Method for separating single TIL cell from liver cancer tissue

Abstract

The invention discloses a method for separating a single TIL cell from a liver cancer tissue, and the method comprises the following specific steps: S1, preparing a tumor tissue single-cell suspension, taking an in-vitro tumor tissue, dispersing the cell through a mechanical physical method to obtain the single-cell suspension, and counting the cell; s2, magnetic bead separation and enrichment: labeling the obtained single-cell suspension with magnetic beads to obtain a CD45 < + > lymphocyte suspension; and S3, single cell selection: adding a fluorescent antibody dye into the CD45 + lymphocyte suspension for dyeing, and picking cells by using a single cell picking instrument under a fluorescent condition to obtain single TIL cells. According to the method for separating the single TIL cells from the liver cancer tissue, the problems that when tumor infiltrating lymphocytes are separated at present, damage to the cells is large, and the single TIL cells in the required form cannot be quickly picked out are solved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a method for isolating single tumor-infiltrating lymphocytes (TILs) from liver cancer tissues. Background Art

[0002] With the development of the medical cause, most diseases have been conquered, and people's physical health has been guaranteed. However, cancer is still one of the important causes of human death.

[0003] Currently, the main methods for treating cancer include chemotherapy, targeted therapy, immunotherapy, etc. Tumor-infiltrating lymphocyte (TILs) therapy is a new emerging immunotherapy for solid tumors in recent years, which has strong specificity and targeting. This therapy requires isolating TILs from tumor tissues for amplification and culture. The interaction between different types of immune cells is complex. Therefore, single tumor-infiltrating lymphocytes are also needed for single-cell sequencing to analyze the heterogeneity and function of TILs to cope with different situations.

[0004] Currently, the main methods for isolating TILs include flow cytometry screening, laser assistance, microfluidics and other technologies, which are all expensive and complex, and have various limitations in use. Among them, flow cytometry screening is fast and sensitive, but it causes greater damage to cells during the isolation process, which has an impact on the subsequent growth of cells or single-cell sequencing; microfluidic chips consume less samples and reagents and have high throughput, but require a large number of living cells and have high requirements for the uniformity of cell size; limited dilution method and most microfluidic technologies follow the Poisson distribution, resulting in multiple cells at each position, which will reduce efficiency and cause data deviation. Therefore, there are still limitations in obtaining single tumor-infiltrating lymphocytes by methods such as flow cytometry and microfluidic chips. When single-cell sequencing of specific single TIL cells is required, the method of flow cytometry sorting cannot quickly select single tumor-infiltrating lymphocytes with the corresponding morphology under the premise of less damage to cells. Summary of the Invention

[0005] Aiming at the above defects, the purpose of the present invention is to propose a method for isolating single TIL cells from liver cancer tissues, so as to solve the problems of greater damage to cells and inability to quickly select single TIL cells with the required morphology during the current isolation of tumor-infiltrating lymphocytes.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] A method for isolating single TIL cells from liver cancer tissues, comprising the following steps:

[0008] S1. Preparation of single-cell suspension of tumor tissue: Take the excised tumor tissue, wash it with culture medium, soak it, cut it into pieces to obtain tumor tissue fragments; further disperse the cells by mechanical and physical methods, filter through a cell sieve to obtain a cell suspension; transfer the cell suspension to a centrifuge tube, centrifuge at a centrifugal force of 200 - 300g for 5 - 10 minutes, discard the supernatant, resuspend the cells with culture medium to obtain a single-cell suspension, and count the cells.

[0009] S2. Magnetic bead sorting and enrichment: After centrifuging the obtained single-cell suspension at 200 - 300g for 5 - 10 minutes, discard the supernatant; perform magnetic bead labeling, resuspend with 80 - 90 μl of MACS Buffer per 10 7 cells, add 20 - 30 μl of CD45+ magnetic beads per 10 7 cells, mix well, incubate at 2 - 8°C for 15 minutes to obtain a magnetically labeled sample solution; then positively select the CD45+ lymphocyte sample solution, centrifuge at 200 - 300g for 5 - 10 minutes, discard the supernatant, and resuspend with culture medium to obtain a CD45+ lymphocyte suspension.

[0010] S3. Single-cell selection: Add fluorescent antibody dye to the CD45+ lymphocyte suspension, mix well, and incubate for 15 - 30 minutes; centrifuge at 200 - 300g for 5 - 10 minutes, then wash with culture medium, and centrifuge at 200 - 300g for 5 - 10 minutes again. After resuspending with culture medium, obtain a stained single-cell suspension, and pick cells with a single-cell picker under fluorescence conditions to obtain single TIL cells.

[0011] Preferably, in step S3, the fluorescent antibody dye is a fluorescent antibody dye conjugated with fluorescein isothiocyanate; under fluorescence conditions, the required single TIL cells are green.

[0012] Preferably, in step S1, the mesh number of the cell sieve is 40 - 100 meshes.

[0013] Preferably, the culture medium is one of DMEM medium, MEM medium, and 1640 basal medium.

[0014] Preferably, in step S2, positive selection includes the following steps:

[0015] ① Install the MS column on the sorter, and wash the MS column with the buffer MACS buffer.

[0016] ② After the buffer MACS buffer has run out, add the magnetically labeled sample solution, and collect the sample solution that flows out first.

[0017] ③ After the sample solution has run out, wash the MS column with the buffer MACS buffer, and collect the sample solution.

[0018] ④ Remove the MS column, add MACS buffer, and quickly push and collect with the plunger matching the MS column to obtain the CD45+ lymphocyte sample solution.

[0019] Further, repeat steps ② - ④ again to improve the purity of the finally obtained CD45+ lymphocyte sample solution;

[0020] Among them, the CD45+ lymphocyte sample solution obtained in step ④ is the sample solution labeled with magnetic beads required for step ② repeated again.

[0021] Preferably, in step S1, the operation of further dispersing cells is to take the syringe plunger and grind the tumor tissue fragments clockwise or counterclockwise with the cross-section of the syringe plunger.

[0022] Further, in step S1, place the cell sieve in a petri dish of appropriate size, cover half of the volume of the cell sieve with the culture medium, transfer the tumor tissue fragments into it, then take the syringe plunger and grind the tumor tissue fragments clockwise or counterclockwise with the cross-section of the syringe plunger.

[0023] Preferably, in step S1, the operation of further dispersing cells is to place the cell sieve in a petri dish of appropriate size, transfer the tumor tissue fragments into it, gently rub the tumor tissue fragments with ophthalmic forceps, and add culture medium to rinse while rubbing until all the tumor tissue fragments are rubbed.

[0024] The technical solution provided by the present invention may include the following beneficial effects:

[0025] Using the mechanical physical method, the tumor tissue is prepared into a single-cell suspension, then CD45+ lymphocytes are enriched by magnetic bead sorting, then labeled and stained with fluorescent antibody dyes, and finally, under fluorescent conditions, it is possible to visually select cells, and a single-cell picker is used to pick TIL cells, with a relatively small negative pressure, thus causing less damage to the cells, and it can be completed within half an hour, and the required time is short. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flowchart of an embodiment of the present invention.

[0027] Figure 2 is a bright-field image of the stained single-cell suspension of an embodiment of the present invention.

[0028] Figure 3 is Figure 2 the image under fluorescent conditions.

[0029] Figure 4 is Figure 2 based on, the bright-field image after picking a single TIL cell.

[0030] Figure 5 is Figure 4 The figure under fluorescence conditions.

[0031] Figure 6 It is the bright-field image of the stained single-cell suspension of another embodiment of the present invention.

[0032] Figure 7 is Figure 6 The figure under fluorescence conditions.

[0033] Figure 8 is based on Figure 6 On this basis, the bright-field image after picking single TIL cells.

[0034] Figure 9 is Figure 8 The figure under fluorescence conditions. Detailed implementation manners

[0035] The technical solution of the present invention will be further described below through specific implementation manners.

[0036] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0038] A method for isolating single TIL cells from liver cancer tissue, comprising the following steps:

[0039] S1. Preparation of tumor tissue single-cell suspension: Take the excised tumor tissue, wash it with culture medium and then soak it, cut it into pieces to obtain tumor tissue fragments; further disperse the cells by mechanical and physical methods, filter through a cell sieve to obtain a cell suspension; transfer the cell suspension to a centrifuge tube, centrifuge it at a centrifugal force of 200-300g for 5-10 minutes, discard the supernatant, and resuspend the cells with culture medium to obtain a single-cell suspension, and count the cells;

[0040] S2. Magnetic bead sorting and enrichment: After centrifuging the obtained single-cell suspension at 200-300g for 5-10 minutes, discard the supernatant; perform magnetic bead labeling, resuspend it with 80-90 μl of MACS Buffer per 10 7 cells, and add 20-30 μl of CD45+ magnetic beads per 10 7Cells were mixed, incubated at 2-8°C for 15 min to obtain a magnetically labeled sample solution; then, CD45+ lymphocyte sample solution was positively sorted, centrifuged at 200-300 g for 5-10 min, the supernatant was discarded, and the cells were resuspended in culture medium to obtain a CD45+ lymphocyte suspension;

[0041] S3. Single-cell selection: Fluorescent antibody dye was added to the CD45+ lymphocyte suspension, mixed well, and incubated for 15-30 minutes; centrifuged at 200-300 g for 5-10 min, then washed with culture medium, and centrifuged again at 200-300 g for 5-10 min. After resuspending in culture medium, a stained single-cell suspension was obtained. Cells were picked using a single-cell picker under fluorescence conditions to obtain single TIL cells.

[0042] To solve the problems existing in the prior art, the technical solution proposed by the present invention provides a method for isolating single TIL cells from liver cancer tissue. Among them, as Figure 1 shown, after preparing a single-cell suspension of tumor tissue, magnetic bead sorting and enrichment were performed. CD45+ magnetic beads can bind to multiple lymphocyte subsets, and a relatively large amount of sorted cells can be obtained. TIL cells are tumor-infiltrating lymphocytes. The CD45+ lymphocyte suspension obtained through the above steps contains the required TIL cells. The obtained CD45+ lymphocyte suspension was subjected to single-cell selection to finally obtain single TIL cells. In the prior art, flow cytometry sorting is usually used. The TIL cells sorted by flow cytometry are a cell population, that is, multiple TIL cells. During subsequent sequencing, the sequencing results will be affected by the heterogeneity between cells. However, the technical solution proposed by the present invention finally isolates single TIL cells, which can effectively avoid the influence of cell heterogeneity on subsequent sequencing results. At the same time, the pressure during flow cytometry sorting is relatively large, about 60.0 PSI, and the cells are damaged under high pressure, and the internal RNA may be degraded. During subsequent sequencing, the incomplete RNA affects the quality of sequencing data. The technical solution proposed by the present invention is to pick cells using a single-cell picker, that is, cells can be picked through capillary action or picked through a relatively low negative pressure, about 5.0 PSI, which is much smaller than the pressure generated by flow cytometry sorting and has less impact on cells.

[0043] Furthermore, using a single-cell picker can directly and rapidly collect cells by aspiration within half an hour, quickly obtaining single TIL cells. In contrast, other separation methods, such as flow cytometry sorting in the prior art, require a certain amount of time for separation (more than half an hour), and need high pressure to push the sheath fluid containing TIL cells to flow, and then pass through a high-voltage electric field to separate them by fluorescence and wavelength differences. Finally, a population of TIL cells is obtained, without the effect of rapid short-time collection of the technical solution of the present invention. Moreover, the cells obtained by separation in the prior art are cell populations rather than single cells. Therefore, the technical solution proposed by the present invention can solve the problems of greater damage to cells and inability to quickly pick out the required single TIL cells when separating tumor-infiltrating lymphocytes currently.

[0044] In addition, in step S1, selecting the mechanical physical method to obtain a single-cell suspension does not require complex equipment and reagents, has a lower cost, causes less damage to cells, reduces the impact on cell viability, makes the biomolecules in the cells more stable, is beneficial to subsequent single-cell sequencing, and provides more accurate sequencing data. The specific mechanical physical method can be grinding, rubbing with a mesh, and cutting into pieces.

[0045] It is worth noting that TIL cells are relatively large in volume. Selecting a relatively low centrifugal force of 200 - 300 g and centrifuging for 5 - 10 min can effectively precipitate the cells, while reducing damage to the cells and removing unnecessary impurities, such as cell debris, unbound CD45+ magnetic beads, unbound fluorescent antibody dyes, and separating CD45+ magnetic beads and cells after sorting, reducing interference with cell picking under fluorescence conditions later. If the centrifugal force is too large and the centrifugation time is too long, it will exert a greater physical pressure on the cells, damage the cells, and cause the cells to aggregate together, affecting the uniform distribution of the cells and increasing the operation difficulty of subsequent cell resuspension.

[0046] At the same time, resuspend with 80 - 90 μl of buffer MACS Buffer per 10 7 cells, add 20 - 30 μl of CD45+ magnetic beads per 10 7 cells, mix well, and incubate at 2 - 8 °C for 15 min. An appropriate amount of buffer MACS Buffer can ensure uniform dispersion of the cells, avoid cell aggregation, thereby increasing the contact probability between subsequent CD45+ magnetic beads and cell surface antigens and enhancing the labeling effect. And 20 - 30 μl of CD45+ magnetic beads per 10 7 cells, incubating at 2 - 8 °C for 15 minutes can ensure sufficient binding of the magnetic beads to the antigens on the cell surface and reduce the occurrence of non-specific binding. In step S3, incubating for 15 - 30 minutes ensures successful staining with the fluorescent antibody dye. If the incubation time is too long, non-specific binding increases, and there will be many interference items when picking cells later, reducing the accuracy of cell picking.

[0047] Preferably, in step S3, the fluorescent antibody dye is a fluorescent antibody dye conjugated with fluorescein isothiocyanate; under fluorescence conditions, the required single TIL cell appears green.

[0048] Specifically, fluorescein isothiocyanate, namely FITC, has a high absorption rate and can effectively absorb the excitation light, thereby generating a stronger fluorescence signal. Under fluorescence conditions, the visualization effect is good. Even when the expression level of CD3 in the obtained CD45+ lymphocyte suspension is low, the presence of CD3 can be accurately detected, thereby identifying the required TIL cells.

[0049] Moreover, the TIL cells labeled with FITC emit bright green fluorescence, making the signals of single cells easier to be detected and distinguished, improving the detection sensitivity, and being more easily distinguishable under fluorescence conditions, thus realizing the picking of single TIL cells.

[0050] Preferably, the fluorescent antibody dye conjugated with fluorescein isothiocyanate is CD3 Antibody, anti - human, FITC, REAfinity produced by Miltenyi Biotec TM dye. This fluorescent antibody dye has high specificity and can specifically recognize and bind to the human CD3 antigen. Due to the characteristics of its recombinant monoclonal antibody, it has a high affinity, can effectively bind to the target antigen, with less non - specific binding, and improves the accuracy of experimental results.

[0051] Preferably, in step S1, the mesh number of the cell sieve is 40 - 100 mesh.

[0052] Specifically, the mesh number of the cell sieve is 40 - 100 mesh, that is, the pore size of the sieve holes is 150 - 425 μm. This range can filter out most cell clusters, cell debris, etc., reduce the influence of other cell clusters and cell debris on subsequent operations, and to a certain extent increase the acquisition amount of TIL cells.

[0053] Preferably, the culture medium is one of DMEM medium, MEM medium, and 1640 basal medium.

[0054] Specifically, the culture medium is one of DMEM medium, MEM medium, and 1640 basal medium. The above three culture media have a high concentration of amino acids and vitamins, which is beneficial to the in vitro culture of cells. Selecting these culture media can wash the surface of the tumor tissue while maintaining a stable pH value, meet the basic metabolic needs of cells, maintain the activity and function of cells, and can reduce the physical damage to cells during centrifugation, protect the integrity and function of cells, and provide an environment for subsequent operations such as centrifugation.

[0055] Preferably, in step S2, positive sorting is performed, including the following steps:

[0056] ① Install the MS column on the sorter and rinse the MS column with the buffer MACS buffer.

[0057] ② After the buffer MACS buffer has run out, add the sample solution labeled with magnetic beads and collect the sample solution that flows out first.

[0058] ③ After the sample solution has run out, wash the MS column with the buffer MACS buffer and collect the sample solution.

[0059] ④ Remove the MS column, add MACS buffer, and quickly push and collect with a plunger matching the MS column to obtain a CD45+ lymphocyte sample solution.

[0060] It should be noted that positive sorting can directly obtain a target cell population with high purity, and the operation is simple, the process is relatively direct, only focusing on the separation of the target cell population, which is fast and efficient.

[0061] Specifically, MACS (Magnetic Activated Cell Sorting) is a magnetic activated cell sorting technology. The MS column is a sorting column used in MACS sorting technology. Compared with other types of sorting columns, the MS column is suitable for positive sorting and can effectively capture target cells, that is, TIL cells after magnetic bead labeling, without being restricted by cell type or sample volume, and only requires fewer magnetic beads, with sufficient antigen epitopes remaining on the cell surface, facilitating subsequent staining, and then picking single TIL cells using a single cell picker under fluorescence conditions.

[0062] Among them, the MACS buffer used is Running Separation Buffer produced by Miltenyi Biotec. This MACS buffer contains bovine serum albumin (BSA), EDTA, and 0.09% sodium azide, which helps to maintain the stability and activity of cells. At the same time, sodium azide has an antiseptic effect, extending the shelf life of the buffer, and this buffer is sterile filtered and can be used directly, reducing errors caused by contamination in the experiment.

[0063] Furthermore, steps ② - ④ are performed again to improve the purity of the finally obtained CD45+ lymphocyte sample solution; among them, the CD45+ lymphocyte sample solution obtained in step ④ is the sample solution labeled with magnetic beads required for step ② to be repeated again.

[0064] Specifically, repeating step ② - step ④ can further improve the purity of the obtained CD45+ lymphocytes, more thoroughly remove unlabeled cells and impurities, and after staining, under fluorescence conditions, it will not cause obvious background interference, and can quickly find the required cells and pick them up.

[0065] Preferably, in step S1, the operation of further dispersing cells is to take the syringe plunger and grind the tumor tissue fragments clockwise or counterclockwise with the cross-section of the syringe plunger.

[0066] In the prior art, enzyme digestion methods are mostly used to disperse cells. However, during the enzyme digestion process, the enzyme may damage the antigens and receptors on the cell surface, affect the activity and function of the cells, may affect the staining of subsequent fluorescent antibody dyes, and after enzyme digestion, the residual enzyme may continue to act on the cells, affecting the subsequent single-cell sequencing results. By the above method, the great influence of enzyme digestion on cells can be avoided, and the tissue can be dispersed into single cells in a relatively short time, reducing the exposure time and damage risk of cells during the processing, and helping to maintain the physiological state and activity of the cells.

[0067] It should be noted that using the same direction for grinding can ensure the uniformity and consistency of grinding, avoid insufficient grinding or damage to TIL cells caused by changes in the grinding direction, until all the tumor tissue fragments are dispersed as much as possible, and a cell suspension is filtered through a cell sieve.

[0068] Preferably, cells are further dispersed by mechanical physical methods and filtered through a cell sieve. The above steps can be repeated 2 - 3 times, that is, after filtering through the cell sieve, culture medium can be added to the remaining tumor tissue fragments for re-grinding so that more cells can be released.

[0069] Furthermore, in step S1, the cell sieve is placed in a petri dish of an appropriate size, the cell sieve is covered with culture medium at half its volume, the tumor tissue fragments are transferred into it, then the syringe plunger is taken, and the tumor tissue fragments are ground clockwise or counterclockwise with the cross-section of the syringe plunger.

[0070] Beneficial effects: Utilizing the combined action of the petri dish and the cell sieve, the petri dish provides a broad and flat plane, enabling the cell sieve to be stably placed on it. This stable plane helps to maintain the balance of the cell sieve during grinding, avoiding uneven grinding or cell damage caused by shaking or tilting. Moreover, performing the grinding operation in the petri dish allows for convenient observation and control of the grinding process, timely adjustment of the grinding force and direction, and ensuring that the tumor tissue fragments are fully ground into a single-cell suspension.

[0071] Preferably, in step S1, the operation of further dispersing the cells is to place the cell sieve in a petri dish of an appropriate size, transfer the tumor tissue fragments thereto, gently rub the tumor tissue fragments with ophthalmic forceps, and add culture medium to rinse while rubbing until all the tumor tissue fragments are rubbed out.

[0072] Among them, while gently rubbing the tumor tissue fragments with ophthalmic forceps, adding culture medium to rinse while rubbing helps the release and collection of cells.

[0073] The technical solution of the present invention will be further described below through specific embodiments.

[0074] Example group

[0075] Example 1

[0076] S1. Preparation of single-cell suspension of tumor tissue: After rinsing about 0.4 cm 3 colorectal cancer tumor tissue with 1640 basal medium, place it in a 100 cm diameter petri dish, add 2 ml of 1640 basal medium near the tumor tissue to infiltrate the tumor tissue, and cut it into tumor tissue fragments with a volume less than 0.1 cm 3 using ophthalmic scissors. Place a 40-mesh cell sieve in another petri dish, add 3 ml of 1640 basal medium to cover half of the volume of the cell sieve and transfer the tumor tissue fragments thereto; take the core of a 2 ml syringe and grind it clockwise in the cell sieve to make the tumor tissue fragments into a cell suspension. Transfer the cell suspension to a 15 ml centrifuge tube, add an appropriate amount of 1640 basal medium to infiltrate the remaining tumor tissue fragments, continue grinding and then transfer the cell suspension to the same centrifuge tube, centrifuge at 300 g for 10 min, discard the supernatant, and resuspend the cells with 1640 basal medium to obtain a single-cell suspension, and perform cell counting.

[0077] S2. Magnetic bead sorting and enrichment: After centrifuging the obtained single-cell suspension at 300 g for 10 min, discard the supernatant; resuspend the cells with MACSbuffer, add 80 μl of MACS buffer to every 10 7 cells, add 20 μl of CD45+ magnetic beads to every 10 7 cells, mix well and place in a 4 °C refrigerator for incubation for 15 min to obtain a sample solution labeled with magnetic beads;

[0078] Install the MS column on the sorter, rinse the sorting column with the buffer MACS buffer to avoid introducing air bubbles;

[0079] After the buffer MACS buffer has drained, add the sample solution labeled with magnetic beads, and collect the unlabeled sample solution that flows out first;

[0080] After the unlabeled sample liquid has flowed out, add the buffer MACS buffer to wash the sorting column, collect the unlabeled sample liquid, and repeat 3 times;

[0081] Remove the sorting column, add 1 μl of MACS buffer, quickly push and collect with the washing plug supporting the sorting column to obtain the CD45+ lymphocyte sample liquid. Centrifuge at 300 g for 10 min, discard the supernatant, and resuspend with 100 μl of 1640 basal medium to obtain the CD45+ lymphocyte suspension;

[0082] S3. Single cell selection: Add an appropriate amount of fluorescent antibody dye to the CD45+ lymphocyte suspension, mix well and incubate for 30 min. The fluorescent antibody dye is anti-human, FITC, REAfinity TM dye (Miltenyi Biotec), and the incubation temperature is 37°C; Centrifuge at 300 g for 5 min, add 1 ml of 1640 basal medium to wash, then centrifuge at 300 g for 5 min, and resuspend with 1640 basal medium to obtain the stained single cell suspension. Add the stained single cell suspension to a 96-well plate, and pick the green fluorescent cells with a single cell picker under fluorescence conditions.

[0083] Example 2

[0084] S1. Preparation of single cell suspension of tumor tissue: Rinse about 0.4 cm 3 colorectal cancer tumor tissue with 1640 basal medium, place it in a 100 cm diameter petri dish, add 2 ml of 1640 basal medium near the tumor tissue to infiltrate the tumor tissue, and cut it into tumor tissue fragments with a volume less than 0.1 cm 3 using ophthalmic scissors. Place a 100-mesh cell sieve in another petri dish, transfer the tumor tissue fragments to the cell sieve, gently rub the tumor tissue fragments with ophthalmic forceps, and rinse with 3 ml of 1640 basal medium while rubbing to make a cell suspension. Transfer the cell suspension to a 15 ml centrifuge tube and continue rubbing. During this period, add an appropriate amount of 1640 basal medium. After rubbing the remaining tumor tissue fragments, transfer the cell suspension to the same centrifuge tube, centrifuge at 300 g for 10 min, discard the supernatant, resuspend the cells with 1640 basal medium to obtain a single cell suspension, and count the cells.

[0085] S2. Magnetic bead sorting and enrichment: After centrifuging the obtained single cell suspension at 300 g for 10 min, discard the supernatant; Resuspend the cells with MACS buffer, add 80 μl of MACS buffer to every 10 7 cells, add 20 μl of CD45+ magnetic beads to every 10 7 cells, mix well and place in a 4°C refrigerator for incubation for 15 min to obtain the sample liquid labeled with magnetic beads;

[0086] Install the MS column on the sorter, rinse the sorting column with the buffer MACS buffer to avoid introducing air bubbles;

[0087] After the buffer MACS buffer has run out, add the sample solution labeled with magnetic beads, and collect the unlabeled sample solution that flows out first;

[0088] After the unlabeled sample solution has run out, wash the sorting column with the buffer MACS buffer, collect the unlabeled sample solution, and repeat 3 times;

[0089] Remove the sorting column, add 1 μl of MACS buffer, quickly push and collect with the washing plug supporting the sorting column to obtain the CD45+ lymphocyte sample solution, centrifuge at 300 g for 10 min, discard the supernatant, and resuspend with 100 μl of 1640 basal medium to obtain the CD45+ lymphocyte suspension;

[0090] S3. Single cell selection: Add an appropriate amount of fluorescent antibody dye to the CD45+ lymphocyte suspension, mix well and incubate for 30 min. The fluorescent antibody dye is anti-human, FITC, REAfinity TM dye (Miltenyi Biotec), and the incubation temperature is 37°C; centrifuge at 300 g for 5 min, add 1 ml of 1640 basal medium for washing, then centrifuge at 300 g for 5 min, and resuspend with 1640 basal medium to obtain the stained single cell suspension. Add the stained single cell suspension to a 96-well plate, and pick the green fluorescent cells with a single cell picker under fluorescent conditions.

[0091] Take pictures to record the bright and dark field pictures before and after picking single TIL cells in Example 1 and Example 2. The picked cells are placed in a 200 μl EP tube and can be used for subsequent single cell sequencing.

[0092] Figure 2 And Figure 3 shows the same area. As Figure 2 shown, under bright field, multiple cells can be seen, and after the separation in step S1, the cells are relatively dispersed. As Figure 3 shown, under dark field, that is, under fluorescent conditions, only the cells within the red circle emit green fluorescence, that is, the cell with the required phenotype of CD45+CD3+ TIL cells. After being picked by the single cell picker, as Figure 4 shown in the bright field and as Figure 5 shown in the dark field, there are no cells in the area delimited by the red circle, indicating that after staining, the single cell picker can quickly pick out the required single TIL cells to obtain the required single TIL cells, avoiding the influence of cell heterogeneity on subsequent sequencing.

[0093] while Figure 6 and Figure 7 are respectively the pictures of the same area where cells were not picked under bright field and dark field. Figure 8 and Figure 9 respectively correspond to Figure 6 and Figure 7 The pictures after picking the required cells. It can be seen that separating cells by mechanical physical method is beneficial to subsequent single cell picking. The cells are relatively dispersed and their morphology is relatively complete.

[0094] In summary, as can be seen from Figures 2 to 9 the technical solution proposed by the present invention is feasible, and can pick out the required single TIL cells. At the same time, under fluorescence conditions, the morphology of the picked single TIL cells can be seen, which is beneficial to selecting the single TIL cells meeting the requirements.

[0095] The technical principle of the present invention has been described above in combination with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted in any way as a limitation on the protection scope of the present invention. Based on the explanations herein, those skilled in the art can think of other specific embodiments of the present invention without creative labor, and these embodiments will fall within the protection scope of the present invention.

Claims

1. A method for isolating a single TIL cell from liver cancer tissue, characterized in that: The following steps are involved: S1. Preparation of single cell suspension of tumor tissue: Take the ex vivo tumor tissue, wash it with culture medium, infiltrate it, cut it into pieces, and obtain tumor tissue fragments; further disperse the cells by mechanical physical method, filter it with cell sieve, and obtain cell suspension; transfer the cell suspension to a centrifuge tube, centrifuge it at a centrifugal force of 200-300g for 5-10min, discard the supernatant, resuspend the cells in culture medium to obtain single cell suspension, and count the cells; S2. Magnetic bead sorting and enrichment: Centrifuge the obtained single cell suspension at 200-300g for 5-10min, discard the supernatant; label with magnetic beads, and use MACS Buffer 80-90μl / 10 7 Resuspend the cells and add 20-30 μl of CD45+ magnetic beads per 10 7 cells, mixed, and incubated at 2-8°C for 15 minutes to obtain a sample solution labeled with magnetic beads; then positively sorted CD45+ lymphocyte sample solution, centrifuged at 200-300g for 5-10 minutes, discarded the supernatant, and resuspended in culture medium to obtain a CD45+ lymphocyte suspension; S3, single cell selection: add fluorescent antibody dye to the CD45+ lymphocyte suspension, mix well, and incubate for 15-30 minutes; The cells were centrifuged at 200-300 g for 5-10 min, washed with culture medium, and centrifuged again at 200-300 g for 5-10 min. The cells were resuspended in culture medium to obtain a stained single cell suspension. Cells were picked with a single cell picker under fluorescent conditions to obtain single TIL cells.

2. The method for isolating a single TIL cell from liver cancer tissue according to claim 1, characterized in that: In step S3, the fluorescent antibody dye is a fluorescent antibody dye combined with fluorescein isothiocyanate; Under fluorescent conditions, the desired single TIL cells appear green.

3. The method for isolating a single TIL cell from liver cancer tissue according to claim 1, characterized in that: In step S1, the mesh size of the cell sieve is 40-100 meshes.

4. The method for isolating a single TIL cell from liver cancer tissue according to claim 1, characterized in that: The culture fluid is one of DMEM culture medium, MEM culture medium and 1640 basal culture medium.

5. The method for isolating a single TIL cell from liver cancer tissue according to claim 1, characterized in that: In step S2, positive sorting is performed, including the following steps: ① Install the MS column on the separator and add MACS buffer to rinse the MS column; ② After the MACS buffer flows out, add the sample solution labeled with magnetic beads and collect the sample solution that flows out first; ③ After the sample liquid flows out, add MACS buffer to clean the MS column and collect the sample liquid; ④ Remove the MS column, add MACS buffer, and quickly push and collect with the wash plug that comes with the MS column to obtain the CD45+ lymphocyte sample solution.

6. The method for isolating a single TIL cell from liver cancer tissue according to claim 5, characterized in that: Performing steps ② to ④ again is used to improve the purity of the final CD45+ lymphocyte sample solution; The CD45+ lymphocyte sample solution obtained in step ④ is the magnetic bead-labeled sample solution required for the repeated step ②.

7. The method for isolating a single TIL cell from liver cancer tissue according to claim 1, characterized in that: In step S1, the operation of further dispersing the cells is to take the syringe needle core and grind the tumor tissue fragments clockwise or counterclockwise with the cross section of the syringe needle core.

8. The method for isolating a single TIL cell from liver cancer tissue according to claim 7, characterized in that: In step S1, the cell sieve is placed in a dish of corresponding size, half of the volume of the cell sieve is covered with culture medium, the tumor tissue fragments are transferred thereto, and then the syringe needle core is taken and the cross section of the syringe needle core is used to grind the tumor tissue fragments clockwise or counterclockwise.

9. The method for isolating a single TIL cell from liver cancer tissue according to claim 1, characterized in that: In step S1, the operation of further dispersing the cells is to place the cell sieve into a dish of corresponding size, transfer the tumor tissue fragments into it, and gently rub the tumor tissue fragments with ophthalmic forceps while adding culture medium to rinse until all the tumor tissue fragments are rubbed.

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