CN112481208A

CN112481208A - Lymphocyte separation liquid and preparation method thereof

Info

Publication number: CN112481208A
Application number: CN202011467201.4A
Authority: CN
Inventors: 赖演媚; 刘灵辉; 赖柔贝
Original assignee: Guangzhou Br Healthcare Medical Technology Co ltd
Current assignee: Guangzhou Br Healthcare Medical Technology Co ltd
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2021-03-12

Abstract

The invention provides a lymphocyte separation solution, which comprises the following components: glycerol, ethanol, sodium chloride, tea saponin and water. The lymphocyte culture solution provided by the invention has the following advantages: 1) low toxicity. 2) The final product is filtered to obtain a low endotoxin product 3) glycerol, the recovery rate of the sample is improved 4) and the cost performance is proper. 5) Is especially suitable for stem cell separation.

Description

Lymphocyte separation liquid and preparation method thereof

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a lymphocyte separation solution and a preparation method thereof.

Background

The cell separation medium is the substance that has been in contact with the cells for the longest time. In standard separation procedures, the cells must be in contact with the separation medium for a minimum of more than 15 minutes to achieve separation. In this process, the components in the separation liquid may adhere to the cell surface or accumulate in the cell through passive diffusion, active transport of the cell to be separated, endocytosis/pinocytosis, surface adhesion, etc., and it is difficult to completely remove the components from the separated target cell by conventional washing, so that the cell separation medium has higher safety requirements.

Many lymphocyte isolate products exist on the market at present, but the application range of the lymphocyte isolate products is different. The separating medium is used for smear or diagnosis, and subsequent culture work is not needed; if the isolated lymphocytes are to be subjected to a drug test, in vitro culture is required; if the isolated lymphocyte stem cells are cultured and then applied to the subsequent treatment of the human body by back transfusion, the requirement on the safety is higher. The main components of the currently commercialized lymphocyte separation solution comprise: dextran (dextran), hydroxyethyl starch, sodium diatrizoate. These ingredients have some toxicity. The common Ficoll separating liquid shows certain toxicity including nausea, vomit, coma, etc. in slow toxicity test based on the data of chemical safety specification. In addition, after the Ficoll separating medium is added into the preservation solution, red blood cells which are not completely lysed by the preservation solution at the layered part of the preservation solution and the sample density separating medium are contacted with the polysucrose to be aggregated into a string shape and deposited at the bottom of the tube to be mixed with the sunken lymphocytes, so that the subsequent culture of the cells is influenced. The hydroxyethyl starch is a compound preparation, and is used with caution for patients with skin pruritus, liver diseases and blood coagulation dysfunction after long-term large-dose use.

Endotoxin is also an important index for measuring cell separation fluid, and is a component in the cell wall of gram-negative bacteria, namely lipopolysaccharide; it is released only when the bacteria die and dissolve or the bacterial cells are destroyed by artificial means. Endotoxins are not proteins and are therefore very heat resistant and not easily removed. Lipopolysaccharide is toxic to the host and can cause symptoms such as fever, microcirculation disturbance, endotoxin shock and disseminated intravascular coagulation, so that the endotoxin of the separation solution applied in culturing cells/reverse transfusing cells of human bodies is quite strict.

Therefore, the search for a lymphocyte separation solution with high safety has become a problem to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a sample density separating medium, and the separated cells can be safe and more active after the separating medium is added into a cell preservation solution.

In order to achieve the purpose, the invention adopts the following technical scheme:

a lymphocyte separation solution, which comprises the following components: glycerol, ethanol, sodium chloride, tea saponin and water. The lymphocyte separation liquid has low toxicity, and adopts nontoxic glycerol, ethanol, sodium chloride and tea saponin as raw materials instead of dextran (dextran) and hydroxyethyl starch with certain toxicity.

The glycerol is selected because of the non-toxicity to cells, and the glycerol can lower the freezing point of the cells, maintain the normal shape of the cells and reduce the cell death in a sample density separating medium mixed system; the glycerol is dissolved in the ethanol water solution and then is used as a medium for separating cells, so that the viscosity of the sample density separation solution can be reduced while the sample density separation solution has a better density gradient, the sinking speed of the cells can be effectively accelerated, and the equivalent cells can be collected in a shorter time; meanwhile, the glycerol in the mixed system can not lead the red blood cells to aggregate and precipitate, and can effectively remove impurities, particularly the interference of the red blood cells.

The tea saponin is selected because the tea saponin can ensure that even red blood cells are reduced into a sample density separating solution, the red blood cells can also have hemolytic reaction with the separating solution to break the cells, and the red blood cells cannot be reduced continuously after being broken into cell fragments. The addition of theasaponin ensures that the cells which eventually sink naturally to the bottom of the tube are free of red blood cells

The ethanol is selected because the ethanol improves the permeability of cells in the separation liquid, and the sodium chloride solution used for adjusting osmotic pressure in the mixed system can accelerate the dehydration speed of the cells in the separation liquid, so that the effective density of diagnostically significant cells is increased, and the diagnostically significant cells can quickly and naturally descend to the bottom of a test tube in the sample density separation liquid.

As a preferred embodiment of the present invention, the lymphocyte separation solution comprises the following components by mass percent: 5-15% of glycerol, 1-10% of ethanol, 1-5% of sodium chloride, 0.1-0.5% of tea saponin and the balance of deionized water, wherein the total amount is 100%. According to the invention, the sodium chloride with the mass percentage of 1-5% is used, so that the separation liquid has higher osmotic pressure, and cells in a sample can be dehydrated and reduced; the ethanol in the ethanol solution with the mass percentage of 1-10% can improve the permeability of cell membranes, so that the dehydration speed of cells can be increased, the effective density of the cells in the sample density separation liquid is increased, the effective density of the cells can be increased in a short tabletting time, and the natural sinking of the cells is accelerated; the tea saponin with the mass percent of 0.1-0.5% can damage erythrocytes of human, so that the erythrocytes generate hemolysis, but can not damage other nucleated cells, even if the erythrocytes are reduced into the density separation liquid of the sample, the erythrocytes are subjected to hemolysis reaction due to the existence of the tea saponin and become cell fragments, so that the density is reduced and the erythrocytes are not reduced to the bottom of the test tube to be mixed with cells with diagnostic significance; the glycerol with the mass percentage of 5-15% can enable the whole mixed system to be in the optimal density gradient, so that the pathological cells for diagnosis can be guaranteed to sink to the bottom of the test tube.

As a preferred embodiment of the present invention, the lymphocyte separation solution comprises the following components by mass percent: 6-10% of glycerol, 2-5% of ethanol, 3-5% of sodium chloride, 0.16-0.3% of tea saponin and the balance of deionized water, wherein the total amount is 100%.

As a preferred embodiment of the present invention, the lymphocyte separation solution comprises the following components by mass percent: 8.3% of glycerol, 3% of ethanol, 4.5% of sodium chloride, 0.16% of tea saponin and the balance of deionized water, wherein the total amount is 100%.

The invention also provides a preparation method of the lymph separation liquid, which comprises the following steps:

s1, mixing the ethanol, sodium chloride, tea saponin and double distilled water according to the formula ratio, stirring and dissolving to obtain a clear solution;

s2, adding glycerol into the biological clear solution obtained in the step S1 at the temperature of 18-22 ℃, and uniformly stirring to obtain the lymph separation liquid.

In a preferred embodiment of the present invention, the ethanol is 1 to 10% by mass, the sodium chloride is 1 to 5% by mass, the tea saponin is 0.1 to 0.5% by mass, and the glycerin is 5 to 15% by mass.

In a preferred embodiment of the present invention, the ethanol is 2 to 5% by mass, the sodium chloride is 3 to 5% by mass, the tea saponin is 0.16 to 0.3% by mass, and the glycerin is 6 to 10% by mass.

In a preferred embodiment of the present invention, the ethanol accounts for 3% by mass, the sodium chloride accounts for 4.5% by mass, the tea saponin accounts for 0.16% by mass, and the glycerol accounts for 8.3% by mass.

The invention has the beneficial effects that: the invention combines the effects of sodium chloride, ethanol, tea saponin and glycerol on cells, and selects proper mass concentration, so that the separating medium can efficiently separate the lymphocytes to obtain high-quality lymphocytes.

Drawings

FIG. 1 is a high power microscope picture of clinical human bone marrow lymph stem cell separated from lymphocyte separating medium product.

FIG. 2 is the low power microscope picture of clinical human bone marrow lymph stem cell separated from lymphocyte separating medium product. The figure shows a low power optical microscope image of the bone marrow lymphocytes isolated by the present invention after 21d culture.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is provided with reference to specific embodiments and accompanying drawings.

The components of the lymph separation liquids of examples 1 to 5 and comparative examples 1 to 4 are shown in Table 1:

table 1: lymph separation liquid composition of examples 1 to 5 and comparative examples 1 to 4

Comparative example 5: focill lymphocyte separation solution

Comparative example 6: percoll lymphocyte separation medium

Example 6

The embodiment provides a preparation method of a lymph separation liquid, which comprises the following steps:

s2, adding glycerol into the biological clear solution obtained in the step S1 at the temperature of 18-22 ℃, and uniformly stirring to obtain the lymph separation liquid;

s3, filtering the lymph separation liquid by a filter element, and filling to form a finished product.

The effect of the separation liquid of examples 1 to 5 and comparative examples 1 to 4 was verified:

1. the product performance of the lymph separation liquid of examples 1-5 was reported by self-test, as shown in table 2:

table 2: lymph separating liquid product performance self-test report

2. The method for separating the bone marrow lymph stem cells by using the lymph separation liquid of the examples 1 to 5 and the comparative examples 1 to 4 comprises the following specific operations:

the whole process of samples, reagents and experimental environment is carried out at 20 +/-2 ℃.

Pretreatment: preparation of 1mL Single cell suspension of bone marrow

(1) Taking a centrifugal tube, adding a separation liquid which is equal to the single-cell suspension of the bone marrow

(2) Carefully sucking the bone marrow single cell suspension with a pipette, adding onto the liquid surface of the separating medium, 600g, and centrifuging for 20-30 min.

(3) After centrifugation, the centrifuge tube is divided into four layers from top to bottom. The first layer is a dilute liquid layer. The second layer is a ring-shaped milky white target cell layer. The third layer is a transparent separation liquid layer. The fourth layer is the red blood cell layer.

(4) Carefully sucking the second annular milky target cell layer into another 15ml centrifuge tube by using a pipette, adding 5ml of washing solution into the centrifuge tube, and uniformly mixing the cells.

(5)600g, centrifuge for 10 min.

(6) The supernatant was discarded.

(7) The resulting cells were resuspended with a pipette with 5ml of wash solution.

(8)600g, centrifuge for 10 min.

(9) (optional) optional repetition of 7, 8, 9

(10) The supernatant was discarded and the cells were resuspended in 0.5ml of DMEM cell culture medium for subsequent culture procedures.

The lymph separation liquids of examples 1 to 5 and comparative examples 1 to 4 had separation effects as shown in Table 3:

table 3: results of separating lymph separation liquids of examples 1 to 5 and comparative examples 1 to 6

As can be seen from table 3, the quality of lymphocytes obtained in comparative examples is low because one of the components of the present invention is absent in comparative examples 1 to 4, and the effect of separating lymphocytes cannot be achieved because comparative examples 1 and 3 do not contain glycerin or sodium chloride, respectively, and thus the results of comparative examples 1 and 3 are zero; however, the quality of the lymphocytes obtained from the separation solutions of examples 1 to 5 was significantly improved, and the cell purity was more than 97%, so that high-quality lymphocytes were obtained. It can be seen that there is some interaction between the components in the separation liquid of the present invention, resulting in synergistic effect. Meanwhile, the separation liquid of the present invention obtained a larger total number of separated cells and the cell survival rate was as high as 99.6% compared to other types of lymphocyte separation liquids (comparative examples 5, 6). Therefore, the lymphocyte separation method can separate the lymphocytes with high efficiency, and the survival rate of the separated lymphocytes can reach 99.6%.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A lymphocyte separation fluid, comprising the following components: glycerol, ethanol, sodium chloride, tea saponin and water.

2. The lymphocyte separation solution according to claim 1, which comprises the following components in percentage by mass: 5-15% of glycerol, 1-10% of ethanol, 1-5% of sodium chloride, 0.1-0.5% of tea saponin and the balance of deionized water, wherein the total amount is 100%.

3. The lymphocyte separation solution according to claim 2, which comprises the following components in percentage by mass: 6-10% of glycerol, 2-5% of ethanol, 3-5% of sodium chloride, 0.16-0.3% of tea saponin and the balance of deionized water, wherein the total amount is 100%.

4. The lymphocyte separation solution according to claim 3, which comprises the following components in percentage by mass: 8.3% of glycerol, 3% of ethanol, 4.5% of sodium chloride, 0.16% of tea saponin and the balance of deionized water, wherein the total amount is 100%.

5. A method for preparing a lymph separation fluid, comprising the steps of:

6. The method according to claim 5, wherein the ethanol is 1 to 10% by mass, the sodium chloride is 1 to 5% by mass, the tea saponin is 0.1 to 0.5% by mass, and the glycerin is 5 to 15% by mass.

7. The method according to claim 6, wherein the ethanol is 2 to 5% by mass, the sodium chloride is 3 to 5% by mass, the tea saponin is 0.16 to 0.3% by mass, and the glycerin is 6 to 10% by mass.

8. The method according to claim 7, wherein the ethanol is 3% by mass, the sodium chloride is 4.5% by mass, the tea saponin is 0.16% by mass, and the glycerin is 8.3% by mass.