Isolation method for isolating peripheral blood immune cells

Abstract

The invention discloses a separation method for separating peripheral blood immune cells, and belongs to the field of biotechnology. The method comprises the following steps: diluting peripheral blood treated by an anticoagulant by using normal saline or a serum-free culture medium to obtain cell suspension; adding a separating medium containing dextran, phospholipid, trehalose and amino acid into a centrifuge tube, wherein the volume of the separating medium is not greater than 1 / 2 of that of the centrifugal tube; and spreading the cell suspension on the separating medium, carrying out centrifugation under the condition that the temperature is 8-25 DEG C, the weight is 200-800g, and the time is 10-30 minutes, and sucking buffy coat cells. According to the separation method disclosed by the invention, when the prepared separating medium is used, an operator can directly spread the cell suspension on the separating medium, the damages to the liquid surface are not needed to be considered too much during spreading, and the requirement on the operation experience of the operator is low; and all the raw materials in a formula provided by the invention are in line with an intravenous injection level raw medicinal material standard, the raw materials have small uncertainty and high safety, and the immune cells obtained by separation have relatively good tumoricidal activity.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a method for isolating peripheral blood immune cells. Background technique

[0002] The current gradient centrifugation method has cumbersome steps and strong skills, and requires relatively high operator experience: gradient centrifugation requires three steps, especially the second step, which requires carefully spreading low-density separation liquid on top of high-density separation liquid. The cell suspension should not be confused with the interface, which greatly tests the experience and patience of the operator, which is time-consuming, laborious, and prone to fatigue. Once the liquid level is mixed, the separation effect will be greatly affected, and even the lymphocyte layer cannot be separated. Moreover, most of the current separation fluids have certain toxicity, which affects the separation effect and the quality of the separated cells, resulting in low separation effici...

Examples

Embodiment 1

[0026] A method for isolating immune cells from peripheral blood, comprising the steps of:

[0027] Step 1: Dilute the anticoagulant-treated peripheral blood with normal saline or serum-free medium to obtain a cell suspension;

[0028] Step 2: Add the separation liquid containing dextran, phospholipid, trehalose and amino acid into the centrifuge tube, the volume of the separation liquid is not more than 1 / 2 of the volume of the centrifuge tube;

[0029] Step 3: Spread the cell suspension on the separation medium, centrifuge at 8-25°C, 200g-800g, 10-30min, and absorb the buffy coat cells.

[0030] Among them, the preparation method of the separation liquid is: in an environment with local 100-level laminar flow purification conditions, at 20 ° C, 4 g of dextran, 3 g of phospholipids, 5 g of trehalose, and ultra-low viscosity alginic acid Sodium 0.5g, Meglumine Diatrizoate 5g, Polydiallyldimethylammonium Chloride 0.1g, L-Valine 0.01g and L-Leucine 0.009g are fully dissolved in...

Embodiment 2

[0032] A method for isolating immune cells from peripheral blood, comprising the steps of:

[0033] Step 1: Dilute the anticoagulant-treated peripheral blood with normal saline or serum-free medium to obtain a cell suspension;

[0034] Step 2: Add the separation liquid containing dextran, phospholipid, trehalose and amino acid into the centrifuge tube, the volume of the separation liquid is not more than 1 / 2 of the volume of the centrifuge tube;

[0035] Step 3: Spread the cell suspension on the separation medium, centrifuge at 8-25°C, 200g-800g, 10-30min, and absorb the buffy coat cells.

[0036]Among them, the preparation method of the separation liquid is: in an environment with local 100-level laminar flow purification conditions, at 20 ° C, 2.5 g of dextran, 0.3 g of phospholipid, 7 g of trehalose, ultra-low viscosity Sodium alginate 1g, diatrizoate meglumine 3g, polydiallyl dimethyl ammonium chloride 0.04g, L-valine 0.009g and L-leucine 0.012g are fully dissolved in ste...

Embodiment 3

[0038] A method for isolating immune cells from peripheral blood, comprising the steps of:

[0039] Step 1: Dilute the anticoagulant-treated peripheral blood with normal saline or serum-free medium to obtain a cell suspension;

[0040] Step 2: Add the separation liquid containing dextran, phospholipid, trehalose and amino acid into the centrifuge tube, the volume of the separation liquid is not more than 1 / 2 of the volume of the centrifuge tube;

[0041] Step 3: Spread the cell suspension on the separation medium, centrifuge at 8-25°C, 200g-800g, 10-30min, and absorb the buffy coat cells.

[0042] Among them, the preparation method of the separation liquid is: in an environment with local 100-level laminar flow purification conditions, at 20 ° C, 2 g of dextran, 2 g of phospholipids, 5.5 g of trehalose, and ultra-low viscosity seaweed Sodium phosphate 0.6g, diatrizoate meglumine 4.5g, polydiallyl dimethyl ammonium chloride 0.09g, L-valine 0.01g and L-leucine 0.011g are fully ...

technical field

[0001] The invention relates to the field of biotechnology, in particular to a method for isolating peripheral blood immune cells. Background technique

[0002] The current gradient centrifugation method has cumbersome steps and strong skills, and requires relatively high operator experience: gradient centrifugation requires three steps, especially the second step, which requires carefully spreading low-density separation liquid on top of high-density separation liquid. The cell suspension should not be confused with the interface, which greatly tests the experience and patience of the operator, which is time-consuming, laborious, and prone to fatigue. Once the liquid level is mixed, the separation effect will be greatly affected, and even the lymphocyte layer cannot be separated. Moreover, most of the current separation fluids have certain toxicity, which affects the separation effect and the quality of the separated cells, resulting in low separation effici...

Examples

Embodiment 1

[0026] A method for isolating immune cells from peripheral blood, comprising the steps of:

[0027] Step 1: Dilute the anticoagulant-treated peripheral blood with normal saline or serum-free medium to obtain a cell suspension;

[0028] Step 2: Add the separation liquid containing dextran, phospholipid, trehalose and amino acid into the centrifuge tube, the volume of the separation liquid is not more than 1 / 2 of the volume of the centrifuge tube;

[0029] Step 3: Spread the cell suspension on the separation medium, centrifuge at 8-25°C, 200g-800g, 10-30min, and absorb the buffy coat cells.

[0030] Among them, the preparation method of the separation liquid is: in an environment with local 100-level laminar flow purification conditions, at 20 ° C, 4 g of dextran, 3 g of phospholipids, 5 g of trehalose, and ultra-low viscosity alginic acid Sodium 0.5g, Meglumine Diatrizoate 5g, Polydiallyldimethylammonium Chloride 0.1g, L-Valine 0.01g and L-Leucine 0.009g are fully dissolved in...

Embodiment 2

[0032] A method for isolating immune cells from peripheral blood, comprising the steps of:

[0033] Step 1: Dilute the anticoagulant-treated peripheral blood with normal saline or serum-free medium to obtain a cell suspension;

[0034] Step 2: Add the separation liquid containing dextran, phospholipid, trehalose and amino acid into the centrifuge tube, the volume of the separation liquid is not more than 1 / 2 of the volume of the centrifuge tube;

[0035] Step 3: Spread the cell suspension on the separation medium, centrifuge at 8-25°C, 200g-800g, 10-30min, and absorb the buffy coat cells.

[0036]Among them, the preparation method of the separation liquid is: in an environment with local 100-level laminar flow purification conditions, at 20 ° C, 2.5 g of dextran, 0.3 g of phospholipid, 7 g of trehalose, ultra-low viscosity Sodium alginate 1g, diatrizoate meglumine 3g, polydiallyl dimethyl ammonium chloride 0.04g, L-valine 0.009g and L-leucine 0.012g are fully dissolved in ste...

Embodiment 3

[0038] A method for isolating immune cells from peripheral blood, comprising the steps of:

[0039] Step 1: Dilute the anticoagulant-treated peripheral blood with normal saline or serum-free medium to obtain a cell suspension;

[0040] Step 2: Add the separation liquid containing dextran, phospholipid, trehalose and amino acid into the centrifuge tube, the volume of the separation liquid is not more than 1 / 2 of the volume of the centrifuge tube;

[0041] Step 3: Spread the cell suspension on the separation medium, centrifuge at 8-25°C, 200g-800g, 10-30min, and absorb the buffy coat cells.

[0042] Among them, the preparation method of the separation liquid is: in an environment with local 100-level laminar flow purification conditions, at 20 ° C, 2 g of dextran, 2 g of phospholipids, 5.5 g of trehalose, and ultra-low viscosity seaweed Sodium phosphate 0.6g, diatrizoate meglumine 4.5g, polydiallyl dimethyl ammonium chloride 0.09g, L-valine 0.01g and L-leucine 0.011g are fully ...