Separation method for improving yield of human lymphocytes

Abstract

The invention belongs to the technical field of biological extraction, and discloses a separation method for improving the yield of human lymphocytes. The method comprises the following steps: collecting blood cells, diluting the blood cells, obtaining lymphocytes and recovering the lymphocytes. The method is characterized in that a blood cell diluent is prepared and comprises human serum albumin, EDTA-2Na (Ethylene Diamine Tetraacetic Acid-2Na) and a calcium-magnesium ion-free PBS (Phosphate Buffer Solution). The method is applied to lymphocyte separation, the osmotic pressure of cells in the separation process can be improved, the normal form of the cells can be maintained, the viscosity between the cells can be reduced, cell adhesion can be prevented, meanwhile, the part which is adhered to form a membrane layer or a cell cluster is separated twice, the lymphocyte separation yield is remarkably increased, and the method is simple to operate and easy to implement. The realization is easy. The method is especially suitable for blood samples of people with immune system diseases, blood samples and blood viscous samples of old and sick people, non-fresh blood samples and the like.

Description

Technical Field

[0001] The invention relates to a method for separating human lymphocytes in vitro, and belongs to the technical field of biological extraction. Background Art

[0002] The isolation and acquisition of lymphocytes is the basis of lymphocyte research and in vitro immune cell culture. After whole blood is collected with EDTA anticoagulant tubes, it will affect cell proliferation during the culture process. Other anticoagulant tubes are usually selected for whole blood collection. If not handled in time, it will affect the effect of mononuclear cell separation. Currently, the commonly used lymphocyte separation methods include density gradient centrifugation, adsorption separation and other special separation methods (Liu Sheng, Wang Chenyang, Huang Mengling, et al. Different methods for isolating mononuclear cells from peripheral blood and their respective characteristics [J]. Chinese Journal of Arteriosclerosis, 2022, 30(09)), (Wu Hongzhen, Zhang Linbo. Research progress in T cell separation methods [J]. Jilin Agriculture, 2011(03))). Currently, density gradient centrifugation is the most commonly used separation method, primarily using human peripheral blood lymphocyte separation buffer to separate blood samples. While this separation is effective for fresh blood samples from healthy individuals, it is often unsatisfactory or very poor for samples from individuals with immune system disorders, the elderly, those with multiple illnesses, those with thick blood, or those with non-fresh blood. This results in very low lymphocyte yields, which can either fail to meet research objectives or result in a low yield of immune cells after culture. Therefore, further research is needed to improve human lymphocyte yield, and the urgent challenge is to find a separation method that can improve this yield. Summary of the Invention

[0003] Based on the current state of the art, the present invention aims to universally solve the problem of low lymphocyte yield and provide a separation method that can increase the lymphocyte yield from various blood samples.

[0004] To achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0005] The separation method for improving the yield of human lymphocytes comprises the following steps:

[0006] (1) Collecting blood cells: Collect human peripheral blood and separate the lower layer of blood cells after centrifugation;

[0007] (2) Blood cell dilution: Dilute the blood cells obtained in step (1) with lymphocyte diluent and mix thoroughly by pipetting.

[0008] (3) Obtaining lymphocytes: Add the diluted blood cells obtained in step (2) to the upper layer of a centrifuge tube containing lymphocyte separation solution, keep the boundary layer clear, and centrifuge at room temperature. After centrifugation, remove the liquid from the centrifuge tube and separate it from the upper layer. The first layer is the diluent (plasma) layer, the second layer is the ring-shaped milky white lymphocyte layer, the third layer is the transparent separation solution layer, and the fourth layer is the red blood cell layer. Discard the top plasma layer, aspirate the white lymphocyte layer, and divide it into centrifuge tubes containing PBS or physiological saline. Centrifuge at room temperature to obtain lymphocytes.

[0009] (4) If there is a white membrane layer or white cell clusters above the red blood cell layer in step (3), gently aspirate the white membrane layer and white cell clusters, and repeat steps (2) and (3) to recover lymphocytes.

[0010] (5) The lymphocytes obtained in steps (3) and (4) are combined to obtain the total separated lymphocytes.

[0011] The lymphocyte diluent is composed of human serum albumin, disodium edetate, and 1×PBS buffer without calcium and magnesium ions. The human serum albumin content in the solution is 0.3-30%, the disodium edetate is 0.5-1.5 mg / mL, and the pH value of the 1×PBS buffer without calcium and magnesium ions is 6.8-7.4.

[0012] Preferably, the centrifugal speed in step (1) is set to 400g-450g for 15min.

[0013] Preferably, the amount of lymphocyte dilution added in step (2) is 1.5 to 2.5 times the volume of blood cells.

[0014] Preferably, in step (3), in order to ensure a clear interface, when adding diluted blood cells, the centrifuge tube of the lymphocyte separation solution is tilted 45° to reduce the impact of blood cells.

[0015] Principle of the present invention: Cell adhesion affects the cell separation rate. Cell adhesion is mainly due to the presence of cell adhesion molecules. The cell adhesion molecules related to lymphocytes are mainly selectins and integrins. Selectins are a class of Ca-dependent 2+ The integrin family is a class of heterophilic cell adhesion molecules that depends on Ca 2+ or Mg 2+ To address the heterophilic cell adhesion molecule, the present invention adds EDTA, a divalent metal ion chelator that chelates calcium and magnesium ions in the blood, to the diluent. Human albumin maintains the stability of the extracellular fluid, preventing cell swelling or shrinkage due to osmotic pressure changes, thereby maintaining normal cell morphology and function, which in turn affects cell adhesion. Furthermore, the use of a buffer solution free of calcium and magnesium ions further prevents cell adhesion.

[0016] Innovations and beneficial effects of the present invention: The present invention provides a diluent that can improve the osmotic pressure of cells during the separation process, maintain the normal morphology of cells, reduce intercellular viscosity, and simultaneously perform a second separation on the parts that have adhered to form membrane layers or cell clusters, thereby significantly improving the lymphocyte separation yield. The method is simple to operate and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a picture of the effect of lymphocytes separated using existing technology; it can be seen that the blood sample lymphocyte layer is not clear, the interface is not obvious, there is an adherent cell membrane layer or cell clusters above the red blood cell layer, and after the lymphocyte layer is separated, the lymphocyte yield is very low.

[0018] Figure 2 This is a diagram showing the separation effect of centrifugally separating lymphocytes using the method of the present invention; it can be seen that the interface of the lymphocyte layer is clearly visible, the adherent cell membrane layer or cell clusters above the red blood cell layer are significantly reduced, and the lymphocyte yield is very high.

[0019] Note: Samples are collected from people with immune system diseases, the elderly and sick, people with thick blood viscosity, or non-fresh blood samples. DETAILED DESCRIPTION

[0020] In order to better illustrate the present invention, the following examples are given:

[0021] Example 1 Selecting the composition ratio range of the diluent of the present invention from the perspective of osmotic pressure

[0022] (1) Optimization of the diluent composition ratio: EDTA-2Na was weighed using an analytical balance, dissolved in 1× PBS buffer (pH 6.8-7.4) without calcium and magnesium ions, sterilized by filtration using a 0.22 μm filter membrane, and human serum albumin (protein concentration 20%) was added. The osmotic pressure was measured using an osmometer. The specific composition ratio and osmotic pressure values ​​are shown in Table 1.

[0023] Table 1: Component ratio and osmotic pressure of diluent

[0024]

[0025]

[0026] Taking into account the effect of osmotic pressure on cell activity, PBMC were suspended in diluent and placed for 1 hour. Too low or too high osmotic pressure will lead to cell lysis. The osmotic pressure was selected in the range of 290-310mOsm / kg for subsequent effect comparison test. The 5th and 8th groups of human albumin single group were selected for test. The test groups were group 1, group 5, group 8, group 10, group 14, group 15, and group 16 respectively.

[0027] (2) Collection of plasma: A 120 mL peripheral blood sample from a sub-healthy client was collected using a sodium heparin anticoagulant tube. The sample was collected in the afternoon and left overnight. The sample was processed the next morning and divided into 7 groups: Group 1, Group 5, Group 8, Group 10, Group 14, Group 15, and Group 16. Each group had 17 mL of sample. After centrifugation at 400 g for 15 min, the upper layer of plasma was separated and transferred to a new centrifuge tube for later use. The lower layer was the blood cell layer.

[0028] (3) Blood cell dilution: Use the prepared diluent according to the instructions of the lymphocyte separation solution, add the prepared diluent to dilute the blood cells at a volume of 1.5 times the volume of blood cells, and use a pipette to gently pipette and mix 5 times.

[0029] (4) Obtaining lymphocytes: 7 groups of diluted blood cells were added to the upper layer of a 50 mL centrifuge tube containing 15 mL of lymphocyte separation solution. The centrifuge tube was tilted at 45° and slowly added to keep the boundary layer clear. The tube was centrifuged at 20°C and 520 g for 22 min (the centrifugal force and time were determined according to the instructions for the separation solution). After centrifugation, the tube was gently removed. The centrifuge tube was divided into four layers from top to bottom. The first layer was the diluent (plasma) layer, the second layer was the ring-shaped milky white lymphocyte layer, the third layer was the transparent separation solution layer, and the fourth layer was the red blood cell layer. The top plasma layer was discarded from all 7 groups, and the white lymphocyte layer was aspirated and divided into centrifuge tubes containing PBS or physiological saline for washing. The tube was gently inverted to mix evenly. Samples were taken for cell counting and centrifuged at room temperature to obtain lymphocytes.

[0030] Experimental test:

[0031] Cell count: 10 μL of the cell suspension was added to a hemocytometer and counted under a microscope. The cell count was calculated using the formula: "Total number of cells in 25 squares x dilution factor x total volume of liquid." The cell count results are shown in Table 2. As can be seen, the lymphocytes isolated using different dilutions in Groups 1, 5, and 8 were essentially identical. Group 10 had a slight increase compared to Group 1, and Groups 14, 15, and 16 had a significant increase compared to Group 1. Therefore, subsequent testing within the dilution ratio range described in the present invention was more conducive to lymphocyte isolation.

[0032] Table 2 Cell counting results and comparison

[0033]

[0034] Example 2

[0035] The separation method for improving the yield of human lymphocytes of the present invention comprises the following steps:

[0036] (1) Blood cell collection: Four peripheral blood samples (80 mL each) were collected from patients with pancreatic atrophy, aplastic anemia, cancer surgery, and sub-health using sodium heparin anticoagulant tubes. The samples were divided into two groups, group 1 and group 2. Each sample in each group was 40 mL. After centrifugation at 400 g for 15 min, the upper plasma layer was separated and transferred to a new centrifuge tube for later use. The lower layer was the blood cell layer.

[0037] (2) Blood cell dilution: Group 1 used an equal volume of PBS to dilute whole blood according to the instructions of a commercial lymphocyte separation solution, and gently pipetted and mixed 5 times; Group 2 used the diluent of the present invention to add 1.5 times the volume of blood cells, filtered and sterilized using a 0.22 μm filter membrane, and gently pipetted and mixed 5 times.

[0038] The diluent of the present invention comprises: 20% human albumin content in the solution, 1.5 mg / mL disodium edetate, and 1×PBS buffer solution without calcium or magnesium ions and having a pH of 6.8-7.4.

[0039] (3) Obtaining lymphocytes: Add the two groups of diluted blood cells to the upper layer of a 50 mL centrifuge tube containing 15 mL of lymphocyte separation solution. Tilt the centrifuge tube at 45° and slowly add the cells to keep the boundary layer clear. Centrifuge at 20°C and 520 g for 22 min (refer to the instructions for the separation solution for centrifugal force and time). Gently remove the cells after centrifugation. The centrifuge tube is divided into four layers from top to bottom. The first layer is the diluent (plasma) layer, the second layer is the ring-shaped milky white lymphocyte layer, the third layer is the transparent separation solution layer, the fourth layer is the white membrane layer or white cell cluster, and the fifth layer is the red blood cell layer. For both groups, the top plasma layer is discarded, the white lymphocyte layer is aspirated, and the cells are divided into centrifuge tubes containing PBS or physiological saline for washing. Gently invert the tube to mix evenly. Samples are taken for cell counting and centrifuged at room temperature to obtain lymphocytes.

[0040] (4) For the two groups of white membrane layers or white cell clusters above the red blood cell layer in step (3), gently aspirate the white membrane layer and white cell clusters, repeat steps (2) and (3) once, and obtain recovered lymphocytes. During washing, take samples for cell counting.

[0041] (5) The lymphocytes obtained in steps (3) and (4) are combined to obtain the total separated lymphocytes.

[0042] Experimental test:

[0043] For cell counting, 10 μL of the cell suspension was added to a hemocytometer and counted under a microscope. The cell number was calculated using the formula "total number of cells in 25 squares x dilution factor x total volume of liquid." The cell count results are shown in Table 3. As can be seen, the number of lymphocytes isolated using this method (Group 2) was significantly higher, increasing by 47.1% to 60.0% compared to the control group (Group 1).

[0044] Table 3 Cell counting results and comparison

[0045]

[0046] The present invention dilutes peripheral blood cells using a calcium-magnesium ion-free PBS diluent containing human albumin and EDTA, thereby improving the osmotic pressure of cells during lymphocyte separation, maintaining normal cell morphology, reducing intercellular viscosity, and preventing cell adhesion. At the same time, a second separation is performed on the part that has adhered to form a membrane layer or cell cluster, significantly improving the lymphocyte separation yield. The method is particularly suitable for blood samples from people with immune system diseases, blood samples from elderly and multi-ill people, and samples such as blood with high viscosity and non-fresh blood.

[0047] The above describes the embodiments of the present invention in detail with reference to the accompanying drawings and tables. It should be noted that several improvements and changes can be made within the knowledge scope of those skilled in the art.

Claims

1. A separation method for improving the yield of human lymphocytes, characterized in that: This is achieved through the following steps: (1) Collect blood cells: collect human peripheral blood and separate the lower layer of blood cells after centrifugation; (2) Blood cell dilution: dilute the blood cells obtained in step (1) with lymphocyte diluent and gently pipette to mix; (3) Obtaining lymphocytes: Add the diluted blood cells obtained in step (2) to the upper layer of a centrifuge tube containing lymphocyte separation solution, slowly add to keep the boundary layer clear, centrifuge at room temperature, and remove after centrifugation; the liquid in the centrifuge tube is layered from top to bottom, the first layer is the plasma layer, the second layer is the lymphocyte layer, the third layer is the separation solution layer, and the fourth layer is the red blood cell layer; discard the top plasma layer, absorb the lymphocyte layer, and divide it into centrifuge tubes containing PBS or physiological saline, centrifuge at room temperature, and obtain lymphocytes; (4) Recovering lymphocytes: If there is a white membrane layer or white cell cluster above the red blood cell layer in step (3), aspirate the white membrane layer or white cell cluster and repeat steps (2) and (3) to recover lymphocytes; (5) The lymphocytes obtained in steps (3) and (4) are combined to obtain the total isolated lymphocytes; The lymphocyte diluent is prepared from human serum albumin, disodium edetate, and 1×PBS buffer solution without calcium and magnesium ions.

2. The method for separating human lymphocytes according to claim 1, wherein: The lymphocyte diluent contains: 0.3-30% human albumin, 0.5-1.5 mg / mL disodium edetate, and 1× PBS buffer solution without calcium and magnesium ions, with a pH of 6.8-7.

4.

3. The method for separating human lymphocytes according to claim 1, wherein: In step (2), the amount of lymphocyte diluent added is 1.5 to 2.5 times the volume of blood cells.

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