CTC single cell extraction, sorting and culture method

Abstract

The invention relates to a CTC single cell related biotechnology, in particular to a CTC single cell extraction, sorting and culture method which comprises the following steps: adding a collected sample into a centrifugal separation tube, and separating a leukocyte layer in the sample into the centrifugal separation tube under the centrifugal action; extracting a leukocyte layer in the centrifugal separation tube into a test tube, and then removing leukocytes to obtain a CTC cell suspension; adding the CTC cell suspension into a cell sorting chip, and sorting out CTC single cells; and adding a culture solution into the cell sorting chip, and culturing each sorted CTC single cell. Compared with the prior art, the cell sorting chip has the advantages that the preserved leukocyte layer can be directly extracted from the centrifugal separation tube, the CTC single cell sorting and culturing process is directly completed in the cell sorting chip, and compared with the prior art, the used equipment is less, the flow is shorter, the working efficiency is improved, and the cost is reduced.

Description

Technical Field

[0001] The present invention relates to CTC single-cell related biotechnology, specifically to a method for extracting, sorting, and culturing CTC single cells. Background Art

[0002] Circulating tumor cells (CTC) are tumor cells that originate from the primary tumor site or are released into the peripheral blood circulation from solid tumors or metastases due to medical procedures. Most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood. A small number of CTCs with high viability and metastatic potential can survive in the circulatory system. When they encounter a suitable stromal environment in organs or tissues, tumor metastasis occurs. This is an important cause of postoperative recurrence and distant metastasis in patients with malignant tumors and an important factor leading to the death of tumor patients. Therefore, how to extract circulating tumor cells has great clinical significance for tumor identification and treatment. Cell sorting and characterization techniques can quickly isolate the required subsets for identification and monitoring for clinical diagnosis. For example, understanding the heterogeneity of a patient's solid tumor at the single-cell level can enable treatment targeting multiple cell subtypes, thereby improving the survival rate. Currently, although there are various techniques for cell extraction, sorting, and culture, there are few full-process techniques for the extraction, sorting, and culture of CTC single cells. Even if existing cell extraction, sorting, and culture techniques are transplanted to CTC single cells, it will result in a too long process flow, a complex equipment system, and too high costs. Summary of the Invention

[0003] In view of the above technical problems, the present invention provides a method for extracting, sorting, and culturing CTC single cells with a relatively simple process flow and less equipment used.

[0004] The technical solution adopted by the present invention to solve the above technical problems is as follows: A method for extracting, sorting, and culturing CTC single cells, which includes the following steps: (1) Add the collected sample into a centrifuge tube, and under the action of centrifugation, separate the white blood cell layer in the sample into the centrifuge tube; (2) Extract the white blood cell layer in the above centrifuge tube into a test tube, and then remove the white blood cells to obtain a CTC cell suspension; (3) Add the above CTC cell suspension into a cell sorting chip to sort out CTC single cells; (4) Add a culture medium into the above cell sorting chip to culture each sorted CTC single cell.

[0005] Preferably, a separation structure is provided in the centrifuge tube, and under the action of centrifugation, the white blood cell layer in the sample is separated through this separation structure.

[0006] Preferably, the separation structure includes a sample chamber and a separation channel communicating with the sample chamber. After the sample and the isolation liquid are added to the sample chamber, the sample is stratified under centrifugation, and the upper leukocyte layer after stratification is separated through the separation channel.

[0007] Preferably, the separation channel is a microfluidic channel. Under centrifugation, the leukocyte layer automatically flows into the accommodation chamber at the bottom of the centrifugal separation tube through the microfluidic channel.

[0008] Preferably, after the leukocyte layer is extracted into a test tube, immunomagnetic beads are added to the test tube, and then the test tube is placed on a shaker for mixing and incubation, and then the leukocytes are removed by attaching a magnetic rack.

[0009] Preferably, the cell sorting chip is provided with a sample injection hole and several sorting holes. The CTC cell suspension injected into the sample injection hole is sorted into single CTC cells through the sorting microfluidic channels arranged in the cell sorting chip, and each single CTC cell flows into the corresponding sorting hole.

[0010] Preferably, a culture solution is dropped into each sorting hole to culture the single CTC cells.

[0011] Preferably, the cell sorting chip is provided with a liquid injection flow channel communicating with each sorting hole. After the culture solution is injected from the sample injection hole, the culture solution flows into each sorting hole through the liquid injection flow channel to culture the single CTC cells.

[0012] Preferably, each sorting hole is a stepped hole with a larger diameter at the upper end and a smaller diameter at the lower end. The sorting microfluidic channels are arranged in the lower layer of the cell sorting chip, and the liquid injection flow channels are arranged in the upper layer of the cell sorting chip; the CTC cell suspension injected into the sample injection hole is sorted into single CTC cells through the sorting microfluidic channels and then flows into the small holes of each stepped hole, and the culture solution injected into the sample injection hole flows into the large holes of each stepped hole through the liquid injection flow channel to culture the single CTC cells in the corresponding small holes.

[0013] Preferably, when injecting the CTC cell suspension into the sample injection hole, the liquid injection flow channel is blocked; when injecting the culture solution into the sample injection hole, the sorting microfluidic channel is blocked.

[0014] As can be seen from the above technical solutions, the present invention separates and preserves the leukocyte layer in the sample through a centrifugal separation tube, and the well-preserved leukocyte layer can be directly extracted from the centrifugal separation tube. Moreover, the sorting and culture processes of single CTC cells are directly completed in the cell sorting chip. Compared with the prior art, fewer devices are used and the process is shorter, which not only improves work efficiency but also reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the separation structure of the present invention.

[0016] Figure 2 It is a partial structural schematic diagram of the cell sorting chip of the present invention.

[0017] Figure 3 It is a partial sectional structural schematic diagram of the cell sorting chip of the present invention. Specific embodiments

[0018] The present invention will be described in detail below with reference to the accompanying drawings. The illustrative embodiments and descriptions of the present invention herein are used to explain the present invention, but not to limit the present invention.

[0019] See Figures 1-3 , the present invention provides a method for extracting, sorting and culturing CTC single cells, which includes the following steps: First, add the collected sample into the centrifugation separation tube 1. The sample can be collected from the pleural effusion, peritoneal effusion, cerebrospinal fluid, lymph fluid, and blood of humans or animals. Then, under the action of centrifugation, the white blood cell layer in the sample is separated and stored in the centrifugation separation tube without separately transferring and storing the white blood cell layer in other utensils. Then, extract the white blood cell layer in the above centrifugation separation tube into a test tube, and then remove the white blood cells to obtain a CTC cell suspension. Specifically, after the white blood cell layer in the sample is extracted into a test tube, add immunomagnetic beads to the test tube, then place the test tube on a shaker and mix and incubate, and then attach a magnetic rack to remove the white blood cells, thereby obtaining a high-purity CTC cell suspension. Add the above high-purity CTC cell suspension into the cell sorting chip 2 to sort out CTC single cells; finally, add a culture medium into the above cell sorting chip to culture each sorted CTC single cell. Thus, the present invention realizes the extraction, sorting and culture of CTC single cells by using a centrifugation separation tube and a cell sorting chip in cooperation with related equipment. This process omits the steps and equipment for separately transferring and storing the white blood cell layer, and does not use separate culture steps and equipment, which not only shortens the process flow and reduces the use of equipment; but also improves work efficiency and saves costs.

[0020] Such as Figure 1, a separation structure 11 is provided inside the centrifugal separation tube 1 of the present invention. Under centrifugal action, the white blood cell layer in the sample is separated through this separation structure. Among them, the separation structure 11 includes a sample chamber 111 and a separation channel 112 communicating with the sample chamber. During implementation, first add an isolation liquid to the sample chamber, then add the sample on the upper layer of the isolation liquid, and then place the centrifugal separation tube on a centrifuge for centrifugation. After centrifugation, due to the different densities of the components in the sample, the white blood cells and red blood cells in the sample in the sample chamber are separated by the isolation liquid. Among them, the supernatant on the upper layer is the white blood cell layer containing CTCs, and the lower layer is the red blood cell layer. Thus, under centrifugal action again, the white blood cell layer on the upper layer is separated through the separation channel. Specifically, the separation channel is a microfluidic channel. Under centrifugal and siphon effects, the white blood cell layer automatically flows into the accommodation chamber 12 at the bottom of the centrifugal separation tube through this microfluidic channel, realizing the separate storage of the white blood cell layer for further use. It can be seen that the entire extraction process of the present invention is not only simple in centrifugal separation, with fewer steps, but also does not require a pipette to extract the supernatant, with simpler operation, shorter process, and higher efficiency.

[0021] As Figure 2 , an injection hole 21 and several sorting holes 22 are provided on the cell sorting chip 2 of the present invention. The CTC cell suspension injected into the injection hole is sorted into CTC single cells through a sorting microchannel 23 provided inside the cell sorting chip, and each CTC single cell flows into the corresponding sorting hole. During implementation, the cross-sectional diameter of the sorting microchannel is designed to be about 30 - 50 micrometers, allowing only single cells of about 30 micrometers to pass through individually. Under the injection pressure of the injection hole, the CTC single cells flow into the sorting holes through the above sorting microchannel, thereby realizing the sorting of CTC single cells. After the CTC single cells are sorted, as a preferred method, a culture solution can be dropped into each sorting hole to culture the CTC single cells. During implementation, the culture solution can be dropped manually one by one, or can be dropped in groups by a machine.

[0022] The present invention also provides another preferred method. A liquid injection channel 24 communicating with each sorting hole is provided inside the cell sorting chip. After injecting the culture solution from the injection hole, the culture solution flows into each sorting hole through the liquid injection channel to culture the CTC single cells. Thus, there is no need to drop the culture solution manually or by machine one by one, improving the work efficiency. As Figure 3 , each sorting hole of the cell sorting chip is a stepped hole with a larger diameter at the upper end and a smaller diameter at the lower end. The sorting microchannel is provided on the lower layer of the cell sorting chip, and the liquid injection channel is provided on the upper layer of the cell sorting chip. Thus, the cell sorting chip of the present invention forms a dual-channel structure, realizing the sorting and culture of CTC single cells and further improving the work efficiency.

[0023] During the implementation process, first inject the CTC cell suspension into the sample injection hole. After the CTC cell suspension is sorted into single CTC cells through the sorting microchannel, it flows into the small hole 221 of each step hole. Then, inject the culture medium into the sample injection hole. The culture medium flows into the large hole 222 of each step hole through the liquid injection flow channel to culture the single CTC cells in the corresponding small holes. Preferably, when injecting the CTC cell suspension into the sample injection hole, the liquid injection flow channel should be blocked to prevent the CTC cell suspension from flowing into the liquid injection flow channel. Similarly, when injecting the culture medium into the sample injection hole, the sorting microchannel should be blocked to prevent the CTC cell suspension from flowing back. And after the injection of the culture medium is completed, the sample injection hole should be filled with a plunger to block both the sorting microchannel and the liquid injection flow channel simultaneously to prevent the CTC cell suspension and the culture medium from flowing back.

Claims

1. A method for CTC single-cell extraction, sorting, and culture, characterized in that, It includes the following steps: (1) Add the collected sample into a centrifuge tube, and separate the white blood cell layer in the sample into the centrifuge tube under the action of centrifugation; (2) Extract the white blood cell layer in the above centrifuge tube into a test tube, then remove the white blood cells to obtain a CTC cell suspension; (3) Add the above CTC cell suspension into a cell sorting chip to sort out single CTC cells; (4) Add a culture medium into the above cell sorting chip to culture each sorted single CTC cell.

2. The CTC single cell extraction, sorting and culture method according to claim 1, wherein: A separation structure is provided in the centrifuge tube, and under the action of centrifugation, the white blood cell layer in the sample is separated through this separation structure.

3. The CTC single cell extraction, sorting and culture method according to claim 2, characterized in that: The separation structure includes a sample chamber and a separation channel communicating with the sample chamber. After the sample and the isolation liquid are added into the sample chamber, the sample is stratified under the action of centrifugation, and the upper white blood cell layer after stratification is separated through the separation channel.

4. The CTC single cell extraction, sorting and culture method according to claim 3, characterized in that: The separation channel is a microfluidic channel. Under the action of centrifugation, the white blood cell layer automatically flows into the accommodation chamber at the bottom of the centrifuge tube through this microfluidic channel.

5. The CTC single cell extraction, sorting and culture method according to claim 1, characterized in that: After the white blood cell layer is extracted into a test tube, add immunomagnetic beads into the test tube, then place the test tube on a shaker and mix and incubate it, and then place it on a magnetic rack to remove the white blood cells.

6. The CTC single cell extraction, sorting and culture method according to any one of claims 1 to 5, characterized in that: The cell sorting chip is provided with a sample injection hole and several sorting holes. The CTC cell suspension injected into the sample injection hole is sorted out into single CTC cells through the sorting microfluidic channels provided in the cell sorting chip, and each single CTC cell flows into the corresponding sorting hole.

7. The CTC single cell extraction, sorting and culture method according to claim 6, characterized in that: Drop a culture medium into each sorting hole to culture the single CTC cells.

8. The CTC single cell extraction, sorting and culture method according to claim 6, characterized in that: The cell sorting chip is provided with a liquid injection flow channel communicating with each sorting hole. After the culture medium is injected from the sample injection hole, the culture medium flows into each sorting hole through the liquid injection flow channel to culture the single CTC cells.

9. The CTC single cell extraction, sorting and culture method according to claim 8, wherein: Each sorting hole is a stepped hole with a larger diameter at the upper end and a smaller diameter at the lower end. The sorting microfluidic channels are arranged in the lower layer of the cell sorting chip, and the liquid injection flow channels are arranged in the upper layer of the cell sorting chip; the CTC cell suspension injected into the sample injection hole is sorted out into single CTC cells through the sorting microfluidic channels and then flows into the small holes of each stepped hole, and the culture medium injected into the sample injection hole flows into the large holes of each stepped hole through the liquid injection flow channels to culture the single CTC cells in the corresponding small holes.

10. The CTC single cell extraction, sorting and culture method according to claim 9, characterized in that: When injecting the CTC cell suspension into the sample injection hole, block the liquid injection flow channel; when injecting the culture medium into the sample injection hole, block the sorting microfluidic channel.

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