CN115896026A - Separation and purification method of circulating tumor cells - Google Patents
Separation and purification method of circulating tumor cells Download PDFInfo
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- CN115896026A CN115896026A CN202211520444.9A CN202211520444A CN115896026A CN 115896026 A CN115896026 A CN 115896026A CN 202211520444 A CN202211520444 A CN 202211520444A CN 115896026 A CN115896026 A CN 115896026A
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- 208000005443 Circulating Neoplastic Cells Diseases 0.000 title claims abstract description 31
- 238000000926 separation method Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000000746 purification Methods 0.000 title abstract description 8
- 210000004369 blood Anatomy 0.000 claims abstract description 25
- 239000008280 blood Substances 0.000 claims abstract description 25
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- 239000007788 liquid Substances 0.000 claims abstract description 13
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 11
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- 239000007853 buffer solution Substances 0.000 claims abstract description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 7
- 210000002751 lymph Anatomy 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims abstract description 6
- 239000006166 lysate Substances 0.000 claims abstract description 5
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- 210000005259 peripheral blood Anatomy 0.000 claims description 13
- 238000005119 centrifugation Methods 0.000 claims description 8
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 8
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Abstract
The invention provides a separation and purification method of circulating tumor cells, which relates to the technical field of biomedicine and comprises the following steps: collecting blood sample and placing the blood sample in an ice box; mixing blood sample and buffer solution, adding lymph separation solution, and centrifuging; collecting lymphocyte layer and transparent separation liquid layer, adding buffer solution, mixing, and centrifuging again; discarding the supernatant to leave the precipitated cells, adding erythrocyte lysate, standing, and centrifuging again; adding fetal calf serum to obtain test solution. The separation and purification method only needs some conventional biochemical reagents and some simple operations, realizes the enrichment and purification of the circulating tumor cells, and reduces the cost.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for separating and purifying circulating tumor cells.
Background
Circulating Tumor Cells (CTCs) in human peripheral blood are rare cells that are rare in number but have important clinical significance, meaning tumor cells that break away from tumor lesions and spread into the human peripheral blood circulation and can develop into tumor metastatic lesions under certain conditions. Generally, circulating tumor cells suggest the presence of tumors and possible metastases in humans. Since more than 90% of cancer deaths are clinically caused by metastasis, and circulating tumor cells provide a source of metastatic foci of tumors in the human body in addition to tumor foci, capturing and detecting circulating tumor cells from blood is becoming increasingly important.
However, the content of circulating tumor cells in peripheral blood is very small, and each 10mL of blood may contain only a few to tens of circulating tumor cells, but has as many as about 1 hundred million white blood cells and 500 hundred million red blood cells, so that the efficient enrichment and separation and sensitive detection of very small amount of CTCs from peripheral blood is an important challenge facing clinical practice at present, and is also a research hotspot in recent years. Accurate quantification of circulating tumor cells mainly involves a two-step core strategy: (1) capture and enrichment, and (2) identification and detection. Capture and enrichment typically involves physical interactions or antibody-antigen interactions of circulating tumor cells and material; detection and identification include various methods and techniques, such as fluorescence microscopy, fluorescence spectroscopy, flow cytometry, surface enhanced raman scatterometry, or electrochemical methods, among others.
In the aspect of circulating tumor cell enrichment, previous researches mostly utilize identification of cell surface specific proteins, and simultaneously, the enrichment and purification of CTCs in peripheral blood are realized by means of magnetic beads, microfluidics, electrodes, nano materials and other auxiliary separation. However, the current clinical application still uses heterogeneous immunomagnetic bead enrichment and other strategies, the process is heterogeneous, the operation is complex and time-consuming, the sample flux is small, the cost is high due to the fact that an antibody is used as an identification probe, and certain biological safety hazards exist.
Disclosure of Invention
In view of this, the present invention aims to provide a method for separating and purifying circulating tumor cells, so as to solve the technical problems that the circulating tumor cell enrichment method adopted in the prior art uses heterogeneous immunomagnetic bead enrichment and other strategies, the process is heterogeneous, the operation is complex and time-consuming, the sample flux is small, the cost is high due to the use of antibodies as recognition probes, and certain biological potential safety hazards are caused.
In order to achieve the above object, the present invention provides a method for separating and purifying circulating tumor cells, comprising the following steps:
s1: collecting blood sample and placing the blood sample in an ice box;
s2: mixing blood sample and buffer solution, adding lymph separation solution, and centrifuging;
s3: collecting the lymphocyte layer and the transparent separation liquid layer, adding a buffer solution, uniformly mixing, and centrifuging again;
s4: discarding the supernatant to leave the precipitated cells, adding erythrocyte lysate, standing and centrifuging again;
s5: adding fetal calf serum to obtain test solution.
According to an alternative embodiment, the blood sample in step S1 is whole blood or peripheral blood.
According to an optional embodiment, when peripheral blood is sampled, a disposable sealed ethylene diamine tetraacetic acid anticoagulation vacuum blood collection tube is adopted.
According to an alternative embodiment, the buffer in step 2 and step 3 is a phosphate buffer.
According to an alternative embodiment, the first layer after centrifugation in step 2 is a plasma layer, the second layer is a layer of cyclic opalescent lymphocytes, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer.
According to an alternative embodiment, the phosphate buffer in step 2 corresponds to the volume of the blood sample, and the layer of lymphocytes and the layer of transparent separation liquid in step 3 correspond to the volume of the phosphate buffer.
According to an alternative embodiment, the centrifugation time in step 2 is 20 minutes, the ramp-up rate is 1 and the ramp-down rate is 1.
According to an alternative embodiment, the centrifugation time in step 3 is 7 minutes, the ramp-up rate is 9 and the ramp-down rate is 9.
The method for separating and purifying the circulating tumor cells has the following technical effects:
the separation and purification method of the circulating tumor cells only needs some conventional biochemical reagents and some simple operations, realizes the enrichment and purification of the circulating tumor cells, reduces the cost, can directly use the processed samples in various quantitative methods, and can replace and use the used reagents and consumables at any time without depending on laboratory conditions.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of an experiment of a method for separating and purifying circulating tumor cells according to an embodiment of the present invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1:
this example isolated and purified A549 cells
1) Collecting peripheral blood by using a disposable closed Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulation vacuum blood collection tube, and placing the collected peripheral blood in an ice box;
2) 4mL of the retrieved blood was mixed with 4mL of Phosphate Buffered Saline (PBS) by pipetting;
3) Two 15mL centrifuge tubes were taken, 4mL of the blood and PBS mixture was added to each centrifuge tube, and carefully added slowly to 4mL of the lymph separation medium (the lymph separation medium was mixed by repeated inversion before use);
4) Centrifuging at 4 ℃ for 20 minutes at 800g with the speed increasing adjusted to 1 and the speed decreasing adjusted to 1; (in this case, the centrifuge tube is divided into 4 layers from top to bottom, namely, the first layer is a plasma layer, the second layer is a ring-shaped milky white lymphocyte layer, the third layer is a transparent separation liquid layer, the fourth layer is a red blood cell layer, and the rising and falling plus centrifugation time is about 40 minutes);
5) Collecting about 4mL of the annular milky white lymphocyte layer of the second layer and the transparent separation liquid layer of the third layer, adding PBS diluent with the same volume, uniformly mixing, centrifuging at 4 ℃ and 2000rpm, regulating the rising speed to 9 and the falling speed to 9, and centrifuging for 7 minutes;
6) Discarding cells of supernatant and remaining precipitates, adding 5-8mL of erythrocyte lysate, and standing for 10 minutes;
7) Separating at 4 deg.C by 500g, regulating the speed to 9 at increasing speed, regulating the speed to 9 at decreasing speed, centrifuging for 10min, discarding supernatant, and precipitating to obtain desired lymphocyte and free A549 cells;
8) The test solution was obtained by adding 1mL of Fetal Bovine Serum (FBS).
Example 2:
this example separated and purified liver cancer SMMC-772 cells
1) Collecting peripheral blood by using a disposable closed Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulation vacuum blood collection tube, and placing the collected peripheral blood in an ice box;
2) 4mL of the retrieved blood was mixed with 4mL of Phosphate Buffered Saline (PBS) by pipetting;
3) Taking two 15mL centrifuge tubes, adding 4mL of the mixed solution of blood and PBS into each centrifuge tube, and carefully and slowly adding the mixed solution into 4mL of lymph separation liquid (the lymph separation liquid is repeatedly turned over and uniformly mixed before being used);
4) Centrifuging at 4 ℃ for 20 minutes at 800g with the speed increasing adjusted to 1 and the speed decreasing adjusted to 1; (in this case, the centrifuge tube is divided into 4 layers from top to bottom, namely, the first layer is a plasma layer, the second layer is a ring-shaped milky white lymphocyte layer, the third layer is a transparent separation liquid layer, the fourth layer is a red blood cell layer, and the rising and falling plus centrifugation time is about 40 minutes);
5) Collecting about 4mL of the annular milky white lymphocyte layer of the second layer and the transparent separation liquid layer of the third layer, adding PBS diluent with the same volume, uniformly mixing, centrifuging at 4 ℃ and 2000rpm, regulating the rising speed to 9 and the falling speed to 9, and centrifuging for 7 minutes;
6) Discarding cells of supernatant and remaining precipitates, adding 5-8mL of erythrocyte lysate, and standing for 10 minutes;
7) Separating at 4 deg.C by 500g, increasing speed to 9, decreasing speed to 9, centrifuging for 10min, discarding supernatant, and precipitating to obtain desired lymphocyte and free hepatocarcinoma SMMC-7721 cells;
8) The test solution was obtained by adding 1mL of Fetal Bovine Serum (FBS).
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (8)
1. A method for separating and purifying circulating tumor cells is characterized by comprising the following steps:
s1: collecting blood sample and placing the blood sample in an ice box;
s2: mixing blood sample and buffer solution, adding lymph separation solution, and centrifuging;
s3: collecting lymphocyte layer and transparent separation liquid layer, adding buffer solution, mixing, and centrifuging again;
s4: discarding the supernatant to leave the precipitated cells, adding erythrocyte lysate, standing and centrifuging again;
s5: adding fetal calf serum to obtain test solution.
2. The method for separating and purifying circulating tumor cells according to claim 1, wherein the blood sample in step S1 is whole blood or peripheral blood.
3. The method for separating and purifying circulating tumor cells according to claim 2, wherein a disposable sealed ethylenediaminetetraacetic acid anticoagulation vacuum blood collection tube is used for sampling peripheral blood.
4. The method for separating and purifying circulating tumor cells according to claim 1, wherein the buffer solution in step 2 and step 3 is phosphate buffer solution.
5. The method of claim 4, wherein the first layer after centrifugation in step 2 is a plasma layer, the second layer is a layer of cyclic opalescent lymphocytes, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer.
6. The method for separating and purifying circulating tumor cells according to claim 5, wherein the volume of the phosphate buffer solution in step 2 is equal to the volume of the blood sample, and the volume of the buffer solution of the lymphocyte layer and the transparent separation liquid layer in step 3 is equal to the volume of the phosphate buffer solution.
7. The method for separating and purifying circulating tumor cells according to claim 1, wherein the centrifugation time in step 2 is 20 minutes, the acceleration rate is 1, and the deceleration rate is 1.
8. The method for separating and purifying circulating tumor cells according to claim 1, wherein the centrifugation time in step 3 is 7 minutes, the acceleration rate is 9, and the deceleration rate is 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108562569A (en) * | 2018-06-04 | 2018-09-21 | 中国人民解放军第二军医大学 | A kind of circulating tumor cell detection method based on Surface enhanced Raman spectroscopy probe |
| CN109856388A (en) * | 2018-11-29 | 2019-06-07 | 北京优迅医学检验实验室有限公司 | The catching method and capture kit of circulating tumor cell |
| CN110079501A (en) * | 2019-05-14 | 2019-08-02 | 兰州大学 | Mouse breast cancer circulating tumor cell system and its method for building up |
| KR102195212B1 (en) * | 2020-02-17 | 2020-12-28 | 주식회사 다우진유전자연구소 | Kit for diagnosis of breast cancer by circulating tumor cell detection and analysis |
| WO2022206133A1 (en) * | 2021-03-30 | 2022-10-06 | 深圳市亚辉龙生物科技股份有限公司 | Microfluidic chip, and automatic separation and detection system and method for circulating tumor cell |
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- 2022-11-29 CN CN202211520444.9A patent/CN115896026A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108562569A (en) * | 2018-06-04 | 2018-09-21 | 中国人民解放军第二军医大学 | A kind of circulating tumor cell detection method based on Surface enhanced Raman spectroscopy probe |
| CN109856388A (en) * | 2018-11-29 | 2019-06-07 | 北京优迅医学检验实验室有限公司 | The catching method and capture kit of circulating tumor cell |
| CN110079501A (en) * | 2019-05-14 | 2019-08-02 | 兰州大学 | Mouse breast cancer circulating tumor cell system and its method for building up |
| KR102195212B1 (en) * | 2020-02-17 | 2020-12-28 | 주식회사 다우진유전자연구소 | Kit for diagnosis of breast cancer by circulating tumor cell detection and analysis |
| WO2022206133A1 (en) * | 2021-03-30 | 2022-10-06 | 深圳市亚辉龙生物科技股份有限公司 | Microfluidic chip, and automatic separation and detection system and method for circulating tumor cell |
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