CN114680102A - Saccharide composition, saccharide-based composition and frozen stock solution - Google Patents

Saccharide composition, saccharide-based composition and frozen stock solution Download PDF

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CN114680102A
CN114680102A CN202111674623.3A CN202111674623A CN114680102A CN 114680102 A CN114680102 A CN 114680102A CN 202111674623 A CN202111674623 A CN 202111674623A CN 114680102 A CN114680102 A CN 114680102A
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parts
sugar
composition
stock solution
based composition
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方日国
于玲玲
袁鹏飞
马奎莹
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Guangzhou Ji Yin Medical Technology Co ltd
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Guangzhou Ji Yin Medical Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Biophysics (AREA)
  • Physiology (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicinal Preparation (AREA)

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

Saccharide composition, saccharide-based composition and frozen stock solution
Technical Field
The application relates to the technical field of biological medicines, in particular to a saccharide composition, a glycosyl composition and a frozen stock solution.
Background
Cryopreservation is a method widely used for preserving biological samples such as cells for a long time. Generally, cryopreservation is performed by using a cryoprotectant to cool a living biological sample to a low temperature (-196 ℃) by a special method, and then storing the sample for a long time. When needed, the biological sample is heated and thawed according to a proper method, and a living biological sample can still be obtained. As immunotherapy and gene therapy become hot research for many researchers at the present stage, the cryopreservation of the related immune cells and stem cells is of great importance.
The main components of the existing cryopreservation protective agent comprise DMSO, dextran, fetal bovine serum and the like, and the existing cryopreservation protective agent has two obvious defects, namely that most of cryopreservation liquid adopts fetal bovine serum FBS as the main raw material of the cryopreservation protective agent, and the biggest defect of the cryopreservation protective agent is that cells have animal serum-derived heterologous proteins, so that the pollution of pathogenic bacteria is difficult to avoid. Secondly, although DMSO is selected as a cryopreservation protective agent which is a gold standard for cell cryopreservation, high-concentration DMSO is not suitable for cell cryopreservation in clinical use, because it may generate toxicity to organisms, and a wide range of adverse events are caused.
Disclosure of Invention
In view of the above-mentioned drawbacks and deficiencies of the prior art, the present application provides a saccharide composition, a saccharide-based composition, and a frozen stock solution. A large amount of water seeps out of organelles when cells are frozen, ice crystals are formed under a low-temperature condition, so that damage is caused to the cells, Ice Recrystallization (IR) is an important reason for inducing cell damage under low-temperature storage, and the cell survival rate is reduced in the process of freezing recovery. The frozen stock solution can recover the cells frozen at low temperature according to a specific program, and can still maintain good cell activity after recovery. In addition, the frozen stock solution can reduce the pollution of pathogenic bacteria and is safer; meanwhile, the incorporation of heterologous proteins is avoided, the use amount of DMSO is reduced, and the clinical use risk can be reduced.
The present application provides the following technical solutions.
1. A saccharide composition comprising at least one of trehalose, sucrose, melibiose and mannan.
2. The saccharide composition according to item 1, wherein the saccharide composition comprises, 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;
0-100 parts of cane sugar, preferably 0.1-50 parts;
0-100 parts of melibiose, preferably 0.1-50 parts;
the mannan is 0-100 parts, preferably 0.5-50 parts.
3. The saccharide composition according to item 1, wherein the saccharide composition consists of trehalose, sucrose, melibiose and mannan.
4. The saccharide composition according to item 3, wherein the saccharide composition comprises, 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;
0-100 parts of cane sugar, preferably 0.1-50 parts;
0-100 parts of melibiose, preferably 0.1-50 parts;
the mannan is 0-100 parts, preferably 0.5-50 parts.
5. A sugar-based composition comprising a sugar-based 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-based composition is the sugar-based composition according to any one of items 1 to 4.
7. The sugar-based composition according to item 5, comprising, based on 100 parts by weight of the sugar-based composition relative to the total weight:
the saccharide composition is 90-100 parts, preferably 95-99.8495 parts;
0-2 parts of vitamin C, preferably 0.05-1 part;
0-2 parts of taurine, preferably 0.05-1 part;
0-2 parts of L-arginine, preferably 0.05-1 part;
0-2 parts of resveratrol, preferably 0.000001-1 part;
the 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 consists of a sugar-based composition and a vitamin C, resveratrol, taurine, L-arginine or Caspase9 inhibitor.
9. The sugar-based composition according to item 8, wherein the sugar-based composition is prepared by mixing the sugar-based composition and the sugar-based composition in an amount of 100 parts by weight relative to the total weight,
the saccharide composition is 90-100 parts, preferably 95-99.8495 parts;
0-2 parts of vitamin C, preferably 0.05-1 part;
0-2 parts of taurine, preferably 0.05-1 part;
0-2 parts of L-arginine, preferably 0.05-1 part;
0-2 parts of resveratrol, preferably 0.000001-1 part;
the Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.
10. A frozen stock solution comprising DMSO, dextran 40, and the sugar-based composition of any one of items 5 to 9.
11. The cryopreservation liquid according to item 10, which comprises, based on 100 parts by weight of the cryopreservation liquid relative to the total weight:
2.5-20 parts of DMSO (dimethyl sulfoxide), preferably 5-10 parts;
2.5-20 parts of dextran 40, preferably 10-20 parts;
the sugar-based composition is 60 to 95 parts, preferably 70 to 85 parts.
12. The cryopreservation solution of item 10, wherein the cryopreservation solution consists of DMSO, dextran 40, a sugar-based composition and physiological saline.
13. The cryopreservation liquid according to item 12, which comprises, based on 100 parts by weight of the cryopreservation liquid relative to the total weight:
2.5-20 parts of DMSO (dimethyl sulfoxide), preferably 5-10 parts;
2.5-20 parts of dextran 40, preferably 10-20 parts;
the sugar-based composition is 60-95 parts, preferably 70-85 parts;
the balance being normal saline.
14. Use of a cryopreservation solution according to any one of claims 10 to 13 in the field of cell cryopreservation.
According to the cryopreservation liquid provided by the application, trehalose, sucrose, melibiose and mannan are combined into a saccharide composition to serve as a basic formula of the cryopreservation liquid to replace Fetal Bovine Serum (FBS) used in a traditional cryopreservation system. Further adding inhibitors of vitamin C, resveratrol, taurine, L-arginine, Caspase9 to obtain sugar-based composition. Adding low dose of DMSO and dextran 40 into saccharide composition or glycosyl composition to obtain frozen stock solution.
The cryopreservation liquid enables cells to be frozen at a low temperature and revived according to a specific program, and can still keep good cell activity, and meanwhile, the problem of heterologous protein pollution or pathogenic bacteria pollution caused by the use of exogenous proteins such as FBS (FBS) and the like is avoided. In addition, the use of low-dose DMSO further reduces the clinical use risk, so that the cryopreservation system is safer. In addition, the cryopreservation solution prepared based on the sugar-based composition further improves the recovery rate of viable cells compared to the cryopreservation solution prepared from the sugar-based composition.
Drawings
FIG. 1 is a comparison of cell viability after cryopreservation recovery of a sugar-based composition (example 4) versus a single sugar formulation (examples 1-3), a commercially available finished cryopreservation solution (Cryostor CS10) (comparative example 3).
FIG. 2 is a comparison of cell viability after cryopreservation recovery of example 5 and the sugar-based composition (example 4) and a commercially available finished cryopreservation solution (Cryostat CS10) (comparative example 3).
FIG. 3 is a comparison of viable cell count after cryopreservation in example 5 with a sugar-based composition (example 4), a commercially available finished cryopreservation solution (Cryostat CS10) (comparative example 3).
FIG. 4 shows the optimized comparison of cell viability after resuscitation for example 5.
Detailed Description
The following description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to assist in understanding, should be taken 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 in the following description for clarity and conciseness.
The present application provides a saccharide composition comprising at least one of trehalose, sucrose, melibiose and mannan.
The saccharide composition may include trehalose.
The saccharide composition may include trehalose and sucrose.
The saccharide composition may include trehalose and melibiose.
The saccharide composition may include trehalose and mannan.
The saccharide composition may include trehalose, sucrose and melibiose.
The saccharide 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 saccharide composition may comprise sucrose.
The saccharide composition may comprise sucrose and melibiose.
The saccharide composition may include sucrose and mannan.
The saccharide composition may include sucrose, melibiose and mannan.
The saccharide composition may comprise melibiose.
The saccharide composition may include melibiose and mannan.
The saccharide composition may comprise mannan.
In the present application, the saccharide composition comprises, 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;
0-100 parts of cane sugar, preferably 0.1-50 parts;
0-100 parts of melibiose, preferably 0.1-50 parts;
the mannan is 0-100 parts, preferably 0.5-50 parts.
In the present application, the saccharide composition consists of trehalose, sucrose, melibiose and mannan. The saccharide composition comprises, 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;
0-100 parts of cane sugar, preferably 0.1-50 parts;
0-100 parts of melibiose, preferably 0.1-50 parts;
the mannan is 0-100 parts, preferably 0.5-50 parts.
In the saccharide composition, the trehalose may be contained in an amount of 0 part, 10 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, 100 parts.
The content of the sucrose can be 0 part, 10 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts and 100 parts.
The content of the melibiose can be 0 part, 10 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts and 100 parts.
The content of the mannan can be 0 part, 10 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts and 100 parts.
The present application provides a method of preparing a saccharide composition comprising: and respectively weighing trehalose, sucrose, melibiose and mannan according to the proportion, and then uniformly mixing the trehalose, the sucrose, the melibiose and the mannan to obtain the saccharide composition.
The present application provides a carbohydrate-based composition comprising a carbohydrate composition and at least one of vitamin C, resveratrol, taurine, L-arginine, or a Caspase9 inhibitor. The saccharide composition herein is the aforementioned saccharide composition.
The sugar-based composition may include a sugar-based composition and vitamin C.
The sugar-based composition may include a sugar-based composition and resveratrol.
The sugar-based composition can include a sugar-based composition and taurine.
The sugar-based composition may include a sugar-based composition and L-arginine.
The carbohydrate-based composition may include a carbohydrate composition and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based 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-based composition, vitamin C, and L-arginine.
The carbohydrate-based composition may include a carbohydrate composition, vitamin C and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition, vitamin C, resveratrol and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition, vitamin C, resveratrol and taurine.
The sugar-based composition may include a sugar-based composition, vitamin C, resveratrol, and L-arginine.
The sugar-based composition may include a sugar-based composition, vitamin C, taurine, and L-arginine.
The sugar-based composition may include a sugar composition, vitamin C, taurine, and a Caspase9 inhibitor.
The carbohydrate-based composition may include a carbohydrate composition, vitamin C, L-arginine, and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition, vitamin C, resveratrol, taurine, and L-arginine.
The sugar-based composition may include a sugar-based composition, vitamin C, resveratrol, taurine, and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition and resveratrol.
The sugar-based composition may include a sugar composition, resveratrol and taurine.
The sugar-based composition may include a sugar-based 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-based 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-based composition, resveratrol, taurine, L-arginine, and a Caspase9 inhibitor.
The sugar-based composition can include a sugar-based composition and taurine.
The sugar-based composition may include a sugar-based composition, taurine, and L-arginine.
The carbohydrate-based composition may include a carbohydrate composition, taurine, and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition, taurine, L-arginine, and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition and L-arginine.
The carbohydrate-based composition may include a carbohydrate composition, L-arginine, and a Caspase9 inhibitor.
The carbohydrate-based composition may include a carbohydrate composition and a Caspase9 inhibitor.
The sugar-based composition may include a sugar-based composition, vitamin C, resveratrol, taurine, L-arginine, and a Caspase9 inhibitor.
In the present application, comprising, based on 100 parts by weight of the sugar-based composition relative to the total weight:
the saccharide composition is 90-100 parts, preferably 95-99.8495 parts;
0-2 parts of vitamin C, preferably 0.05-1 part;
0-2 parts of taurine, preferably 0.05-1 part;
0-2 parts of L-arginine, preferably 0.05-1 part;
0-2 parts of resveratrol, preferably 0.000001-1 part;
the Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.
In the present application, the sugar-based composition consists of a sugar-based composition and vitamin C, resveratrol, taurine, L-arginine or Caspase9 inhibitors.
In the present application, the sugar-based composition is used in an amount of 100 parts by weight relative to the total weight,
the saccharide composition is 90-100 parts, preferably 95-99.8495 parts;
0-2 parts of vitamin C, preferably 0.05-1 part;
0-2 parts of taurine, preferably 0.05-1 part;
0-2 parts of L-arginine, preferably 0.05-1 part;
0-2 parts of resveratrol, preferably; 0.000001-1 part
The Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.
In the sugar-based composition, the content of the sugar-based composition may be 90 parts, 91 parts, 92 parts, 93 parts, 94 parts, 95 parts, 95.5 parts, 96 parts, 96.5 parts, 97 parts, 97.5 parts, 98 parts, 98.5 parts, 99 parts, 99.5 parts, or 100 parts.
The vitamin C may be contained in an amount of 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 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 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, 0.37 part, 0.38 part, 0.39 part, 0.54 part, 0.65 part, 0.75 part, 0.54 part, 0.73 part, 0.54 part, 0.73 part, 0.71 part, 0.65 part, 0.75 part, 0.54 part, 0.71 part, 0.73 part, 0.75 part, 0.71 part, 0.65 part, 0.75 part, 0.7 part, 0.73 part, 0.75 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.73 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.23 part, 0.7 part, 0.73 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0, 0.83 part, 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 part and 2 parts.
The taurine may be contained in an amount of 0.0 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 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 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, 0.65 part, 0.75 part, 0.54 part, 0.73 part, 0.54 part, 0.73 part, 0.54 part, 0.71 part, 0.65 part, 0.75 part, 0.73 part, 0.75 part, 0.71 part, 0.75 part, 0.73 part, 0.75 part, 0.23 part, 0.71 part, 0.73 part, 0.23 part, 0.75 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.7 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.23 part, 0.7 part, 0.73 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.23, 0.83 part, 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 part and 2 parts.
The L-arginine may be contained in an amount of 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 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 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, 0.37 part, 0.38 part, 0.54 part, 0.75 part, 0.54 part, 0.65 part, 0.75 part, 0.54 part, 0.73 part, 0.53 part, 0.54 part, 0.73 part, 0.71 part, 0.73 part, 0.75 part, 0.71 part, 0.75 part, 0.53 part, 0.7 part, 0.73 part, 0.23 part, 0.53 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.82 part, 0.83 part, 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 parts and 2 parts.
The resveratrol may be contained in 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 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 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, 0.37 part, 0.38 part, 0.39 part, 0.54 part, 0.65 part, 0.54 part, 0.73 part, 0.54 part, 0.71 part, 0.73 part, 0.53 part, 0.73 part, 0.71 part, 0.75 part, 0.71 part, 0.53 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.75 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.53 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.7 part, 0.73 part, 0.7 part, 0.73 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.73 part, 0.7 part, 0.23 part, 0.73 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.83 part, 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 part and 2 parts.
The Caspase9 inhibitor may be contained in an amount of 0.0 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 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 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, 0.36 part, 0.37 part, 0.38 part, 0.54 part, 0.65 part, 0.54 part, 0.73 part, 0.54 part, 0.71 part, 0.65 part, 0.73 part, 0.75 part, 0.65 part, 0.75 part, 0.73 part, 0.71 part, 0.75 part, 0.71 part, 0.65 part, 0.73 part, 0.71 part, 0.75 part, 0.65 part, 0.75 part, 0.73 part, 0.71 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.73 part, 0.23 part, 0.54 part, 0.23 part, 0.73 part, 0.54 part, 0.75 part, 0.23 part, 0.73 part, 0.54 part, 0.73 part, 0.23 part, 0.54 part, 0.23 part, 0.75 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.75 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.73 part, 0.23 part, 0.54 part, 0.75 part, 0.23 part, 0.7 part, 0.54 part, 0.73 part, 0.75 part, 0.23 part, 0.7 part, 0.73 part, 0.23 part, 0.7 part, 0.23 part, 0.7 part, 0.23 part, 0.83 part, 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 part and 2 parts.
The application provides a preparation method of a glycosyl composition, which comprises the steps of weighing the carbohydrate composition and an inhibitor of vitamin C, resveratrol, taurine, L-arginine or Caspase9 according to the proportion, and then uniformly mixing the carbohydrate composition and the inhibitor of vitamin C, resveratrol, taurine, L-arginine or Caspase9 to obtain the glycosyl composition.
The application provides a freezing stock solution, which comprises DMSO, dextran 40 and the glycosyl composition.
In the present application, the frozen stock solution comprises the following components in parts by weight relative to 100 parts by weight of the total weight:
2.5-20 parts of DMSO (dimethyl sulfoxide), preferably 5-10 parts;
2.5-20 parts of dextran 40, preferably 10-20 parts;
the sugar-based composition is 60 to 95 parts, preferably 70 to 85 parts.
In the present application, the freezing medium is composed of DMSO, dextran 40, sugar-based composition and physiological saline.
In the present application, the frozen stock solution is prepared by mixing, based on 100 parts by weight of the frozen stock solution relative to the total weight,
2.5-20 parts of DMSO (dimethyl sulfoxide), preferably 5-10 parts;
2.5-20 parts of dextran 40, preferably 10-20 parts;
the sugar-based composition is 60-95 parts, preferably 70-85 parts;
the balance being normal saline.
The application provides a preparation method of a frozen stock solution, which comprises the following steps: respectively preparing the dextran 40 and the glycosyl composition into storage solutions, then weighing DMSO, the dextran 40 storage solution, the glycosyl composition storage solution and normal saline according to the proportion, and then uniformly mixing the DMSO, the dextran 40 storage solution, the glycosyl composition storage solution and the normal saline to obtain the frozen stock solution.
The application provides an application of the cryopreservation solution in the field of cell cryopreservation.
Examples
The experimental methods used in the following examples are all conventional methods, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
K562 cells were purchased from ATCC cell banks.
Cryostor CS10 frozen stock solution: purchased from stem cell corporation.
Apcannexin v: purchased from BioLegend.
A: preparing a first storage liquid:
weighing trehalose: 5.07g, sucrose: 10.25g, melibiose: 5.07g, mannan: 5g, dextran 40: 10g, the weighed ingredients were transferred into 50ml centrifuge tubes, respectively. And adding normal saline for injection, and respectively metering to 50 ml. Then respectively adopting 0.22 mu m filter membrane to sterilize and filter, subpackaging and transferring to a sterile reagent bottle to prepare trehalose stock solution, sucrose stock solution, melibiose stock solution, mannan stock solution and dextran 40 stock solution. Stored at 4 ℃ and used within 6 months. The method is suitable for the embodiments 1-3 (the volume of the frozen stock solution prepared by the embodiments 1-3 is 100 ml).
B: preparation of the second stock solution
Weighing trehalose: 7.56g, sucrose: 6.84g, melibiose: 6.84g, mannan: 10g of the mixture was mixed and dissolved in water, and the dissolved mixture was transferred to a 100ml volumetric flask, and then physiological saline for injection was added thereto to a constant volume of 100 ml. Then sterilizing and filtering by adopting a 0.22 mu m filter membrane, subpackaging and transferring to a sterile reagent bottle to obtain the saccharide composition stock solution. Stored at 4 ℃ and used within 6 months.
40 g of dextran is weighed and dissolved in 20g of dextran, and then the volume is adjusted to 100ml by using physiological saline. Then a filter membrane with the diameter of 0.22 mu m is adopted for sterilization and filtration, and the dextran 40 storage solution can be obtained. Stored at 4 ℃ and used within 6 months.
Weighing vitamin C, dissolving 0.5g of vitamin C, then using normal saline to fix the volume to 5ml, and then adopting a filter membrane with the diameter of 0.22 mu m to perform aseptic filtration to obtain the vitamin C stock solution.
Weighing 2.28mg of resveratrol, dissolving, then using normal saline to fix the volume to 10ml, and then adopting a 0.22 mu m filter membrane to perform aseptic filtration to obtain resveratrol stock solution.
Weighing 0.5g of L-arginine to dissolve, then using normal saline to fix the volume to 5ml, and then adopting a 0.22 mu m filter membrane to perform aseptic filtration to obtain L-arginine stock solution.
Weighing 0.25 of taurine, dissolving, then fixing the volume to 5ml by using normal saline, and then sterilizing and filtering by adopting a 0.22 mu m filter membrane to obtain taurine stock solution.
Weighing 1mg of Caspase9 inhibitor, adding 213 ul of sterile DMSO, and dissolving to obtain Caspase9 inhibitor stock solution.
Second stock solution was applied to example 4, example 5, example 6, example 7, example 8, example 9, example 10, example 11, example 12, example 13, comparative example 2 and comparative example 3, and the volume of the prepared stock solution was 10 ml.
Example 1
10ml of trehalose stock solution, 2.5ml of DMSO, 50ml of dextran 40 stock solution and 37.5ml of physiological saline for injection were mixed to obtain a frozen stock solution.
Example 2
A10 ml sucrose stock solution was taken, and 5ml DMSO, 50ml dextran 40, and 37.5ml physiological saline for injection were added thereto and mixed to obtain a frozen stock solution.
Example 3
A frozen stock solution was prepared by collecting 10ml of melibiose stock solution, adding 5ml of DMSO, 50ml of dextran 40, and 37.5ml of physiological saline for injection, and mixing.
Example 4
1ml of the saccharide composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, and 3.5ml of physiological saline for injection were mixed to obtain a frozen stock solution.
Example 5
1ml of the saccharide composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 100. mu.l of vitamin C stock solution, 100. mu.l of resveratrol stock solution, 100. mu. l L-arginine stock solution, 200. mu.l of taurine stock solution, 30. mu.l of Caspase9 inhibitor stock solution and 2.97ml of physiological saline for injection are mixed to obtain a frozen stock solution.
Example 6
1ml of the saccharide composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 100. mu.l of vitamin C stock solution, and 3.4ml of physiological saline for injection were mixed to obtain a frozen stock solution.
Example 7
Mixing 1ml saccharide composition stock solution, 0.5ml DMSO, 5ml dextran 40 stock solution, 100 μ l resveratrol stock solution, and 3.4ml physiological saline for injection to obtain frozen stock solution.
Example 8
1ml of the saccharide composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 100. mu. l L-arginine stock solution, and 3.4ml of physiological saline for injection were mixed to obtain a frozen stock solution.
Example 9
1ml of the saccharide composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 30. mu.l of Caspase9 inhibitor stock solution, and 3.47ml of physiological saline for injection were mixed to obtain a frozen stock solution.
Example 10
Mixing 1ml saccharide composition, 0.5ml DMSO, 5ml dextran 40 stock solution, 100 μ l vitamin C stock solution, 100 μ l resveratrol stock solution, and 3.3ml physiological saline for injection to obtain frozen stock solution.
Example 11
Mixing 1ml saccharide composition stock solution, 0.5ml DMSO, 5ml dextran 40 stock solution, 100 μ l vitamin C stock solution, 100 μ l resveratrol stock solution, 100 μ l L-arginine stock solution, and 3.2ml physiological saline for injection to obtain frozen stock solution.
Example 12
Mixing 1ml saccharide composition stock solution, 0.5ml DMSO, 5ml dextran 40 stock solution, 100 μ l resveratrol stock solution, 100 μ l L-arginine stock solution, 30 μ l Caspase9 inhibitor stock solution, and 3.27ml physiological saline for injection to obtain frozen stock solution.
Example 13
Mixing 1ml saccharide composition stock solution, 0.5ml DMSO, 5ml dextran 40 stock solution, 100 μ l resveratrol stock solution, 100 μ l L-arginine stock solution, and 3.3ml physiological saline for injection to obtain frozen stock solution.
Comparative example 1
0.5ml of DMSO, 5ml of dextran 40 stock solution and 4.5ml of physiological saline for injection were mixed to obtain a frozen stock solution.
Comparative example 2
Mixing 1ml of glycosyl composition stock solution, 0.5ml of DMSO, 5ml of dextran 40 stock solution, 100 mul of resveratrol stock solution, 100 mul of taurine stock solution, 30 mul of Caspase9 inhibitor stock solution and 3.27ml of normal saline for injection to obtain a frozen stock solution.
Comparative example 3 is Cryostor CS10 frozen stock solution from STEMCELL corporation.
Table 1 is a comparative table of raw material compositions of examples and comparative examples
Figure BDA0003451271740000131
Figure BDA0003451271740000141
Test examples
1. Rate of cell viability
And (4) selecting the K562 cells after amplification culture for cryopreservation. The frozen stock solutions of examples 1 to 13 and comparative examples 1 to 3 were subjected to freezing experiments. In particular, the frozen stock solution is used for resuspension400g, 5 minutes room temperature centrifugation of K562 cells, using 1.8ml of the frozen tube frozen, cell density of 1X 107Each volume is 1ml, and the frozen volume is 1 ml. The cells and the frozen stock solution are uniformly mixed and then added into a freezing tube, and then the mixture can be transferred to a refrigerator with the temperature of-80 ℃ for cooling, frozen in the refrigerator with the temperature of-80 ℃ for overnight and then transferred into a liquid nitrogen tank for storage (for 7 days or 14 days in the liquid nitrogen tube). And (3) a recovery process, namely opening the constant-temperature water bath kettle, heating the water to 37.5 +/-0.5 ℃, taking the cells out of the liquid nitrogen tank, and transferring the cells to the side of the water bath kettle by using a container filled with liquid nitrogen. The cells were rapidly transferred to a water bath and thawed rapidly by gentle shaking, the entire thawing process was controlled within 2 minutes, and the results are shown in table 2. The cell viability rate is measured by flow cytometry and PI and Annexin V are simultaneously detected, wherein the PI and Annexin V double-negative cell population is a viable cell population, and the ratio of the PI and Annexin V double-negative cell population is a viable cell ratio. The number of viable cells according to the present invention was obtained by counting analysis using a cell counter (Nexcelom model: K2) after staining with AO/PI, and the results are shown in Table 2 and FIGS. 1 to 4.
TABLE 2 survival rate and number of viable cells of K562 cells after freezing test using the freezing solutions of each example and comparative example
Figure BDA0003451271740000142
Figure BDA0003451271740000151
And (3) knotting: as can be seen from the above table and FIGS. 1-4, after K562 cells were cryopreserved for 14 days, and then resuscitated in a 37 ℃ water bath, the cell viability rate (81.58% + -2.42%) of example 4 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 that of the commercial cryopreserved solution (comparative example 1) (72.38% + -10.86%) and also higher than that of the single-saccharide formulation (example 1: 61.25% + -9.28%, example 2: 69.54% + -3.86%, example 3: 65.81% + -4.40%). Although there is no significant difference in the recovery rate of the cells compared with example 4 in example 5, the recovery rate of the recovered cells represented by example 5 is higher when the number of the recovered cells in example 5 is greater than that in example 4 under the condition of the same number of the frozen cells. By performing single or multiple removals of the added vitamin C, resveratrol, L-arginine, taurine, Caspase9 inhibitors in the frozen stock solution of example 5 (examples 10-20), the cell viability was reduced for each group at different time points after removals compared to example 5.
While embodiments of the present application have been described above in connection with specific embodiments thereof, the present application is not limited to the above-described embodiments and fields of application, which are intended to be illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto and changes may be made without departing from the scope of the invention as defined by the appended claims.

Claims (14)

1. A saccharide composition comprising at least one of trehalose, sucrose, melibiose and mannan.
2. The saccharide composition according to claim 1, wherein the saccharide composition comprises, 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;
0-100 parts of cane sugar, preferably 0.1-50 parts;
0-100 parts of melibiose, preferably 0.1-50 parts;
the mannan is 0-100 parts, preferably 0.5-50 parts.
3. The saccharide composition according to claim 1, wherein the saccharide composition consists of trehalose, sucrose, melibiose and mannan.
4. The saccharide composition according to claim 3, wherein the saccharide composition is 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;
0-100 parts of cane sugar, preferably 0.1-50 parts;
0-100 parts of melibiose, preferably 0.1-50 parts;
the mannan is 0-100 parts, preferably 0.5-50 parts.
5. A carbohydrate-based composition comprising a carbohydrate composition and at least one of vitamin C, resveratrol, taurine, L-arginine, or a Caspase9 inhibitor.
6. The sugar-based composition according to claim 5, wherein the sugar-based composition is according to any one of claims 1 to 4.
7. The sugar-based composition according to claim 5, comprising, based on 100 parts by weight of the sugar-based composition relative to the total weight:
the saccharide composition is 90-100 parts, preferably 95-99.8495 parts;
0-2 parts of vitamin C, preferably 0.05-1 part;
0-2 parts of taurine, preferably 0.05-1 part;
0-2 parts of L-arginine, preferably 0.05-1 part;
0-2 parts of resveratrol, preferably 0.000001-1 part;
the 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 consists of a sugar-based composition and an inhibitor of vitamin C, resveratrol, taurine, L-arginine or Caspase 9.
9. The sugar-based composition according to claim 8, wherein the sugar-based composition is present in an amount of 100 parts by weight based on the total weight of the sugar-based composition,
the saccharide composition is 90-100 parts, preferably 95-99.8495 parts;
0-2 parts of vitamin C, preferably 0.05-1 part;
0-2 parts of taurine, preferably 0.05-1 part;
0-2 parts of L-arginine, preferably 0.05-1 part;
0-2 parts of resveratrol, preferably 0.000001-1 part;
the Caspase9 inhibitor is 0-2 parts, preferably 0.0005-1 part.
10. A frozen stock solution comprising DMSO, dextran 40, and the sugar-based composition of any one of claims 5 to 9.
11. The cryopreservation liquid according to claim 10, comprising, based on 100 parts by weight of the cryopreservation liquid relative to the total weight:
2.5-20 parts of DMSO (dimethyl sulfoxide), preferably 5-10 parts;
2.5-20 parts of dextran 40, preferably 10-20 parts;
the sugar-based composition is 60 to 95 parts, preferably 70 to 85 parts.
12. The cryopreservation solution of claim 10, wherein the cryopreservation solution consists of DMSO, dextran 40, a sugar-based composition and physiological saline.
13. The cryopreservation liquid according to claim 12, comprising, based on 100 parts by weight of the cryopreservation liquid relative to the total weight:
2.5-20 parts of DMSO (dimethyl sulfoxide), preferably 5-10 parts;
2.5-20 parts of dextran 40, preferably 10-20 parts;
the sugar-based composition is 60-95 parts, preferably 70-85 parts;
the balance being normal saline.
14. Use of a cryopreservation solution according to any one of claims 10 to 13 in the field of cell cryopreservation.
CN202111674623.3A 2020-12-31 2021-12-31 Saccharide composition, saccharide-based composition and frozen stock solution Pending CN114680102A (en)

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Publication number Priority date Publication date Assignee Title
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115777689A (en) * 2022-11-09 2023-03-14 武汉赛维尔生物科技有限公司 Serum-free and protein-free non-programmed cell cryopreservation solution

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