CN101469310B - Method for enriching target cells from biology specimen and method for removing leucocyte - Google Patents

Abstract

The invention relates to a method for enriching target cells from biological samples and a method for removing white blood cells from the biological samples. The invention practically provides the method for enriching the target cells from the biological samples, which comprises: using a mode based on mol osmotic pressure concentration to remove red blood cells, and using immune affinity chromatography to remove the white blood cells. The method not only has low cost but also highly efficiently removes the white blood cells and the red blood cells, so as to enrich the target cells and be easy for subsequent detection. The invention also provides the method for removing the white blood cells from the biological samples, which comprises: adopting Affi-Gel 10 coupled by CD45 antibodies to remove the white blood cells.

Description

Method for enriching target cells from biological sample and method for removing leukocytes

technical field

The present invention relates to a method for enriching target cells from a biological sample and a method for removing leukocytes.

Background technique

Cells including stem cells, fetal cells, circulating tumor cells (CTCs), immune cells, etc. have great applications in the prevention, detection, diagnosis and treatment of diseases.

Stem cells refer to a class of pluripotent cells with self-replicating ability, including bone marrow stem cells, adult stem cells, umbilical cord blood stem cells, skin stem cells, etc. Under certain conditions, stem cells can differentiate into a variety of functional cells, so stem cells have a wide range of applications in treating many difficult diseases, providing organ transplantation, rehabilitation, health care, reducing aging, and restoring youthful vitality.

Fetal cells have been shown to be detectable in the maternal circulation as early as five weeks of gestation. At present, the methods for obtaining fetal cells for prenatal diagnosis mainly include amniocentesis, chorionic villi biopsy and umbilical vessel puncture. These methods have certain risks to the fetus and may cause miscarriage. Isolation of fetal cells from maternal blood promises to obtain fetal cells only through a simple venipuncture route. Clinical applications of fetal cells will include early diagnosis of fetal chromosomal or genetic abnormalities.

Numerous reports suggest that circulating tumor cells (CTCs) can be found in patients even before the primary tumor is detected by imaging analysis. In addition to their potential role in early diagnosis and prediction, CTCs may also play a major role in characterizing genetic and immunophenotypic changes with tumor progression, thereby helping to guide individualized therapy.

The number of immune cells (such as T lymphocytes, B lymphocytes, K lymphocytes or NK lymphocytes) due to diseases of the immune system or other systems, or due to immunization or certain clinical treatment measures and certain external environmental factors Or function can change. Therefore, the detection of cellular immunity is of great significance for the diagnosis and pathogenesis research of certain diseases, the evaluation of the effect of immunotherapy or vaccination, and the influence of environmental factors on the immune function of the body.

However, the stem cells, fetal cells, circulating tumor cells (CTCs), and immune cells are rare in biological samples, such as blood, bone marrow, spinal cord, or surgical fluid, and are rare cells, so enriching these target cells is the first priority. question.

Although various techniques have been used to enrich, isolate and characterize target cells, many of them share a similar principle, antibody-based positive selection. Due to the heterogeneous expression of cell surface markers, these strategies for target cell detection are clearly limited by the availability, sensitivity and specificity of antibodies against biomarkers on different target cells. Other strategies include negative depletion of red blood cells (RBC), white blood cells (WBC). Smaller RBCs and WBCs are currently removed primarily based on size filtering, but since some target cells such as CTCs can be as small as WBCs, such size filtering based methods risk losing target cells.

For the method for removing leukocytes, although there is a method for removing leukocytes using anti-CD45 coated 30nm magnetic beads (Miltenyi product, Germany) in the prior art, this method is not only expensive (RMB600-700/test), time-consuming (more than 4 hours /test), and the WBC removal efficiency is not high (WBC removal rate is about 2-3logs).

Contents of the invention

In order to overcome the defects of the prior art, the present invention creatively combines the method based on osmolarity with immunoaffinity chromatography to remove RBC and WBC respectively, so as to enrich target cells, especially rare purpose of cells. This method is not only low in cost, but also efficiently enriches target cells and is easy for subsequent detection. The target cells include stem cells, fetal cells, circulating tumor cells (CTCs), immune cells and the like.

The invention also provides a method for removing leukocytes, which not only has low cost, saves time, but also has high efficiency.

Detailed ways

In a specific embodiment of the present invention, the present invention provides a method for enriching target cells from a biological sample, the method comprising:

Red blood cells were removed by an osmolarity-based approach and white blood cells were removed by immunoaffinity chromatography.

The present invention creatively combines the removal of red blood cells based on osmolarity and the removal of white blood cells by immunoaffinity chromatography to enrich target cells. There is no particular sequence requirement for the removal of red blood cells by means of osmolarity and the removal of white blood cells by immunoaffinity chromatography. For example, affinity chromatography can be used to remove white blood cells first, and then red blood cells can be removed by osmolarity-based method; or red blood cells can be removed by osmolarity-based method, and then white blood cells can be removed by affinity chromatography. In order to allow leukocytes to pass through the affinity chromatography column more quickly and efficiently, in a preferred embodiment of the present invention, erythrocytes are removed based on osmolarity and then leukocytes are removed by affinity chromatography.

In a specific embodiment of the present invention, the biological sample is a body fluid; preferably, the body fluid is blood, bone marrow, spinal cord or surgical fluid. In one embodiment of the present invention, blood is taken as an example to verify the effectiveness of the method of the present invention. At the same time, because blood, bone marrow, spinal cord or surgical fluid have similar densities and target cells exist therein, the method of the present invention can be used to enrich target cells in bone marrow, spinal cord or surgical fluid.

To collect blood, a human or any other animal blood sample is collected in anticoagulant collection tubes or tubes containing any anticoagulant including ethylenediaminetetraacetic acid (EDTA), dextrose citrate ( ACD) and so on. In order to collect bone marrow, the donor's ilium can be punctured to collect the bone marrow; hematopoietic stem cells from the bone marrow and other parts can also be mobilized to release a large number of hematopoietic stem cells into the peripheral blood, and then collected from the donor's arm vein. The spinal cord can be obtained by, for example, a spinal tap. The surgical fluid can be a body fluid obtained by surgery, such as peritoneal or pleural effusion, which includes some target cells and proteins described in the present invention.

In a preferred embodiment of the present invention, the blood used is taken on the day of blood collection, preferably within 20 hours of blood collection, more preferably within 10 hours of blood collection, further preferably within 5 hours of blood collection or within 5 hours of blood collection. Within 3 hours or within 1 hour of blood collection, it is best to choose fresh blood obtained immediately after blood collection. The erythrocytes in the above fresh blood are sensitive to the RBC lysis buffer, while the target cells are not sensitive to the RBC lysis buffer.

In a specific embodiment of the present invention, the size of the immune particles used in the immunoaffinity chromatography is 1 μm-20 μm, more preferably 2 μm-15 μm, further preferably 5 μm-10 μm. In a specific embodiment of the present invention, it is further preferred that the immune particles are reproducible. Immune particles can be washed and reused with buffers such as Tris-Glycine, saving costs.

In one embodiment of the invention, red blood cells are removed using an osmolarity-based approach. In a preferred embodiment of the present invention, said osmolarity-based approach includes sedimentation and lysis of red blood cells. The pellet is preferably centrifuged at 500-3000 rpm, more preferably 100-2000 rpm, for 1-6 minutes, preferably 2-4 minutes, to precipitate RBCs, and preferably remove plasma at the same time. The collected RBCs were gently vortexed in RBC lysis buffer for 5-20 minutes at room temperature. The obtained solution is then centrifuged at 500-3000 rpm, more preferably 100-2000 rpm, for 1-10 minutes, more preferably 3-6 minutes to pellet unlysed cells including white blood cells and rare cells.

In a specific embodiment of the present invention, the immune particles for removing leukocytes are located in an affinity chromatography column and combined with a specific binder targeting leukocytes. Preferably said specific binder is an antibody recognizing an antigen expressed on leukocytes. Since CD45 (also known as "leukocyte surface common antigen") is the most recognized and accepted leukocyte surface antigen, it is more preferred that the antibody is an anti-CD45 antibody. Most preferably the anti-CD45 mAb (CD45 mAb) is coupled to the Affi-Gel 10 matrix. Chromatography can be performed using affinity chromatography columns known to those skilled in the art. In a preferred embodiment of the present invention, in order to remove leukocytes, the solution containing leukocytes is mixed at a flow rate of about 0.5-8.5ml/min, preferably at a flow rate of about 0.75-3.5ml/min, more preferably at a flow rate of about 1-2.5ml /min flow through the affinity chromatography column.

The target cells obtained by the method of the present invention are suitable for subsequent analysis, operation or application. Of course, after the target cells of the present invention are obtained, the proteins and/or genes in the target cells can be subjected to subsequent analysis, operation or application. In a specific embodiment of the present invention, the subsequent analysis, operation or application is one of the following: flow cytometry, polymerase chain reaction (PCR), immunofluorescence, immunocytochemistry, image analysis, enzymatic assay , inflammation studies, gene expression profiling, therapeutic efficacy testing, cell culture and cell therapy, mass spectrometry and other cell and/or protein-related research, etc.

In a specific embodiment of the present invention, the target cells enriched by the method of the present invention include at least one of stem cells, fetal cells, circulating tumor cells (CTCs), and immune cells. On the basis of using the method of the present invention to enrich the above-mentioned target cells, further separation and purification methods can be used to obtain circulating tumor cells, stem cells (the stem cells in the present invention are bone marrow stem cells, adult stem cells, umbilical cord blood stem cells or skin stem cells), Fetal cells, immune cells (including T lymphocytes, B lymphocytes, K lymphocytes and NK lymphocytes), etc., and these purified circulating tumor cells, stem cells, fetal cells, immune cells can be further used for prediction, detection, diagnosis and treat.

In a specific embodiment of the invention, the method of the invention further comprises removing plasma proteins. In order to save time and reduce operating steps, the step of removing plasma proteins can be performed simultaneously with the step of precipitating red blood cells. For example, in one embodiment of the invention, whole blood is centrifuged at 1500°C for 3 minutes to pellet red blood cells and remove plasma including plasma proteins.

In a specific embodiment of the present invention, it is preferred to recover leukocytes and/or plasma proteins removed by the method of the present invention and further use them for subsequent analysis, manipulation or application, for example, flow cytometry, polymerization Enzyme chain reaction (PCR), immunofluorescence, immunocytochemistry, image analysis, enzymatic assays, inflammation studies, gene expression profiling, therapeutic efficacy testing, cell culture, cell therapy, mass spectrometry and other cell and/or protein-related research etc. For example, further separation and purification methods can be used to obtain white blood cells or plasma proteins, and these purified white blood cells or plasma proteins can be further used for prediction, detection, diagnosis and treatment.

In the present invention, the term "enrichment" is used in its broadest sense to make a substance more concentrated than it is under the original environmental conditions, which includes "accumulation", "concentration", "extraction", "separation", etc. meaning inside.

The present invention also provides a method for removing leukocytes, the method comprising using anti-CD45 antibody-coupled Affi-Gel 10 to remove leukocytes. Chromatography can be performed using affinity chromatography columns known to those skilled in the art. In a preferred embodiment of the present invention, in order to remove leukocytes, the solution containing leukocytes is mixed at a flow rate of about 0.5-8.5ml/min, preferably at a flow rate of about 0.75-3.5ml/min, more preferably at a flow rate of about 1-2.5ml /min flow through the affinity chromatography column. Immune particles can be washed and reused with buffers such as Tris-Glycine, saving costs. This method is not only low cost, time saving, but also high efficiency.

The present invention is further described in the form of examples below, which are not intended to limit the scope of the present invention.

Example

Embodiment 1, the method for removing leukocyte from biological sample

Dilute human blood in PBS containing 5mM EDTA, add 3 times the volume of RBC lysis buffer (see below for specific components), and gently rotate and shake at room temperature for 12 minutes. The samples were spun at 1500 rpm for 5 minutes to collect the cell pellet. The lysed RBCs in the supernatant were discarded, and the cell pellet was resuspended in 3ml-5ml of PBS containing 5mM EDTA, and passed through anti-CD45 antibody-conjugated Affi-Gel 10 (Pierce, IL, US). and a chromatography column through which the cells were passed in PBS at a flow rate of 2 ml/min to remove WBC, and the flow-through was collected. Compared with utilizing Miltenyi's kit to remove leukocytes in the prior art, the method for removing leukocytes of the present invention significantly reduces cost (the cost of the separation method of the present invention is RMB 80-150/test; while the cost of Multiny's kit is RMB 600-700 / test), shortened time (each test of the separation method of the present invention needs to finish removing leukocytes in less than 40 minutes; and Multiny's kit removes leukocytes and needs more than 4 hours/test), and efficient (the WBC of the inventive method removes The rate can reach about 3-4logs; while the WBC removal rate of Multiny's kit is only about 2-3logs).

Preparation of anti-CD45 antibody-conjugated Affi-Gel 10: Since Affi-Gel 10 purchased from Pierce (IL, US) contains chemical groups available for activation, mix anti-CD45 antibody with Affi-Gel 10 for about 15 minutes , the coupling of anti-CD45 antibody to Affi-Gel 10 can be completed.

Embodiment 2, the method for enriching target cell from biological sample

5-10 ml of human blood was collected in a test tube containing EDTA and spun at 1500 rpm for 3 minutes to pellet RBC and remove plasma including plasma proteins. The collected RBCs were gently vortexed and shaken in 10 ml of NH ₄ Cl-based RBC lysis buffer (specific components as above) for 15 minutes at room temperature. The obtained solution was then spun centrifuged at 1500 rpm for 5 minutes to pellet unlysed cells including WBC and rare cells. The resulting unlysed cell pellet was resuspended in 5 ml PBS, and passed through a 0.5 ml anti-CD45 antibody-coupled Affi-Gel 10 (Pierce, IL, US) affinity chromatography column to remove WBC, wherein the cells were separated in 2 ml /min flow rate in PBS flow through the affinity chromatography column, and collect the flow-through. The collected flow-through containing rare cells was centrifuged at 900g for more than 5 minutes. Cell pellets were resuspended for subsequent analysis. After enrichment, the affinity column can be washed with 10 ml Tris-Glycine (pH 2.5) to remove all bound molecules and then neutralized with PBS (pH 7.4) for reuse.

Embodiment 3, the yield of the enrichment method of the present invention is verified by admixture research

Target cells (such as HeLa cells or MCF-7 cells available from ATCC) were prelabeled with 4,6-diamino-2-phenylindole (DAPI) and then spiked into human blood using the method described in Example 1. Methods to enrich spiked cells. The average recovery was about 70% to 80%. Compared with the enrichment method utilizing Miltenyi's kit to remove leukocytes in the prior art, the enrichment method of the present invention significantly reduces cost (the cost of the enrichment method of the present invention is RMB 100-200/test; while utilizing Multinyi's kit Only the cost of removing leukocytes is RMB 600-700/test), shortened time (each test of the enrichment method of the present invention needs less than 1 hour to complete the removal of erythrocytes and leukocytes; while Multiny's kit only needs to remove leukocytes More than 4 hours/test), and high efficiency (the WBC removal rate of the method of the present invention can reach about 3-4logs; while the WBC removal rate of Multiny's kit is only about 2-3logs).

Claims (9)

1. A method for enriching target cells from a biological sample, wherein the biological sample is a body fluid, and the target cells include at least one of stem cells, fetal cells, circulating tumor cells, and immune cells, the The stem cells are bone marrow stem cells, adult stem cells, umbilical cord blood stem cells or skin stem cells, and the method includes: Removal of red blood cells by osmolarity-based means and white blood cells by immunoaffinity chromatography; Wherein the method based on osmolality includes precipitation and lysing red blood cells; wherein the precipitation is centrifuged at 500-3000rpm for 1-6 minutes to precipitate RBCs, and the collected RBCs are gently rotated and oscillated in RBC lysis buffer at room temperature 5 to 20 minutes, and then centrifuge the obtained solution at 500 to 3000 rpm for 1 to 10 minutes to precipitate unlysed cells, which include white blood cells and rare cells; Wherein, immune particles are used in the immunoaffinity chromatography, and the size of the immune particles is 1 μm to 20 μm, and the immune particles are located in the affinity chromatography column and combined with specific binders targeting white blood cells; the specific The conjugate is an antibody that recognizes an antigen expressed on leukocytes, the antibody is an anti-CD45 antibody, and the anti-CD45 antibody is coupled to the Affi-Gel10 matrix; wherein the solution containing leukocytes is mixed at 0.5-8.5ml/min flow rate through the affinity chromatography column. the 2. The method of claim 1, wherein said bodily fluid is blood, bone marrow, spinal cord or surgical fluid. the 3. The method of claim 2, wherein said blood is fresh blood on the day of blood collection. the 4. The method of claim 1, wherein the size of the immune particles used in the immunoaffinity chromatography is 5 μm to 10 μm. the 5. The method of claim 1 or 4, wherein the immune particles used in the immunoaffinity chromatography are reproducible. the 6. The method of claim 1, wherein the CD45 antibody is an anti-CD45 mAb. the 7. The method according to claim 1, the target cells obtained by the method are suitable for subsequent analysis, operation or application, and the subsequent analysis, operation or application is one of the following: flow cytometry, polymerase chain reaction , immunofluorescence, immunocytochemistry, image analysis, enzymatic assays, inflammation studies, gene expression profiling, therapeutic efficacy testing, cell culture, and cell therapy. the 8. The method of claim 1, further comprising removing plasma proteins. the 9. The method of claim 8, further comprising recovering the removed leukocytes and/or the removed plasma proteins. the