CN112369408A - Immune cell storage liquid and preparation and application methods thereof - Google Patents

Abstract

An immune cell storage solution, wherein each 1000ml of the storage solution comprises the following components in percentage by weight: 25-75g of cane sugar; 50-150ml of glycerol; 20-30g of albumin; 5-10g of sodium bicarbonate; 0.2-0.4g of cholesterol; 5-8% mg of propylene glycol; 0.6-1.1g of vitamin E; 0.6-0.9mg of vitamin C; EDTA 0.01-0.4% mg; 65-95% ml of phosphate buffer solution; sodium selenite 0.01-0.5% mg; the balance being cell culture medium. The storage liquid which can still maintain the survival rate of more than 80 percent of cells and the adherence rate of more than 70 percent after 48 hours in the storage or transportation process can meet the short-distance transportation requirement of the fresh mesenchymal stem cells, and the storage liquid uses clinical medicines with definite components, is safe to human bodies, can be directly injected after being recovered to room temperature, and is greatly convenient for the clinical application of the mesenchymal stem cell preparation after the short-distance transportation.

Description

Immune cell storage liquid and preparation and application methods thereof

Technical Field

The invention relates to the field of immune cell storage, in particular to immune cell storage liquid and a preparation method and an application method thereof.

Background

Immune cells are a group of cells involved in or associated with immune responses, and mainly include lymphocyte populations, monocytes, macrophages, dendritic cells and the like, which are collectively referred to as peripheral blood mononuclear cells. They play an important role in stabilizing the health and well-being of the human body, and play roles in immune defense, immune stabilization and immune surveillance of the human body.

In recent years, the immunotherapy technology of tumors and viruses is applied to clinic, and the immunity of patients can be improved by stimulating, culturing, amplifying and then returning autologous immune cells in vitro. However, the immunity of the human body reaches the peak at the age of 20 years, the immune system gradually goes down the slope with the age of the book, and at the age of 40 years, the immunity is only 1/2 at the peak; by age 70, 1/10 with peak is left. In case of failure to obtain healthy immune cells, the therapeutic effect is reduced. If healthy immune cells are stored in advance, it is more likely that accurate cells can be found to fight the risk of cancer, and at the same time, anti-aging beauty can be achieved.

The immune cell therapy is mainly from the patient's own peripheral blood, and the peripheral blood mononuclear cells are obtained by separation and collection. However, in general, the population receiving immune cell therapy is mainly tumor or virus infected people, and the immune cell function of the people is very low, and the activity of the isolated immune cells is low. Therefore, when the activity of the autoimmune cells is strong, people can store healthy cells in advance so as to treat diseases and meet the need of resisting aging and beautifying.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides an immune cell storage solution and a preparation and application method thereof.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: an immune cell storage solution, wherein each 1000ml of the storage solution comprises the following components in percentage by weight:

25-75g of cane sugar;

50-150ml of glycerol;

20-30g of albumin;

5-10g of sodium bicarbonate;

0.2-0.4g of cholesterol;

5-8% mg of propylene glycol;

0.6-1.1g of vitamin E;

0.6-0.9mg of vitamin C;

EDTA 0.01-0.4% mg;

65-95% ml of phosphate buffer solution;

sodium selenite 0.01-0.5% mg;

the balance being cell culture medium.

The improvement of the invention is that every 1000ml of storage liquid comprises the following components and contents:

55g of cane sugar;

100ml of glycerol;

25g of albumin;

7g of sodium bicarbonate;

0.3g of cholesterol;

6% mg of propylene glycol;

vitamin E1 g;

vitamin C0.7mg;

EDTA 0.2% mg;

the phosphate buffer solution is 75% ml;

sodium selenite 0.2% mg;

the balance being cell culture medium.

In the improvement of the invention, the cell culture medium is DMEM/F12 medium.

The invention further provides a preparation method of the immune cell storage solution, which comprises the following steps:

step 1, mixing the materials according to the proportion of any one of claims 1 to 3, uniformly mixing, and precooling at 2-5 ℃ to obtain stock solution;

and 2, centrifuging the stock solution at 3000r/min of 2000-3000r/min for 10-20min, and then taking the supernatant to obtain the immune cell storage solution.

The invention further provides a using method of the immune cell storage solution, which comprises the following steps:

step 1, washing cells for 2-3 times by using normal saline, uniformly coating the cells in a culture dish, placing the culture dish in a shaking table at 37 ℃ and 80rpm, and incubating for 10 min;

step 2, sucking out residual digestive juice by using a pipette, adding the storage solution in any one of claims 1 to 3 to an infiltration state, placing the mixture in a shaking table at 37 ℃ and 70rpm, and incubating for 100min to promote cell adherence;

step 3, after the cells adhere to the wall, adding a small amount of storage liquid, culturing in an environment with the concentration and content of CO2 of 5% and the humidity of 60-80% at 37 ℃, and observing and recording the separation and growth conditions of the cells;

and 4, replacing the working culture solution once in 2-3 days.

(III) advantageous effects

Compared with the prior art, the invention provides an immune cell storage solution and a preparation and application method thereof, and the immune cell storage solution has the following beneficial effects: the storage solution with novel proportion is a storage solution which can still maintain the survival rate of more than 80% and the anchorage rate of more than 70% of cells after 48 hours in storage or transportation, can meet the short-distance transportation requirement of fresh mesenchymal stem cells, is a clinical medicine with definite use components, is safe to human bodies, can be directly injected after being recovered to room temperature, and is greatly convenient for clinical application after the short-distance transportation of mesenchymal stem cell preparations.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

Example 1

The invention provides immune cell storage liquid, wherein each 1000ml of storage liquid comprises the following components in percentage by weight:

25g of cane sugar;

50ml of glycerol;

20g of albumin;

5g of sodium bicarbonate;

0.2g of cholesterol;

propylene glycol 5% mg;

vitamin E0.6g;

vitamin C0.6mg;

EDTA 0.01% mg;

the phosphate buffer solution is 65% ml;

sodium selenite 0.01% mg;

the balance being cell culture medium.

Example 2

The invention provides immune cell storage liquid, wherein each 1000ml of storage liquid comprises the following components in percentage by weight:

55g of cane sugar;

100ml of glycerol;

25g of albumin;

7g of sodium bicarbonate;

0.3g of cholesterol;

6% mg of propylene glycol;

vitamin E1 g;

vitamin C0.7mg;

EDTA 0.2% mg;

the phosphate buffer solution is 75% ml;

sodium selenite 0.2% mg;

the balance being cell culture medium.

Example 3

The invention provides immune cell storage liquid, wherein each 1000ml of storage liquid comprises the following components in percentage by weight:

75g of cane sugar;

150ml of glycerol;

30g of albumin;

10g of sodium bicarbonate;

0.4g of cholesterol;

8% mg of propylene glycol;

vitamin E1.1g;

vitamin C0.9 mg;

EDTA 0.4% mg;

the phosphate buffer solution is 95% ml;

sodium selenite 0.5% mg;

the balance being cell culture medium.

In three examples, the effect of each ingredient is as follows:

albumin (Albumin), which is also an impermeable cryoprotectant, primarily regulates intracellular water homeostasis and maintains the osmotic pressure of the cell solution.

Propylene Glycol (1, 2-Propanediol), osmotic cryoprotectant, can diffuse and permeate cell membrane and permeate into cells, balance the osmotic pressure inside and outside cells, reduce the concentration of electrolyte in the solution inside and outside cells, avoid excessive dehydration of cells and protect cells from being damaged by high-concentration electrolyte.

Cholesterol (Cholesterol) is an important component of cell membrane, and can interfere with the ordering at low temperature, prevent the formation of ice crystals, maintain the fluidity and keep the normal physiological function and activity of cells.

EDTA: is a divalent metal chelating agent, an anticoagulant, and can inhibit nuclease activity.

Phosphate buffer solution: is a commonly used buffer solution for biological studies to help maintain a constant pH, maintain system osmolality and ionic concentration, usually close to human pH (isotonic).

Sodium selenite: selenium is a component of glutathione peroxidase, maintains the function of cell membranes through oxidation, and increases the yield of endogenous antioxidants of the lipid nature of proteins. Participate in energy conversion, influence metabolism, and play an extremely important role in fat emulsification, fat absorption and multivitamin absorption. Meanwhile, the enzyme also participates in the synthesis of coenzyme A and coenzyme Q, and influences the functions of other biological enzyme systems. Has effects on amino acid metabolism, protein synthesis, carbohydrate metabolism, and biological oxidation.

The components are chemical reagents or biological products and bulk drugs commonly used in the field of biological medicine, when the components are used for injecting human bodies, medicinal products approved by the national food and drug administration are preferably used, and human serum albumin is preferably used as albumin.

The invention also provides a preparation method of the immune cell storage solution, which comprises the following steps:

step 1, according to the formula of the 3 embodiments, uniformly mixing, and precooling at 2-5 ℃ to obtain a stock solution;

and 2, centrifuging the stock solution at 3000r/min of 2000-3000r/min for 10-20min, and then taking the supernatant to obtain the immune cell storage solution.

The invention further provides a using method of the immune cell storage solution, which comprises the following steps:

step 1, washing cells for 2-3 times by using normal saline, uniformly coating the cells in a culture dish, placing the culture dish in a shaking table at 37 ℃ and 80rpm, and incubating for 10 min;

step 2, sucking out residual digestive juice by using a pipette, adding the storage solution of the 3 embodiments to an infiltration state, placing the mixture in a shaking table at 37 ℃ and 70rpm, and incubating for 100min to promote cell adherence;

step 3, after the cells adhere to the wall, adding a small amount of storage liquid, culturing in an environment with the concentration and content of CO2 of 5% and the humidity of 60-80% at 37 ℃, and observing and recording the separation and growth conditions of the cells;

and 4, replacing the working culture solution once in 2-3 days.

Sampling is carried out at 0h, 6h, 15h, 24h and 48h respectively, the survival rate of the interstitial cells (adopting trypan blue dye exclusion method) and the adherence rate are detected respectively, the adherence rate is adherent cells/(adherent cells + dead cells), and the samples of 48h are taken for plating to carry out adipogenic differentiation detection, which is shown in the following table:

the prepared cell preparation is injected into 5 mice in tail vein, each injection is 1mL, and the abnormal reaction of the mice is observed for one week.

The activity after cell recovery is shown in the following table:

it can be seen through experiments that the cell culture solution of the present invention also maintains high activity after cell recovery.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (5)

1. An immune cell stock solution, comprising: every 1000ml of storage solution comprises the following components and contents:

25-75g of cane sugar;

50-150ml of glycerol;

20-30g of albumin;

5-10g of sodium bicarbonate;

0.2-0.4g of cholesterol;

5-8% mg of propylene glycol;

0.6-1.1g of vitamin E;

0.6-0.9mg of vitamin C;

EDTA 0.01-0.4% mg;

65-95% ml of phosphate buffer solution;

sodium selenite 0.01-0.5% mg;

the balance being cell culture medium.

2. The immune cell storage solution of claim 1, wherein: every 1000ml of storage solution comprises the following components and contents:

55g of cane sugar;

100ml of glycerol;

25g of albumin;

7g of sodium bicarbonate;

0.3g of cholesterol;

6% mg of propylene glycol;

vitamin E1 g;

vitamin C0.7mg;

EDTA 0.2% mg;

the phosphate buffer solution is 75% ml;

sodium selenite 0.2% mg;

the balance being cell culture medium.

3. The immune cell storage solution of claim 1, wherein: the cell culture medium is DMEM/F12 medium.

4. A preparation method of immune cell stock solution is characterized by comprising the following steps:

step 1, mixing the materials according to the proportion of any one of claims 1 to 3, uniformly mixing, and precooling at 2-5 ℃ to obtain stock solution;

and 2, centrifuging the stock solution at 3000r/min of 2000-3000r/min for 10-20min, and then taking the supernatant to obtain the immune cell storage solution.

5. The method for using the immune cell stock solution is characterized by comprising the following steps:

step 1, washing cells for 2-3 times by using normal saline, uniformly coating the cells in a culture dish, placing the culture dish in a shaking table at 37 ℃ and 80rpm, and incubating for 10 min;

step 2, sucking out residual digestive juice by using a pipette, adding the storage solution in any one of claims 1 to 3 to an infiltration state, placing the mixture in a shaking table at 37 ℃ and 70rpm, and incubating for 100min to promote cell adherence;

step 3, after the cells adhere to the wall, adding a small amount of storage liquid, culturing in an environment with the concentration and content of CO2 of 5% and the humidity of 60-80% at 37 ℃, and observing and recording the separation and growth conditions of the cells;

and 4, replacing the working culture solution once in 2-3 days.