阅读说明：本技术 细胞运输保护液及制备方法和应用 (Cell transportation protective solution, preparation method and application ) 是由 焦晓敏 杜宏武 于 2019-11-21 设计创作，主要内容包括：本发明提供了一种细胞运输保护液及制备方法和应用,涉及生物技术领域,本发明提供的细胞运输保护液包括保护剂、基础培养基、白蛋白、抗生素和水。其中,保护剂包括纤维素醚、环糊精、聚合醇或泊洛沙姆188,以上述各物质作为保护剂,成本低,且能够减缓细胞生长速度,避免细胞生长过密,影响细胞活性。并且,上述保护剂还能够增加细胞运输保护液的粘稠度,有效避免运输过程中因震动摇晃引起的细胞物理损伤。同时,基础培养基中富含细胞代谢必需成分,白蛋白也可为细胞提供营养,从而有效保持细胞的活性。此外,抗生素能够起到良好的抑菌杀菌作用,维持细胞无菌生长环境。(The invention provides a cell transportation protective solution, a preparation method and application thereof, and relates to the technical field of biology. The protective agent comprises cellulose ether, cyclodextrin, polyalcohol or poloxamer 188, and the substances are used as the protective agent, so that the cost is low, the cell growth speed can be reduced, and the influence on the cell activity caused by over-dense cell growth can be avoided. Moreover, the protective agent can also increase the viscosity of the cell transportation protective solution, and effectively avoid physical damage to cells caused by vibration and shaking in the transportation process. Meanwhile, the basic culture medium is rich in essential components for cell metabolism, and the albumin can also provide nutrition for the cells, so that the activity of the cells is effectively maintained. In addition, the antibiotic can play a good role in bacteriostasis and sterilization and maintain the aseptic growth environment of cells.)

1. A cell transport protection solution, comprising:

protective agent, basic culture medium, albumin, antibiotic and water;

the protective agent comprises cellulose ether, cyclodextrin, polymeric alcohol, or poloxamer 188.

2. The solution of claim 1, wherein the cellulose ether comprises hydroxypropyl cellulose or sodium carboxymethyl cellulose;

preferably, the carboxymethyl cellulose sodium salt comprises a low or medium viscosity carboxymethyl cellulose sodium salt.

3. The cell transport protection solution of claim 1, wherein the cyclodextrin comprises α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, 2-hydroxypropyl- β -1-cyclodextrin, 2-hydroxypropyl- γ -cyclodextrin, or methyl- β -cyclodextrin.

4. A cell transport protection solution according to claim 1, wherein the polymeric alcohol comprises polyvinyl alcohol or a polyol, preferably the polyol is polyethylene glycol.

5. The cell transport protection solution of claim 1, wherein the base medium comprises MEM or DMEM;

preferably, the albumin is from human or other animal source, preferably fetal bovine serum.

6. The solution according to claim 1, wherein the antibiotics comprise penicillin and streptomycin;

preferably, the mass ratio of penicillin to streptomycin is 1-2.5:1, more preferably 1: 1;

preferably, the water is sterile deionized water.

7. The cellular transport protection solution of claim 1, wherein each 1L of the cellular transport protection solution comprises:

2-15g of protective agent, 0.1-0.3L of basic culture medium, 0.01-0.03L of albumin, 0.2-1.5g of antibiotic and the balance of water;

preferably, each 1L of said cell trafficking protection solution comprises:

3-12g of protective agent, 0.12-0.25L of basal medium, 0.012-0.028L of albumin, 0.3-1.2g of antibiotic and the balance of water;

preferably, each 1L of said cell trafficking protection solution comprises:

5-10g of protective agent, 0.15-0.2L of basic culture medium, 0.015-0.025L of albumin, 0.4-1g of antibiotic and the balance of water.

8. The cell transportation protective solution of any one of claims 1-7, wherein the cells comprise adherent cells;

preferably, the cells comprise mesenchymal stem cells.

9. A process for preparing a protective solution for cell trafficking according to any of claims 1-8 wherein the protective agent, basal medium, albumin, antibiotic and water are taken and mixed to homogeneity to obtain the protective solution for cell trafficking.

10. Use of a cell trafficking protection solution of any one of claims 1-8 in cell trafficking.

Technical Field

The invention relates to the technical field of biology, in particular to a cell transportation protective solution, a preparation method and application thereof.

Background

With the development of medicine, in the clinical application and scientific research practice process, the requirements of people on preservation of isolated organs, tissues, cells and the like are continuously improved, the standards are continuously improved, and higher requirements on cell survival media are also provided in the preservation, transportation and other processes of some organs or tissues to be transplanted. Not only must the viability of the organ, tissue or cell be ensured, but also the original biological activity of the organ, tissue or cell must be retained to the maximum extent, and at the same time, contamination by bacteria and the like and death of the organ, tissue or cell must be prevented. The properties of various organs, tissues or cells are different, and the preservation medium of the organ, tissue or cell has different requirements in the processes of preservation, transportation and the like.

Stem cells (Stem cells), which are highly undifferentiated and have the potential function of regenerating various tissues and organs and the human body, are called "universal cells" in the medical field, and under certain conditions, can be differentiated into various functional cells. The stem cells have differentiation potential, are directionally induced to proliferate and differentiate into corresponding tissues and organs under proper conditions, and have extraordinary significance in clinical treatment, such as the functions of repairing damaged tissue cells and replacing damaged cells, or the function of stimulating the regeneration of body self cells. Therefore, the research and application of stem cells are greatly diversified in the medical field. Particularly, mesenchymal stem cells have been developed rapidly in recent years.

Mesenchymal Stem Cells (mesnchymal Stem Cells) are derived from the mesoderm and are multipotent Stem Cells of a non-hematopoietic origin. The mesenchymal stem cells are characterized by weak immunogenicity and difficult immunological rejection with a host, so the mesenchymal stem cells become ideal stem cell seed sources in multiple fields. In the technical field of tissue engineering, tissue engineering materials carrying stem cells are used for repairing diseased tissues, and in clinical application of treatment of infantile cerebral palsy and myocardial infarction, the functional reconstruction of necrotic tissues by injecting mesenchymal stem cells is achieved successfully. Therefore, the mesenchymal stem cells have great application prospect.

At present, in order to ensure the activity of mesenchymal stem cells in the transportation process, the cells are generally transported by using liquid nitrogen or dry ice after being frozen, the transportation condition is very harsh, the transportation process needs to be ensured to be in a low-temperature state, and the cost is high. And if the cells are repeatedly frozen and thawed in the transportation process, the survival rate of the cells can be influenced. In addition, the state of the received cells cannot be directly observed, and no guarantee is provided for subsequent research experiments.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is a first object of the present invention to provide a cell transport protective solution that alleviates at least one of the technical problems of the prior art.

The second purpose of the invention is to provide a preparation method of the cell transportation protective solution.

The third purpose of the invention is to provide the application of the cell transportation protective solution in cell transportation.

The invention provides a cell transportation protective solution, which comprises:

protective agent, basic culture medium, albumin, antibiotic and water;

the protective agent comprises cellulose ether, cyclodextrin, polymeric alcohol, or poloxamer 188.

Further, the cellulose ether comprises hydroxypropyl cellulose or sodium carboxymethyl cellulose;

preferably, the carboxymethyl cellulose sodium salt comprises a low or medium viscosity carboxymethyl cellulose sodium salt.

Further, the cyclodextrin includes α -cyclodextrin, β -cyclodextrin, gamma-cyclodextrin, 2-hydroxypropyl- β -1-cyclodextrin, 2-hydroxypropyl-gamma-cyclodextrin or methyl- β -cyclodextrin.

Further, the polymeric alcohol comprises polyvinyl alcohol or a polyol, preferably the polyol is polyethylene glycol.

Further, the basal medium comprises MEM or DMEM;

preferably, the albumin is from human or animal origin, preferably fetal bovine serum.

Further, the antibiotics include penicillin and streptomycin;

preferably, the mass ratio of penicillin to streptomycin is 1-2.5:1, more preferably 1: 1;

preferably, the water is sterile deionized water.

Further, each 1L of the cell transport protective solution comprises:

2-15g of protective agent, 0.1-0.3L of basic culture medium, 0.01-0.03L of albumin, 0.2-1.5g of antibiotic and the balance of water;

preferably, each 1L of said cell trafficking protection solution comprises:

3-12g of protective agent, 0.12-0.25L of basal medium, 0.012-0.028L of albumin, 0.3-1.2g of antibiotic and the balance of water;

preferably, each 1L of said cell trafficking protection solution comprises:

5-10g of protective agent, 0.15-0.2L of basic culture medium, 0.015-0.025L of albumin, 0.4-1g of antibiotic and the balance of water.

Further, the cells comprise adherent cells;

preferably, the cells comprise mesenchymal stem cells.

The invention also provides a preparation method of the cell transportation protective solution, which comprises the steps of taking the protective agent, the basal medium, the albumin, the antibiotic and the water, and uniformly mixing to obtain the cell transportation protective solution.

In addition, the invention also provides application of the cell transportation protective solution in cell transportation.

Compared with the prior art, the invention has the following beneficial effects:

the cell transportation protective solution provided by the invention comprises a protective agent, a basic culture medium, albumin, antibiotics and water. The protective agent comprises cellulose ether, cyclodextrin, polyalcohol or poloxamer 188, and the substances are used as the protective agent, so that the cost is low, the cell growth speed can be reduced, and the influence on the cell activity caused by over-dense cell growth can be avoided. Moreover, the protective agent can also increase the viscosity of the cell transportation protective solution, and effectively avoid the damage of the liquid to cells caused by vibration or shaking in the transportation process. Meanwhile, the basic culture medium is rich in components necessary for cell metabolism, so that the activity of the cells can be effectively maintained. Albumin may also provide nutrients to the cells. In addition, the antibiotic can play a good role in bacteriostasis and sterilization and maintain the aseptic growth environment of cells.

The preparation method of the cell transportation protective solution provided by the invention has the advantages of simple process and convenient operation, and the cell transportation protective solution can be obtained by dissolving and uniformly mixing the protective agent, the basic culture medium, the albumin, the antibiotic and the water. The preparation method does not need to use expensive instruments, can effectively save cost, and is suitable for large-scale production and application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other results can be obtained according to the drawings without creative efforts for those skilled in the art.

FIG. 1 is a graph showing the results of cell survival after passage using the cell transport protective solution provided in example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are a part of the present invention, and not all of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the environment of normal temperature or 37 ℃, the cells grow actively, the biochemical reaction in the cells is active, the metabolism is vigorous, the oxygen consumption is high, and the energy consumption is large. After the cells grow too densely, growth inhibition can occur, the cells are stimulated to secrete adverse factors, and changes in environmental pH and osmotic pressure can be caused, so that the cells are swelled and killed. This problem is more pronounced in cell transport, so cells are usually transported after freezing with liquid nitrogen or dry ice. And (3) carrying out non-cell culture state transportation at low temperature of liquid nitrogen or dry ice, dissociating adherent cells, freezing and storing, and then placing in the liquid nitrogen or the dry ice for low temperature transportation. In the frozen state, the cells stop vital activities and metabolism and go "dormant". However, the requirements of the refrigerated transportation on transportation conditions are very strict, the cost is high, and the cell viability is low, so that the refrigerated transportation is difficult to be widely adopted in the practical application process.

Based on this, the invention provides a cell transport protective solution, comprising:

protective agent, basic culture medium, albumin, antibiotic and water;

the protective agent comprises cellulose ether, cyclodextrin, polymeric alcohol, or poloxamer 188.

The cellulose ether, cyclodextrin, polyalcohol or poloxamer 188 is used as a protective agent, so that the cost is low, the cell growth speed can be effectively slowed down, and the influence on the cell activity caused by over-dense cell growth is avoided. Moreover, the protective agent can also increase the viscosity of the cell transportation protective solution, and avoid cell damage caused by vibration or shaking in the transportation process.

By cellulose ether is meant a group that is converted by one or more hydroxyl groups present on one or more anhydroglucose repeat units in the cellulose polymer to provide one or more ether linkages on the cellulose polymer, thereby forming a cellulose ether. To further save costs on the basis of ensuring a protective effect, in some preferred embodiments the cellulose ether may be selected from hydroxypropyl cellulose (HPC) or different grades of sodium carboxymethyl cellulose. Preferably, the different grades of carboxymethyl cellulose sodium salt may be selected from low viscosity carboxymethyl cellulose sodium salt (CMC-LV) or medium viscosity carboxymethyl cellulose sodium salt (CMC-MV).

In some preferred embodiments, the cyclodextrin can be selected from α -cyclodextrin (α -CD), β -cyclodextrin (β -CD), gamma-cyclodextrin (gamma-CD), 2-hydroxypropyl- β -1-cyclodextrin (HBC), 2-hydroxypropyl-gamma-cyclodextrin (HGC) or methyl- β -cyclodextrin (MBC). each of the above cyclodextrins can effectively increase the viscosity of the cell transport protective solution, and increase the stability of the cell transport protective solution on the basis of ensuring the survival rate of cells.

Typical polymeric alcohols may be selected from polyvinyl alcohol (PVA) or polyols, polyvinyl alcohol and polyols, wherein the polyol is preferably polyethylene glycol.

In addition, poloxamer 188(Polox188) can be selected as a protective agent in the cell transportation protective solution, and poloxamer 188 not only slows down the growth speed of cells and increases the viscosity of the cell transportation protective solution, but also is beneficial to the maintenance of various growth factors after the cells are stored.

In the cell transportation protective solution provided by the invention, the basic culture medium is rich in components necessary for cell metabolism, so that the activity of cells can be effectively maintained. The type of the basic culture medium is not limited, and any basic substance which can supply cell nutrition and promote cell reproduction and proliferation in cultured cells can be used as the basic culture medium in the cell transportation protective solution.

In some preferred embodiments, the basal medium comprises MEM or DMEM. The phosphate and carbonate buffer system in the MEM can effectively keep the stability of the cell environment, and the MEM is rich in inorganic salt ions, amino acids, sugar, vitamins and other important components necessary for cell metabolism, and can effectively keep the activity of cells. DMEM is a medium containing various amino acids and glucose, which is a medium modified based on MEM medium and is also effective in maintaining cell activity.

Albumin is added into the cell transportation protective solution provided by the invention, so that nutrition can be provided for cells.

In some preferred embodiments, fetal bovine serum or human serum albumin may be selected as the albumin. Since the fetal calf is not in contact with the outside, the fetal calf serum has high quality, and contains few components harmful to cells such as antibodies and complements.

In addition, in the cell transportation protective solution provided by the invention, the antibiotic can play a good role in bacteriostasis and sterilization, and maintain a growth environment without cell pollution.

In some preferred embodiments, the antibiotic comprises penicillin and streptomycin. Penicillin can effectively inhibit and kill gram-positive bacteria, streptomycin can effectively inhibit and kill gram-negative bacteria, and the penicillin and the streptomycin are used in combination, so that the effects of preventing pollution and killing bacteria are better.

Preferably, the mass ratio of penicillin to streptomycin is 1-2.5:1, for example, but not limited to 1:1, 1.2:1, 1.5:1, 1.8:1 or 2:1, more preferably 1: 1. When the mass ratio of the streptomycin to the streptomycin is in the range, the effect of bacteriostasis and sterilization is better.

In order to further improve the matching effect of the components in the cell transportation protective solution provided by the invention, the proportion of the components is defined as follows:

each 1L of the cell trafficking protection solution comprises:

2-15g of protective agent, 0.1-0.3L of basic culture medium, 0.01-0.03L of albumin, 0.2-1.5g of antibiotic and the balance of water.

Wherein, the content of the protective agent in each 1L of the cell transportation protective solution can be, but is not limited to, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g or 15 g; the amount of basal medium in each 1L of the cell transport protective solution can be, for example, but not limited to, 0.1L, 0.2L, or 0.3L; the amount of albumin in the cell trafficking protection solution per 1L may be, for example, but not limited to, 0.01L, 0.02L, or 0.03L; the amount of antibiotic in the cell transport protective solution per 1L may be, for example, but not limited to, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1g, 1.1g, 1.2g, 1.3g, 1.4g or 1.5 g. The balance of water means that every 1L of the cell transportation protective solution comprises water except protective agents, basic culture media, albumin, antibiotics and other optional auxiliary agents or additives. Preferably, the water is sterile deionized water.

Preferably, each 1L of said cell trafficking protection solution comprises:

3-12g of protective agent, 0.12-0.25L of basal medium, 0.012-0.028L of albumin, 0.3-1.2g of antibiotic and the balance of water;

preferably, each 1L of said cell trafficking protection solution comprises:

5-10g of protective agent, 0.15-0.2L of basic culture medium, 0.015-0.025L of albumin, 0.4-1g of antibiotic and the balance of water.

By further adjusting and optimizing the proportion, the cell transportation protective solution provided by the invention can more effectively maintain the activity of cells.

Because the adherent cells are usually digested in the traditional cell transportation process, and then the adherent cells are transported at a low temperature after being frozen, the digestion and re-suspension steps can affect the state and the survival rate of the cells. The cell transportation protective solution provided by the invention can be directly applied to adherent cells, the cells in an adherent state are transported, the survival rate and the state of the cells are ensured, the growth speed of the cells in the adherent transportation protective solution is reduced, and the situation that the cells grow too densely is avoided. The cells in the growth state can be transported and the state can be directly observed after the cells are received, and the subsequent research experiment is guaranteed.

Preferably, the cell comprises a mesenchymal stem cell, and a typical mesenchymal stem cell may be a dental pulp mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell or a bone marrow mesenchymal stem cell.

The invention also provides a preparation method of the cell transportation protective solution, which comprises the steps of dissolving the protective agent, the basic culture medium, the albumin, the antibiotic and water and uniformly mixing to obtain the cell transportation protective solution.

The preparation method of the cell transportation protective solution provided by the invention has the advantages of simple process and convenient operation, and the cell transportation protective solution can be obtained by dissolving and uniformly mixing the protective agent, the basic culture medium, the albumin, the antibiotic and the water. The preparation method does not need to use expensive instruments, can effectively save cost, and is suitable for large-scale production and application.

In some specific embodiments, taking 1L of cell transportation protective solution as an example, under aseptic conditions, 0.01 to 0.03L of albumin is added into 0.1 to 0.3L of basic culture medium, 2 to 15g of protective agent and 0.2 to 1.5g of antibiotic are added, and after stirring and dissolving, sterile deionized water is added to 1L of constant volume, and the mixture is stirred uniformly to obtain the cell transportation protective solution.

In addition, the invention also provides application of the cell transportation protective solution in cell transportation.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.