A method for extracting, sorting and culturing CTC single cells

Abstract

The present invention relates to CTC single cell related biotechnology, specifically a CTC single cell extraction, sorting, and culture method, which includes adding a collected sample into a centrifuge tube, separating the white blood cell layer in the sample into the centrifuge tube under centrifugal action; extracting the white blood cell layer in the centrifuge tube into a test tube, then removing the white blood cells to obtain a CTC cell suspension; adding the CTC cell suspension to a cell sorting chip to sort out CTC single cells; adding culture medium to the cell sorting chip, and culturing each sorted CTC single cell. The present invention can directly extract the preserved white blood cell layer from the centrifuge tube, and the sorting and culture process of the CTC single cell is directly completed in the cell sorting chip. Compared with the existing technology, it uses less equipment and has a shorter process, which not only improves work efficiency but also reduces costs.

Description

Technical Field

[0001] The present invention relates to CTC single cell related biotechnology, specifically a CTC single cell extraction, sorting and culture method. Background Art

[0002] Circulating tumor cells (CTCs) originate from the primary tumor site or are released from solid tumors or metastases into the peripheral bloodstream during diagnostic and therapeutic procedures. Most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood. However, a small number of CTCs with high motility and metastatic potential can survive in the circulation and, upon encountering a suitable stromal environment in organs and tissues, metastasize to the tumor. This is a major cause of postoperative recurrence and distant metastasis in patients with malignant tumors, as well as a significant factor in cancer mortality. Therefore, the ability to isolate circulating tumor cells is of immense clinical significance for tumor identification and treatment. Cell sorting and characterization technologies can rapidly isolate desired subpopulations for identification and monitoring for clinical diagnosis. Understanding the heterogeneity of a patient's solid tumor at the single-cell level, for example, can enable therapies tailored to multiple cell subtypes, thereby improving survival rates. Currently, despite a wide variety of cell extraction, sorting, and culture technologies, few fully integrated technologies for the extraction, sorting, and culture of single CTCs exist. Even if existing cell extraction, sorting, culture and other technologies are transplanted to CTC single cells, it will result in a long process flow, complex equipment system and high cost. Summary of the Invention

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

[0004] The technical solution adopted by the present invention to solve the above technical problems is: a method for extracting, sorting and culturing single CTC cells, which comprises the following steps:

[0005] (1) adding the collected sample into a centrifugal separation tube, and separating the white blood cell layer in the sample into the centrifugal separation tube under centrifugal action;

[0006] (2) extracting the white blood cell layer in the above centrifugation tube into a test tube, and then removing the white blood cells to obtain a CTC cell suspension;

[0007] (3) Add the above CTC cell suspension to the cell sorting chip to sort out CTC single cells;

[0008] (4) Add culture medium to the above-mentioned cell sorting chip and culture each sorted CTC single cell.

[0009] Preferably, a separation structure is provided in the centrifugal separation tube, and the white blood cell layer in the sample is separated through the separation structure under the action of centrifugation.

[0010] Preferably, the separation structure includes a sample cavity and a separation channel connected to the sample cavity. After the sample and the isolation fluid are added to the sample cavity, the sample is stratified under centrifugal action, and the upper white blood cell layer after stratification is separated through the separation channel.

[0011] Preferably, the separation channel is a microfluidic channel, and under the action of centrifugation, the white blood cell layer automatically flows from the microfluidic channel into the accommodating cavity at the bottom of the centrifugal separation tube.

[0012] Preferably, after the white blood cell 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 attached to a magnetic rack to remove white blood cells.

[0013] Preferably, the cell sorting chip is provided with an injection hole and several sorting holes. The CTC cell suspension injected into the injection hole is sorted into CTC single cells through a sorting microchannel provided in the cell sorting chip, and each CTC single cell flows into the corresponding sorting hole.

[0014] Preferably, culture medium is added dropwise to each sorting well to culture the CTC single cells.

[0015] Preferably, the cell sorting chip is provided with an injection channel connected to each sorting hole. After the culture fluid is injected from the injection hole, the culture fluid flows into each sorting hole through the injection channel to culture the CTC single cell.

[0016] Preferably, each sorting hole is a stepped hole with a large diameter at the upper end and a small diameter at the lower end. The sorting microfluidic channel is arranged in the lower layer of the cell sorting chip, and the injection channel is arranged in the upper layer of the cell sorting chip. The CTC cell suspension injected into the injection hole is sorted out by the sorting microfluidic channel to obtain CTC single cells and then flows into the small holes of each stepped hole. The culture fluid injected into the injection hole flows into the large holes of each stepped hole through the injection channel to culture the CTC single cells in the corresponding small holes.

[0017] Preferably, when the CTC cell suspension is injected into the injection hole, the injection channel is blocked; when the culture fluid is injected into the injection hole, the sorting microchannel is blocked.

[0018] From the above technical solutions, it can be seen that the present invention separates and preserves the white blood cell layer in the sample through a centrifugal separation tube, and the preserved white blood cell layer can be directly extracted from the centrifugal separation tube. In addition, the CTC single cell sorting and culture process is directly completed in the cell sorting chip. Compared with the existing technology, it uses less equipment and has a shorter process, which not only improves work efficiency but also reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the separation structure of the present invention.

[0020] Figure 2 It is a partial structural schematic diagram of the cell sorting chip of the present invention.

[0021] Figure 3 It is a schematic diagram of the partial cross-sectional structure of the cell sorting chip of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below with reference to the accompanying drawings. The exemplary embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0023] See also Figure 1-3 The present invention provides a method for extracting, sorting and culturing single cells of CTCs, which comprises the following steps:

[0024] First, add the collected sample into the centrifuge tube 1. The sample can be collected from pleural effusion, peritoneal effusion, cerebrospinal fluid, lymph fluid, and blood of humans or animals. Then, the white blood cell layer in the sample is separated and stored in the centrifuge tube under centrifugal action. There is no need to transfer the white blood cell layer to other devices. Then, extract the white blood cell layer in the centrifuge 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 the 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 a magnetic rack is attached to remove the white blood cells, thereby obtaining a high-purity CTC cell suspension. The above-mentioned high-purity CTC cell suspension is added to the cell sorting chip 2 to sort out CTC single cells; finally, culture medium is added to the above-mentioned cell sorting chip, and each sorted CTC single cell is cultured. Therefore, the present invention utilizes centrifugal separation tubes and cell sorting chips in conjunction with related equipment to achieve CTC single-cell extraction, sorting, and culture. This process omits the steps and equipment for separate transfer and storage of the white blood cell layer, and does not use separate culture steps and equipment. This not only shortens the process flow and reduces the use of equipment; it also improves work efficiency and saves costs.

[0025] like Figure 1The centrifuge tube 1 of the present invention is provided with a separation structure 11, and under the action of centrifugation, the white blood cell layer in the sample is separated through the separation structure. The separation structure 11 includes a sample cavity 111 and a separation channel 112 connected to the sample cavity. During implementation, an isolation fluid is first added to the sample cavity, and then the sample is added to the upper layer of the isolation fluid, and then the centrifuge tube is placed 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 cavity are separated by the isolation fluid, wherein the supernatant in the upper layer is the white blood cell layer containing CTCs, and the lower layer is the red blood cell layer. Thus, under the action of centrifugation, the upper white blood cell layer is separated through the separation channel. Specifically, the separation channel is a microfluidic channel, and under the action of centrifugation and siphoning, the white blood cell layer automatically flows from the microfluidic channel into the holding cavity 12 at the bottom of the centrifuge tube, so that the white blood cell layer can be stored separately for standby use. It can be seen that the entire extraction process of the present invention is not only simple in centrifugation and has fewer steps, but also does not require the use of a pipette to extract the supernatant, resulting in simpler operation, shorter process and higher efficiency.

[0026] like Figure 2 The cell sorting chip 2 of the present invention is provided with an injection hole 21 and several sorting holes 22. The CTC cell suspension injected into the injection hole is sorted into CTC single cells through the sorting microchannel 23 provided in 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 approximately 30-50 microns, which is only for a single cell of about 30 microns to pass through. Under the injection pressure of the injection hole, the CTC single cell flows into the sorting hole through the above-mentioned sorting microchannel, thereby realizing CTC single cell sorting. After the CTC single cell is sorted out, as a preferred method, a culture medium can be added dropwise to each sorting hole to culture the CTC single cell. During implementation, the culture medium can be added dropwise manually one by one, or it can be added dropwise in groups by a machine.

[0027] The present invention also provides another preferred embodiment, wherein the cell sorting chip is provided with an injection channel 24 connected to each sorting hole. After the culture fluid is injected from the injection hole, the culture fluid flows into each sorting hole through the injection channel to culture the CTC single cell, thereby eliminating the need for manual or machine dripping of the culture fluid one by one, thereby improving work efficiency. Figure 3 Each sorting hole in the cell sorting chip is a stepped hole with a larger diameter at the top and a smaller diameter at the bottom. The sorting microfluidic channel is located in the lower layer of the cell sorting chip, and the injection channel is located in the upper layer of the cell sorting chip. Thus, the cell sorting chip of the present invention forms a dual-channel structure, enabling the sorting and culture of single CTCs, further improving work efficiency.

[0028] During implementation, the CTC cell suspension is first injected into the injection hole. After the CTC single cells are sorted out by the sorting microfluidic channel, the CTC cell suspension flows into the small hole 221 of each step hole. Then, the culture fluid is injected into the injection hole. The culture fluid flows into the large hole 222 of each step hole through the injection flow channel to culture the CTC single cells in the corresponding small hole. Preferably, when the CTC cell suspension is injected into the injection hole, the injection flow channel should be blocked to prevent the CTC cell suspension from flowing into the injection flow channel. Similarly, when the culture fluid is injected into the injection hole, the sorting microfluidic channel should be blocked to prevent the CTC cell suspension from flowing back. And after the culture fluid injection is completed, the injection hole should be filled with a plunger to simultaneously block the sorting microfluidic channel and the injection flow channel to prevent the CTC cell suspension and the culture fluid from flowing back.

Claims

1. A method for extracting, sorting and culturing single CTC cells, characterized in that: The following steps are involved: (1) adding the collected sample into a centrifugal separation tube, and separating the white blood cell layer in the sample into the centrifugal separation tube under centrifugal action; (2) extracting the white blood cell layer in the above centrifugation tube into a test tube, and then removing the white blood cells to obtain a CTC cell suspension; (3) Add the above CTC cell suspension to the cell sorting chip to sort out CTC single cells; (4) Adding culture medium to the cell sorting chip and culturing each sorted CTC single cell; The cell sorting chip is provided with an injection hole and several sorting holes. The CTC cell suspension injected into the injection hole is sorted into CTC single cells through a sorting microchannel arranged in the cell sorting chip, and each CTC single cell flows into the corresponding sorting hole; the cell sorting chip is provided with an injection channel connected to each sorting hole. After the culture fluid is injected from the injection hole, the culture fluid flows into each sorting hole through the injection channel to culture the CTC single cell; each sorting hole is a stepped hole with a large diameter at the upper end and a small diameter at the lower end. The sorting microchannel is arranged in the lower layer of the cell sorting chip, and the injection channel is arranged in the upper layer of the cell sorting chip; the CTC cell suspension injected into the injection hole is sorted into CTC single cells through the sorting microchannel and then flows into the small hole of each stepped hole, and the culture fluid injected into the injection hole flows into the large hole of each stepped hole through the injection channel to culture the CTC single cell in the corresponding small hole.

2. The method for extracting, sorting, and culturing single CTCs according to claim 1, wherein: The centrifugal separation tube is provided with a separation structure, and under the action of centrifugation, the white blood cell layer in the sample is separated through the separation structure.

3. The method for extracting, sorting, and culturing single CTCs according to claim 2, wherein: The separation structure includes a sample cavity and a separation channel connected to the sample cavity. After the sample and the isolation fluid are added to the sample cavity, the sample is separated into layers under centrifugal action, and the upper white blood cell layer after separation is separated through the separation channel.

4. The method for extracting, sorting, and culturing single CTCs according to claim 3, wherein: The separation channel is a microfluidic channel. Under the action of centrifugation, the white blood cell layer automatically flows from the microfluidic channel into the accommodating cavity at the bottom of the centrifugal separation tube.

5. The method for extracting, sorting, and culturing single CTCs according to claim 1, wherein: After the white blood cell 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 attached to a magnetic rack to remove white blood cells.

6. The method for single-cell CTC extraction, sorting, and culture according to claim 1, characterized in that: Culture medium is added to each sorting well to culture CTC single cells.

7. The method for single-cell CTC extraction, sorting, and culture according to claim 1, characterized in that: When the CTC cell suspension is injected into the injection hole, the injection channel is blocked; when the culture fluid is injected into the injection hole, the sorting microchannel is blocked.

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