680102A - A kind of sugar composition, sugar-based composition and freezing liquid - Google Patents

Abstract

The application discloses a saccharide composition comprising at least one of trehalose, sucrose, melibiose and mannan. The application also provides a sugar-based composition, a frozen stock solution and application of the frozen stock solution. The cryopreservation solution provided by the application selects low-dose DMSO and dextran 40, and the combination of trehalose, sucrose, melibiose and mannan replaces Fetal Bovine Serum (FBS) to be used as a basic formula of the cryopreservation solution for composition, and the inhibitors of vitamin C, resveratrol, taurine, L-arginine and Caspase9 are added, so that cells frozen at low temperature can still keep good cell activity after being recovered according to a specific program. Not only can reduce the pollution of pathogenic bacteria, but also is safer; meanwhile, the method avoids the incorporation of heterologous proteins, reduces the usage amount of DMSO, and can reduce the clinical use risk.

Description

A kind of sugar composition, sugar-based composition and freezing liquid

technical field

The present application relates to the technical field of biomedicine, and in particular to a carbohydrate composition, a carbohydrate-based composition and a freezing solution.

Background technique

Cryopreservation is a widely used method for long-term preservation of biological samples such as cells. Usually cryopreservation is to use a cryoprotectant and a special method to cool the living biological samples to a low temperature (-196°C), and then store them for a long time. When needed, the biological sample is heated and thawed according to a suitable method, and a living biological sample can still be obtained. As immunotherapy and gene therapy have become the research hotspots of many researchers at this stage, the cryopreservation of related immune cells and stem cells is particularly important.

The main components of the existing cryopreservation agent are composed of DMSO, dextran, fetal bovine serum, etc., which have two obvious shortcomings. One is that most of the cryopreservation solutions use fetal bovine serum FBS as the cryopreservation agent. The main raw material of the cryopreservation agent is that the biggest defect of the cryoprotectant is that the cells contain heterologous proteins derived from animal serum, which is difficult to avoid the contamination of pathogenic bacteria. Second, although the choice of DMSO as a cryoprotectant is the gold standard for cryopreservation of cells, high concentrations of DMSO are not suitable for cryopreservation of cells for clinical use, because it may be toxic to the body and lead to a wide range of adverse events.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a carbohydrate composition, a carbohydrate-based composition and a freezing solution. During cryopreservation, a large amount of water leaks out of the organelles, and ice crystals are formed under low temperature conditions, thereby causing damage to the cells themselves. Ice recrystallization (IR) is an important reason for the induction of cell damage under cryopreservation, which leads to cryopreservation recovery. Decreased cell viability during the process. The cryopreservation solution can recover the cryopreserved cells according to a specific procedure, and can still maintain good cell viability after the recovery. In addition, the use of the cryopreservation solution described in this application can reduce the contamination of pathogenic bacteria and is safer; meanwhile, the incorporation of heterologous proteins can be avoided, and the amount of DMSO used can be reduced, which can reduce the risk of clinical use.

The present application provides the following technical solutions.

1. A saccharide composition, characterized in that it comprises at least one of trehalose, sucrose, melibiose and mannan.

2. The saccharide composition according to item 1, characterized in that the saccharide composition, in terms of 100 parts by weight of the saccharide composition relative to the total weight, comprises:

Trehalose is 0-100 parts, preferably 0.1-50 parts;

Sucrose is 0-100 parts, preferably 0.1-50 parts;

Melibiose is 0-100 parts, preferably 0.1-50 parts;

Mannan is 0-100 parts, preferably 0.5-50 parts.

3. The saccharide composition according to item 1, wherein the saccharide composition is composed of trehalose, sucrose, melibiose and mannan.

4. The saccharide composition according to item 3, characterized in that, based on 100 parts by weight of the saccharide composition relative to the total weight,

Trehalose is 0-100 parts, preferably 0.1-50 parts;

Sucrose is 0-100 parts, preferably 0.1-50 parts;

Melibiose is 0-100 parts, preferably 0.1-50 parts;

Mannan is 0-100 parts, preferably 0.5-50 parts.

5. A sugar-based composition, characterized in that it comprises a sugar composition and at least one of vitamin C, resveratrol, taurine, L-arginine or a Caspase9 inhibitor.

6. The sugar-based composition according to item 5, wherein the sugar composition is the sugar composition according to any one of items 1-4.

7. The sugar-based composition according to item 5, characterized in that, based on 100 parts by weight of the sugar-based composition relative to the total weight, it comprises:

The carbohydrate composition is 90-100 parts, preferably 95-99.8495 parts;

Vitamin C is 0-2 parts, preferably 0.05-1 part;

Taurine is 0-2 part, preferably 0.05-1 part;

L-arginine is 0-2 parts, preferably 0.05-1 part;

Resveratrol is 0-2 parts, preferably 0.000001-1 parts;

Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.

8. The sugar-based composition according to item 5, wherein the sugar-based composition is composed of a sugar composition and vitamin C, resveratrol, taurine, L-arginine or a Caspase9 inhibitor .

9. The sugar-based composition according to item 8, characterized in that, based on 100 parts by weight of the sugar-based composition relative to the total weight,

The carbohydrate composition is 90-100 parts, preferably 95-99.8495 parts;

Vitamin C is 0-2 parts, preferably 0.05-1 part;

Taurine is 0-2 part, preferably 0.05-1 part;

L-arginine is 0-2 parts, preferably 0.05-1 part;

Resveratrol is 0-2 parts, preferably 0.000001-1 parts;

Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.

10. A freezing solution, characterized in that it comprises DMSO, dextran 40 and the sugar-based composition according to any one of items 5-9.

11. The freezing solution according to item 10, characterized in that, in terms of 100 parts by weight of the freezing solution relative to the total weight, it comprises:

DMSO is 2.5-20 parts, preferably 5-10 parts;

Dextran 40 is 2.5-20 parts, preferably 10-20 parts;

The sugar-based composition is 60-95 parts, preferably 70-85 parts.

12. The cryopreservation solution according to item 10, characterized in that, the cryopreservation solution is composed of DMSO, dextran 40, a sugar-based composition and physiological saline.

13. The freezing solution according to item 12, characterized in that, in terms of 100 parts by weight of the freezing solution relative to the total weight, it comprises:

DMSO is 2.5-20 parts, preferably 5-10 parts;

Dextran 40 is 2.5-20 parts, preferably 10-20 parts;

The sugar-based composition is 60-95 parts, preferably 70-85 parts;

Normal saline is the balance.

14. Use of the cryopreservation solution according to any one of items 10-13 in the field of cell cryopreservation.

The cryopreservation solution provided by the application firstly combines trehalose, sucrose, melibiose and mannan into a carbohydrate composition, which is used as the basic formula of the cryopreservation solution to replace the fetal bovine serum used in the traditional cryopreservation system. (FBS). Further adding vitamin C, resveratrol, taurine, L-arginine, and inhibitor of Caspase 9 to the sugar composition to obtain a sugar composition. Adding low doses of DMSO and dextran 40 to the sugar composition or sugar-based composition to obtain a cryopreservation solution.

The cryopreservation solution described in this application enables cells to maintain good cell viability after being cryopreserved at low temperature and resuscitated according to specific procedures, while avoiding the problem of heterologous protein contamination or pathogenic bacteria contamination caused by the use of exogenous proteins such as FBS. In addition, the use of low-dose DMSO further reduces the risk of clinical use and makes the cryopreservation system safer. In addition, the cryopreservation solution formulated based on the sugar-based composition further improves the recovery rate of viable cells compared to the cryopreservation solution formulated with the sugar-based composition.

Description of drawings

Figure 1 shows the comparison of cell viability after cryopreservation and resuscitation between the sugar-based composition (Example 4), the single sugar formula (Examples 1-3), and the commercial finished product cryopreservation solution (Cryostor CS10) (Comparative Example 3).

Figure 2 is a comparison of cell viability after cryopreservation and resuscitation between Example 5 and the sugar-based composition (Example 4) and the commercially available finished product cryopreservation solution (Cryostor CS10) (Comparative Example 3).

Figure 3 is a comparison of the number of viable cells after cryopreservation and resuscitation between Example 5 and the sugar-based composition (Example 4) and the commercially available finished product cryopreservation solution (Cryostor CS10) (Comparative Example 3).

FIG. 4 is the optimization and comparison of cell viability after resuscitation of Example 5. FIG.

Detailed ways

Exemplary embodiments of the present application are described below, including various details of the embodiments of the present application to facilitate understanding and should be considered as exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application. Also, descriptions of well-known functions and constructions are omitted from the following description for clarity and conciseness.

The present application provides a carbohydrate composition comprising at least one of trehalose, sucrose, melibiose and mannan.

The carbohydrate composition may include trehalose.

The carbohydrate composition may include trehalose and sucrose.

The saccharide composition may include trehalose and melibiose.

The carbohydrate composition may include trehalose and mannan.

The saccharide composition may include trehalose, sucrose, and melibiose.

The carbohydrate composition may include trehalose, sucrose and mannan.

The saccharide composition may include trehalose, mannan and melibiose.

The saccharide composition may include trehalose, sucrose, melibiose, and mannan.

The carbohydrate composition may include sucrose.

The carbohydrate composition may include sucrose and melibiose.

The carbohydrate composition may include sucrose and mannan.

The saccharide composition may include sucrose, melibiose, and mannan.

The saccharide composition may include melibiose.

The carbohydrate composition may include melibiose and mannan.

The carbohydrate composition may include mannan.

In this application, the saccharide composition, based on 100 parts by weight of the saccharide composition relative to the total weight, includes:

Trehalose is 0-100 parts, preferably 0.1-50 parts;

Sucrose is 0-100 parts, preferably 0.1-50 parts;

Melibiose is 0-100 parts, preferably 0.1-50 parts;

Mannan is 0-100 parts, preferably 0.5-50 parts.

In the present application, the carbohydrate composition consists of trehalose, sucrose, melibiose and mannan. Based on 100 parts by weight of the saccharide composition relative to the total weight, the components of the saccharide composition include,

Trehalose is 0-100 parts, preferably 0.1-50 parts;

Sucrose is 0-100 parts, preferably 0.1-50 parts;

Melibiose is 0-100 parts, preferably 0.1-50 parts;

Mannan is 0-100 parts, preferably 0.5-50 parts.

In the saccharide composition, the content of trehalose may be 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 parts.

The content of the sucrose can be 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 parts.

The content of the melibiose can be 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 parts.

The content of the mannan can be 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 parts.

The present application provides a preparation method of a carbohydrate composition, comprising: weighing trehalose, sucrose, melibiose and mannan respectively according to the above ratio, and then mixing the trehalose, sucrose, melibiose and mannan evenly to obtain the saccharide composition.

The present application provides a sugar-based composition, comprising a sugar composition and at least one of vitamin C, resveratrol, taurine, L-arginine or a Caspase9 inhibitor. The saccharide composition here is the aforementioned saccharide composition.

The sugar-based composition may include a sugar composition and vitamin C.

The sugar-based composition may include a sugar composition and resveratrol.

The sugar-based composition may include a sugar composition and taurine.

The sugar-based composition may include a sugar-based composition and L-arginine.

The glycosyl composition may include a carbohydrate composition and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, vitamin C and resveratrol.

The sugar-based composition may include a sugar composition, vitamin C, and taurine.

The sugar-based composition may include a sugar composition, vitamin C, and L-arginine.

The sugar-based composition may include a sugar-based composition, vitamin C, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, vitamin C, resveratrol, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, vitamin C, resveratrol, and taurine.

The sugar-based composition may include a sugar composition, vitamin C, resveratrol, and L-arginine.

The sugar-based composition may include a sugar composition, vitamin C, taurine, and L-arginine.

The sugar-based composition may include a sugar composition, vitamin C, taurine, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, vitamin C, L-arginine, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, vitamin C, resveratrol, taurine, and L-arginine.

The sugar-based composition may include a sugar composition, vitamin C, resveratrol, taurine, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition and resveratrol.

The sugar-based composition may include a sugar composition, resveratrol, and taurine.

The sugar-based composition may include a sugar composition, resveratrol, and L-arginine.

The sugar-based composition may include a sugar composition, resveratrol, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, resveratrol, taurine, and L-arginine.

The sugar-based composition may include a sugar composition, resveratrol, taurine, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, resveratrol, taurine, L-arginine, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition and taurine.

The sugar-based composition may include a sugar composition, taurine and L-arginine.

The glycosyl composition may include a carbohydrate composition, taurine and a Caspase9 inhibitor.

The glycosyl composition may include a carbohydrate composition, taurine, L-arginine, and a Caspase9 inhibitor.

The sugar-based composition may include a sugar-based composition and L-arginine.

The glycosyl composition may include a carbohydrate composition, L-arginine, and a Caspase9 inhibitor.

The glycosyl composition may include a carbohydrate composition and a Caspase9 inhibitor.

The sugar-based composition may include a sugar composition, vitamin C, resveratrol, taurine, L-arginine, and a Caspase9 inhibitor.

In this application, based on 100 parts by weight of the sugar-based composition relative to the total weight, it includes:

The carbohydrate composition is 90-100 parts, preferably 95-99.8495 parts;

Vitamin C is 0-2 parts, preferably 0.05-1 part;

Taurine is 0-2 part, preferably 0.05-1 part;

L-arginine is 0-2 parts, preferably 0.05-1 part;

Resveratrol is 0-2 parts, preferably 0.000001-1 parts;

Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.

In the present application, the sugar-based composition consists of a sugar composition and vitamin C, resveratrol, taurine, L-arginine or a Caspase9 inhibitor.

In this application, based on 100 parts by weight of the sugar-based composition relative to the total weight,

The carbohydrate composition is 90-100 parts, preferably 95-99.8495 parts;

Vitamin C is 0-2 parts, preferably 0.05-1 part;

Taurine is 0-2 part, preferably 0.05-1 part;

L-arginine is 0-2 parts, preferably 0.05-1 part;

Resveratrol is 0-2 parts, preferably; 0.000001-1 parts

Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.

In the sugar-based composition, the content of the sugar composition may be 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5 servings, 98 servings, 98.5 servings, 99 servings, 99.5 servings or 100 servings.

The content of the vitamin C can be 0 part, 0.05 part, 0.06 part, 0.07 part, 0.08 part, 0.09 part, 0.1 part, 0.11 part, 0.12 part, 0.13 part, 0.14 part, 0.15 part, 0.16 part, 0.17 part, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34 , 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51 part, 0.52 part, 0.53 part, 0.54 part, 0.55 part, 0.56 part, 0.57 part, 0.58 part, 0.59 part, 0.6 part, 0.61 part, 0.62 part, 0.63 part, 0.64 part, 0.65 part, 0.66 part, 0.67 part, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84 , 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.5 servings, 2 servings.

The content of the taurine can be 0 part, 0.05 part, 0.06 part, 0.07 part, 0.08 part, 0.09 part, 0.1 part, 0.11 part, 0.12 part, 0.13 part, 0.14 part, 0.15 part, 0.16 part, 0.17 part , 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34 part, 0.35 part, 0.36 part, 0.37 part, 0.38 part, 0.39 part, 0.4 part, 0.41 part, 0.42 part, 0.43 part, 0.44 part, 0.45 part, 0.46 part, 0.47 part, 0.48 part, 0.49 part, 0.5 part, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67 , 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84 part, 0.85 part, 0.86 part, 0.87 part, 0.88 part, 0.89 part, 0.9 part, 0.91 part, 0.92 part, 0.93 part, 0.94 part, 0.95 part, 0.96 part, 0.97 part, 0.98 part, 0.99 part, 1 part, 1.5 servings, 2 servings.

The content of the L-arginine can be 0 part, 0.05 part, 0.06 part, 0.07 part, 0.08 part, 0.09 part, 0.1 part, 0.11 part, 0.12 part, 0.13 part, 0.14 part, 0.15 part, 0.16 part, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33 , 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5 servings, 0.51 servings, 0.52 servings, 0.53 servings, 0.54 servings, 0.55 servings, 0.56 servings, 0.57 servings, 0.58 servings, 0.59 servings, 0.6 servings, 0.61 servings, 0.62 servings, 0.63 servings, 0.64 servings, 0.65 servings, 0.66 servings, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83 , 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1 servings, 1.5 servings, 2 servings.

The content of the resveratrol can be 0 part, 0.05 part, 0.06 part, 0.07 part, 0.08 part, 0.09 part, 0.1 part, 0.11 part, 0.12 part, 0.13 part, 0.14 part, 0.15 part, 0.16 part, 0.17 part part, 0.18 part, 0.19 part, 0.2 part, 0.21 part, 0.22 part, 0.23 part, 0.24 part, 0.25 part, 0.26 part, 0.27 part, 0.28 part, 0.29 part, 0.3 part, 0.31 part, 0.32 part, 0.33 part, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5 , 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67 servings, 0.68 servings, 0.69 servings, 0.7 servings, 0.71 servings, 0.72 servings, 0.73 servings, 0.74 servings, 0.75 servings, 0.76 servings, 0.77 servings, 0.78 servings, 0.79 servings, 0.8 servings, 0.81 servings, 0.82 servings, 0.83 servings, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1 , 1.5 servings, 2 servings.

The content of the Caspase9 inhibitor can be 0 part, 0.05 part, 0.06 part, 0.07 part, 0.08 part, 0.09 part, 0.1 part, 0.11 part, 0.12 part, 0.13 part, 0.14 part, 0.15 part, 0.16 part, 0.17 part , 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34 part, 0.35 part, 0.36 part, 0.37 part, 0.38 part, 0.39 part, 0.4 part, 0.41 part, 0.42 part, 0.43 part, 0.44 part, 0.45 part, 0.46 part, 0.47 part, 0.48 part, 0.49 part, 0.5 part, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67 , 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84 part, 0.85 part, 0.86 part, 0.87 part, 0.88 part, 0.89 part, 0.9 part, 0.91 part, 0.92 part, 0.93 part, 0.94 part, 0.95 part, 0.96 part, 0.97 part, 0.98 part, 0.99 part, 1 part, 1.5 servings, 2 servings.

The application provides a preparation method of a sugar-based composition, comprising weighing the sugar composition and vitamin C, resveratrol, taurine, L-arginine or Caspase9 inhibitor according to the above ratio, and then adding the The saccharide composition and vitamin C, resveratrol, taurine, L-arginine or Caspase9 inhibitor are mixed uniformly to obtain the saccharide composition.

The present application provides a freezing solution comprising DMSO, dextran 40 and the sugar-based composition.

In this application, based on the total weight of 100 parts by weight of the cryopreservation solution, it includes:

DMSO is 2.5-20 parts, preferably 5-10 parts;

Dextran 40 is 2.5-20 parts, preferably 10-20 parts;

The sugar-based composition is 60-95 parts, preferably 70-85 parts.

In the present application, the cryopreservation solution is composed of DMSO, dextran 40, sugar-based composition and physiological saline.

In this application, based on 100 parts by weight of the cryopreservation solution relative to the total weight,

DMSO is 2.5-20 parts, preferably 5-10 parts;

Dextran 40 is 2.5-20 parts, preferably 10-20 parts;

The sugar-based composition is 60-95 parts, preferably 70-85 parts;

Normal saline is the balance.

The present application provides a method for preparing a freezing solution, comprising: preparing dextran 40 and the sugar-based composition into storage solutions respectively, and then measuring DMSO, dextran 40 storage solution, and sugar-based composition storage solution according to the above proportions. and physiological saline, and then mix DMSO, dextran 40 stock solution, glycosyl composition stock solution and physiological saline to obtain the cryopreservation solution.

The present application provides a use of the cryopreservation solution in the field of cell cryopreservation.

Example

The experimental methods used in the following examples are conventional methods unless otherwise required.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

K562 cells were purchased from ATCC cell bank.

Cryostor CS10 freezing medium: purchased from STEMCELL Company.

APCAnnexinV: purchased from BioLegend.

A: Preparation of the first stock solution:

Weigh trehalose: 5.07 g, sucrose: 10.25 g, melibiose: 5.07 g, mannan: 5 g, and dextran 40: 10 g, and transfer the weighed components into 50 ml centrifuge tubes. And add normal saline for injection, respectively, to 50ml. Then, after sterilization and filtration with 0.22 μm filter membrane respectively, they are transferred to sterile reagent bottles, and they can be formulated into trehalose storage solution, sucrose storage solution, melibiose storage solution, mannan storage solution and dextran 40 stock solution. Store at 4°C and use within 6 months. It is applicable to Example 1-Example 3 (the volume of cryopreservation solution prepared in Example 1-3 is 100ml).

B: Configuration of the second storage solution

Weigh trehalose: 7.56g, sucrose: 6.84g, melibiose: 6.84g, mannan: 10g and mix, and add water to dissolve, then transfer the dissolved mixture to a 100ml volumetric flask, and then add for injection Normal saline, dilute to 100ml. Then, after sterilization and filtration with a 0.22 μm filter membrane, the saccharide composition storage solution can be obtained by sub-packaging and transferring it to a sterile reagent bottle. Store at 4°C and use within 6 months.

Weigh dextran 40, dissolve 20 g, and then use normal saline to dilute to 100 ml. Then, the dextran 40 stock solution can be obtained by sterilizing and filtration using a 0.22 μm filter membrane. Store at 4°C and use within 6 months.

Weigh vitamin C, dissolve 0.5 g, then dilute the volume to 5 ml with physiological saline, and then use a 0.22 μm filter to sterilize and filter to obtain a vitamin C storage solution.

2.28 mg of resveratrol was weighed and dissolved, and then the volume was adjusted to 10 ml with physiological saline, and then sterilized and filtered through a 0.22 μm filter to obtain a resveratrol stock solution.

0.5 g of L-arginine was weighed and dissolved, and then the volume was adjusted to 5 ml with physiological saline, and then sterilized and filtered through a 0.22 μm filter to obtain an L-arginine stock solution.

Weigh 0.25% of taurine to dissolve, then dilute the volume to 5ml with physiological saline, and then sterilize and filter through a 0.22μm filter to obtain a taurine stock solution.

Weigh 1 mg of Caspase9 inhibitor, add 213 μl of sterile DMSO to dissolve, and obtain a Caspase9 inhibitor stock solution.

The second stock solution is suitable for Example 4, Example 5, Example 6, Example 7, Example 8, Example 9, Example 10, Example 11, Example 12, Example 13, Comparative Example 2 and In Comparative Example 3, the volume of the prepared freezing solution was 10 ml.

Example 1

Take 10 ml of trehalose stock solution, 2.5 ml of DMSO, 50 ml of dextran 40 stock solution, and 37.5 ml of normal saline for injection, and mix them to obtain a freezing solution.

Example 2

Take 10 ml of sucrose stock solution, add 5 ml of DMSO, 50 ml of dextran 40, and 37.5 ml of normal saline for injection into it, and mix to obtain a cryogenic solution.

Example 3

Take 10 ml of melibiose stock solution, add 5 ml of DMSO, 50 ml of dextran 40, and 37.5 ml of normal saline for injection into it, and mix to obtain a cryopreservation solution.

Example 4

Take 1 ml of saccharide composition stock solution, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, and 3.5 ml of normal saline for injection, and mix them to obtain a freezing solution.

Example 5

Take 1ml carbohydrate composition stock solution, 0.5ml DMSO, 5ml dextran 40 stock solution, 100µl vitamin C stock solution, 100µl resveratrol stock solution, 100µl L-arginine stock solution, 200µl taurine stock solution, 30µl The Caspase9 inhibitor stock solution and 2.97 ml of normal saline for injection were mixed to obtain a cryopreserved solution.

Example 6

Take 1 ml of carbohydrate composition stock solution, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, 100 μl of vitamin C stock solution, and 3.4 ml of normal saline for injection, and mix to obtain a cryopreservation solution.

Example 7

Take 1 ml of saccharide composition stock solution, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, 100 μl of resveratrol stock solution, and 3.4 ml of normal saline for injection, and mix to obtain a cryopreservation solution.

Example 8

Take 1 ml of carbohydrate composition stock solution, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, 100 μl of L-arginine stock solution, and 3.4 ml of normal saline for injection, and mix to obtain a cryopreservation solution.

Example 9

Take 1 ml of saccharide composition stock solution, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, 30 μl of Caspase9 inhibitor stock solution, and 3.47 ml of normal saline for injection, and mix them to obtain a cryogenic solution.

Example 10

Mix 1 ml of carbohydrate composition, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, 100 μl of vitamin C stock solution, 100 μl of resveratrol stock solution, and 3.3 ml of normal saline for injection to obtain a cryopreservation solution.

Example 11

Take 1ml of carbohydrate composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 100µl of vitamin C stock solution, 100µl of resveratrol stock solution, 100µl of L-arginine stock solution, and 3.2ml of normal saline for injection, Mix to obtain a cryopreservation solution.

Example 12

Take 1ml carbohydrate composition stock solution, 0.5ml DMSO, 5ml dextran 40 stock solution, 100µl resveratrol stock solution, 100µl L-arginine stock solution, 30µl Caspase9 inhibitor stock solution, 3.27ml normal saline for injection , and mixed to obtain a freezing solution.

Example 13

Take 1 ml of saccharide composition stock solution, 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, 100 μl of resveratrol stock solution, 100 μl of L-arginine stock solution, and 3.3 ml of normal saline for injection, and mix to obtain a cryopreservation solution.

Comparative Example 1

Take 0.5 ml of DMSO, 5 ml of dextran 40 stock solution, and 4.5 ml of normal saline for injection, and mix them to obtain a cryopreservation solution.

Comparative Example 2

Take 1ml of glycosyl composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 100µl of resveratrol stock solution, 100µl of taurine stock solution, 30µl of Caspase9 inhibitor stock solution, and 3.27ml of normal saline for injection, mix Obtain cryopreservation.

Comparative Example 3 is Cryostor CS10 cryopreservation solution purchased from STEMCELL Company.

Table 1 is the raw material composition comparison table of each embodiment and comparative example

Test example

1. Cell viability

The K562 cells that were expanded and cultured were selected for cryopreservation. The cryopreservation experiments of Example 1-Example 13 and Comparative Example 1-Comparative Example 3 were performed. Specifically, the K562 cells that had been centrifuged at 400 g at 400 g and centrifuged at room temperature for 5 minutes were resuspended with the above freezing solution, and frozen in a 1.8 ml cryovial with a cell density of 1×10 ⁷ cells/ml and a freezing volume of 1 ml. After the cells are mixed with the cryopreservation solution, they can be transferred to a -80°C refrigerator to cool down. After being frozen at -80°C overnight, they are transferred to a liquid nitrogen tank for storage (stored in a liquid nitrogen tube for 7 days or 14 days). ). In the recovery process, the constant temperature water bath was turned on, and when the water temperature was heated to 37.5 ± 0.5 °C, the cells were taken out from the liquid nitrogen tank, and the cells were transferred to the water bath with a container filled with liquid nitrogen. The cells were quickly transferred to a water bath, and the cells were thawed quickly by shaking gently. The whole thawing process was controlled within 2 minutes. The results are shown in Table 2. The cell viability rate described herein is the simultaneous detection of PI and Annexin V by flow cytometry, wherein the PI and Annexin V double-negative cell population is a viable cell population, and the ratio thereof is the viable cell ratio. The number of viable cells described in the present invention is obtained by counting and analyzing with a cell counter (Nexcelom model: K2) after AO/PI staining. The results are shown in Table 2 and Figures 1-4.

Table 2 The viability and the number of viable cells of K562 cells after the cryopreservation test using the cryopreservation solution of each embodiment and the comparative example

Summary: It can be seen from the above table and Figures 1-4 that the cell viability of Example 4 (81.58% ± 2.42%) was significantly higher than that of Comparative Example 2 (44.97% ± 8.7%) and Comparative Example 3 (70.67% ± 2.09%) and also higher than the commercial cryopreservation (Comparative Example 1) (72.38% ± 10.86%), and also higher than the single sugar formula group (Example 1: 61.25 %±9.28%, Example 2: 69.54%±3.86%, Example 3: 65.81%±4.40%). Comparing Example 5 with Example 4, although there is no significant difference in cell resuscitation viability, the number of viable cells after resuscitation in Example 5 is more than that in Example 4 when the same number of cells are cryopreserved, which represents the resuscitation of Example 5. The percentage of viable cells recovered is higher. By removing vitamin C, resveratrol, L-arginine, taurine, and Caspase9 inhibitor added in the cryopreservation solution of Example 5 by single removal or multiple removal (Examples 10-20), the same as in Example 5 5 Compared with the removal, the cell viability was decreased in each group at different time points.

Although the embodiments of the present application are described above in combination, the present application is not limited to the above-mentioned specific embodiments and application fields, and the above-mentioned specific embodiments are only illustrative and instructive, rather than restrictive. Under the inspiration of this specification and without departing from the scope of protection of the claims of this application, those of ordinary skill in the art can also make many forms, which all belong to the protection of this application.

Claims (14)

1. A carbohydrate composition, characterized in that it comprises at least one of trehalose, sucrose, melibiose and mannan. 2. The saccharide composition according to claim 1, wherein the saccharide composition, in terms of the saccharide composition of 100 parts by weight relative to the total weight, comprises: Trehalose is 0-100 parts, preferably 0.1-50 parts; Sucrose is 0-100 parts, preferably 0.1-50 parts; Melibiose is 0-100 parts, preferably 0.1-50 parts; Mannan is 0-100 parts, preferably 0.5-50 parts. 3. The saccharide composition according to claim 1, wherein the saccharide composition is composed of trehalose, sucrose, melibiose and mannan. 4. The saccharide composition according to claim 3, characterized in that, based on the saccharide composition of 100 parts by weight relative to the total weight, Trehalose is 0-100 parts, preferably 0.1-50 parts; Sucrose is 0-100 parts, preferably 0.1-50 parts; Melibiose is 0-100 parts, preferably 0.1-50 parts; Mannan is 0-100 parts, preferably 0.5-50 parts. 5. A sugar-based composition, characterized in that it comprises a sugar composition and at least one of vitamin C, resveratrol, taurine, L-arginine or a Caspase9 inhibitor. 6. The sugar-based composition of claim 5, wherein the sugar composition is the sugar composition of any one of claims 1-4. 7. The sugar-based composition according to claim 5, characterized in that, in terms of the sugar-based composition relative to the total weight of 100 parts by weight, comprising: The carbohydrate composition is 90-100 parts, preferably 95-99.8495 parts; Vitamin C is 0-2 parts, preferably 0.05-1 part; Taurine is 0-2 part, preferably 0.05-1 part; L-arginine is 0-2 parts, preferably 0.05-1 part; Resveratrol is 0-2 parts, preferably 0.000001-1 parts; Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part. 8. The sugar-based composition according to claim 5, wherein the sugar-based composition is composed of a sugar composition and vitamin C, resveratrol, taurine, L-arginine or a Caspase9 inhibitor composition. 9. The sugar-based composition according to claim 8, characterized in that, based on the sugar-based composition of 100 parts by weight relative to the total weight, The carbohydrate composition is 90-100 parts, preferably 95-99.8495 parts; Vitamin C is 0-2 parts, preferably 0.05-1 part; Taurine is 0-2 part, preferably 0.05-1 part; L-arginine is 0-2 parts, preferably 0.05-1 part; Resveratrol is 0-2 parts, preferably 0.000001-1 parts; Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part. 10. A cryopreservation solution, characterized in that it comprises DMSO, dextran 40 and the sugar-based composition according to any one of claims 5-9. 11. cryopreservation solution according to claim 10 is characterized in that, with respect to the cryopreservation solution of 100 parts by weight of total weight, comprising: DMSO is 2.5-20 parts, preferably 5-10 parts; Dextran 40 is 2.5-20 parts, preferably 10-20 parts; The sugar-based composition is 60-95 parts, preferably 70-85 parts. 12 . The cryopreservation solution according to claim 10 , wherein the cryopreservation solution is composed of DMSO, dextran 40, a sugar-based composition and physiological saline. 13 . 13. The cryopreservation solution according to claim 12, characterized in that, in terms of the cryopreservation solution of 100 parts by weight relative to the total weight, comprising: DMSO is 2.5-20 parts, preferably 5-10 parts; Dextran 40 is 2.5-20 parts, preferably 10-20 parts; The sugar-based composition is 60-95 parts, preferably 70-85 parts; Normal saline is the balance. 14. A use of the cryopreservation solution according to any one of claims 10-13 in the field of cell cryopreservation.

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