Method for extracting tumor infiltrating lymphocytes from liver tumor tissue cut off through operation

Abstract

The invention discloses a method for extracting tumor infiltrating lymphocytes from liver tumor tissue cut by an operation. The method comprises the following steps of: cleaning liver tumor tissues cut off by an operation, cutting into small blocks, oscillating and digesting for 45-60 minutes at 37 DEG C by using type IV collagenase, resuspending cells by using a lymphocyte culture medium, inoculating into a culture bottle, culturing for 24-48 hours in a 5% CO2 incubator at 37 DEG C, carrying out secondary digestion for 45-60 minutes at 37 DEG C by using a mixture of hyaluronidase and heparanase, and taking out by using an incubator at 37 DEG C; the obtained cells are subjected to directional culture according to uses, and obvious cell colonies can be seen in 3-7 days, namely, the cells are successfully separated and amplified. By adopting the secondary digestion of the combination of hyaluronidase and heparanase, the main component glycosaminoglycan of an extracellular matrix can be effectively decomposed, the extracellular matrix of tumor tissues can be effectively destroyed, the release of tumor infiltrating lymphocytes can be promoted, the separation efficiency of cells can be improved, and more living cells can be obtained.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, in particular to a method for extracting tumor-infiltrating lymphocytes from surgically removed liver tumor tissue, providing an efficient and reliable cell extraction method for immune research on liver tumors. Background Art

[0002] Liver tumors, especially hepatocellular carcinoma (HCC), are one of the most common malignant tumors with a high mortality rate worldwide. According to statistics, hundreds of thousands of deaths are caused by HCC each year, which has brought a huge burden to the public health and medical systems.

[0003] In the development of liver tumors, the tumor microenvironment plays a vital role, among which infiltrating lymphocytes (such as T cells, B cells and natural killer cells) play a key role in regulating tumor immune surveillance and promoting or inhibiting tumor progression. These lymphocytes affect tumor growth and metastasis by releasing cytokines, directly killing tumor cells, and interacting with tumor cells. Therefore, in-depth research on the characteristics and functions of lymphocytes in the liver tumor microenvironment is of great significance for understanding the immune escape mechanism of hepatocellular carcinoma and developing new immunotherapy strategies.

[0004] Due to the complexity of the tumor microenvironment, the successful separation of tumor-infiltrating lymphocytes has always been a difficult problem. Traditional cell extraction methods usually use DNA enzymes in combination with multiple other enzymes to dissociate tissues for a long time. Although this method can decompose the extracellular matrix, excessive enzymatic hydrolysis may cause cell membrane rupture or cell structure damage, affecting cell survival and function. In addition, this operation has many steps, is difficult to operate, and has high requirements for personnel skills. Therefore, the development of an efficient and reliable extraction method can not only improve the separation efficiency of tumor-infiltrating lymphocytes, but also provide important cell models for related basic research, clinical diagnosis and treatment, which has significant clinical and scientific research value. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for extracting tumor infiltrating lymphocytes with simpler operation, shorter reaction time and less cell damage, thereby increasing the probability of successful separation and amplification of tumor infiltrating lymphocytes.

[0006] The present invention is implemented as follows: a method for extracting tumor-infiltrating lymphocytes from surgically removed liver tumor tissue, wherein the surgically removed liver tumor tissue is washed and cut into small pieces, and then digested once with a type IV collagenase solution at 37°C for 45-60 minutes by shaking, and the cells are resuspended with a lymphocyte culture medium and then inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator for 24-48 hours, and then digested again with a mixed solution of hyaluronidase and heparanase at 37°C for 45-60 minutes, and the obtained cells are subjected to directional culture, and obvious cell colonies can be seen in 3-7 days, that is, the cells are successfully separated and amplified.

[0007] In the mixed solution of hyaluronidase and heparanase, the mass volume ratio concentration of hyaluronidase is 0.075%-0.15%, and the mass volume ratio concentration of heparanase is 0.01%-0.05%.

[0008] The primary digestion was performed using type IV collagenase solution in a 37°C air bath constant temperature oscillator at a low speed of 100-200 rpm for 45-60 minutes.

[0009] The secondary digestion was performed by oscillating the mixture of hyaluronidase and heparanase at 37°C in an air bath constant temperature oscillator at a low speed of 100-200 rpm for 45-60 minutes.

[0010] After the secondary digestion, the digested mixed solution is filtered using a sieve with a pore size of 70-100 um.

[0011] Preferably, the method comprises the following steps:

[0012] (1) Sample preparation: After the liver tumor tissue was removed during surgery, it was cleaned and cut into several small pieces of 2-5 mm3 using sterile scissors;

[0013] (2) Primary digestion with collagenase: 15 to 30 cut tumor tissue blocks were placed in 2 to 5 mL of type IV collagenase solution and mixed thoroughly; the solution was oscillated at a low speed of 100 to 200 rpm for 45 to 60 minutes in a 37°C air bath constant temperature oscillator; the mass volume ratio of the type IV collagenase solution was 0.075% to 0.15%;

[0014] (3) Cell collection and culture: After digestion, resuspend the cells in 10-20 mL of 1640 medium, balance, and centrifuge at 300 g for 5-8 minutes; discard the supernatant after centrifugation, resuspend the cells in complete lymphocyte medium, inoculate 15-30 mL of each aliquot into a culture flask, and culture in a 37°C, 5% CO2 incubator for 24-48 hours;

[0015] (4) Secondary digestion with hyaluronidase and heparanase: The cell suspensions were recovered and centrifuged at 300 g for 5-8 minutes, the supernatant was discarded, the precipitate was flicked, 2-5 mL of hyaluronidase and heparanase mixture was added to each portion, mixed thoroughly, and oscillated at a low speed of 100-200 rpm for 45-60 minutes in a 37°C air bath constant temperature oscillator;

[0016] (5) Cell collection and re-culture: After digestion, resuspend the cells in 10-20 mL of 1640 medium for each portion. After sufficient pipetting and mixing, filter the digested mixture through a 70-100 um cell sieve, collect the filtrate, and centrifuge at 300g for 5-8 minutes. After centrifugation, discard the supernatant, resuspend the cells in complete lymphocyte medium, inoculate them in a culture flask, and culture them in a directed manner. Cell colonies can be seen in 3-7 days.

[0017] The cells were resuspended in complete lymphocyte medium, wherein the medium was X-VIVO 15 medium containing 5%-15% fetal bovine serum and 500-6000 IU / mL IL-2 in a volume ratio.

[0018] The advantages and technical effects of the present invention are as follows: the type IV collagenase of the present invention can dissolve collagen in tumor tissue, destroy its reticular framework, and release the extracellular matrix and cells in the tumor tissue together. After 24-48 hours of culture, the effect of a single digestion on the cells is reduced, and the cell state is restored. Since the extracellular matrix is ​​viscous and often wraps around the cells, making it impossible for the cells to receive stimulation signals for normal amplification, secondary digestion using hyaluronidase and heparanase can effectively decompose glycosaminoglycans, the main component of the extracellular matrix, thereby effectively destroying the extracellular matrix of tumor tissue, promoting the release of tumor-infiltrating lymphocytes, improving the separation efficiency of cells, and obtaining more living cells. This method can provide an important cell model for studying the immune microenvironment of liver cancer and provide a basis for the technical development and optimization of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The liver tumor tissue removed by surgery and stored in a collection tube according to Example 1 of the present invention;

[0020] Figure 2 This is a microscopic observation picture of the inoculation culture 24 hours after the first digestion of Example 1 of the present invention (4 times magnification);

[0021] Figure 3 This is a microscopic observation picture of the inoculation culture 24 hours after the first digestion of Example 1 of the present invention (10 times magnification);

[0022] Figure 4 This is a microscopic observation picture of cells cultured on day 6 after the second digestion in Example 1 of the present invention (4x magnification);

[0023] Figure 5 This is a microscopic observation picture of cells cultured on day 6 after the second digestion in Example 1 of the present invention (10 times magnification);

[0024] Figure 6 This is a cell proliferation curve diagram of Example 1 of the present invention;

[0025] Figure 7 This is a flow cytometry result diagram of cells cultured on day 21 in Example 1 of the present invention;

[0026] Figure 8 The liver tumor tissue removed by surgery and stored in a collection tube according to Example 2 of the present invention;

[0027] Fig. 9 This is a microscopic observation picture of the inoculation culture 24 hours after the first digestion of Example 2 of the present invention (4 times magnification);

[0028] Fig.10 This is a microscopic observation picture of the inoculation culture 24 hours after the first digestion of Example 2 of the present invention (10 times magnification);

[0029] Fig.11 This is a microscopic observation picture of cells cultured on day 6 after the second digestion in Example 2 of the present invention (4x magnification);

[0030] Fig.12 This is a microscopic observation picture of cells cultured on day 6 after the second digestion in Example 2 of the present invention (10x magnification);

[0031] Fig.13 This is a cell proliferation curve diagram of Example 2 of the present invention;

[0032] Fig.14 This is a flow cytometry result diagram of cells cultured on day D23 in Example 2 of the present invention;

[0033] Fig.15 The liver tumor tissue removed by surgery and stored in the collection tube in the comparative example of the present invention;

[0034] Fig.16 This is a microscopic observation picture after cell inoculation in the comparative example of the present invention (4 times magnification);

[0035] Fig.17 This is a microscopic observation picture after cell inoculation in the comparative example of the present invention (10 times magnification);

[0036] Fig.18 This is a microscopic observation picture of cells inoculated and cultured on day 7 in a comparative example of the present invention (4x magnification);

[0037] Fig.19 This is a microscopic observation picture of cells inoculated and cultured on day 7 in a comparative example of the present invention (10 times magnification);

[0038] Fig. 20 is a cell proliferation curve diagram in a comparative example of the present invention;

[0039] Fig.21 It is a flow cytometry result diagram of cells cultured on day D23 in a comparative example of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] The method for extracting tumor-infiltrating lymphocytes from surgically removed liver tumor tissue of the present invention comprises the following steps: washing the surgically removed liver tumor tissue and cutting it into small pieces, digesting it once with a type IV collagenase solution at 37°C for 45-60 minutes by shaking, resuspending the cells with a lymphocyte culture medium and then inoculating them in a culture bottle, culturing them in a 37°C, 5% CO2 incubator for 24-48 hours, and then digesting them again with a mixed solution of hyaluronidase and heparanase at 37°C for 45-60 minutes. The obtained cells are subjected to directional culture, and obvious cell colonies can be seen in 3-7 days, that is, the cells are successfully separated and amplified.

[0042] In the mixed solution of hyaluronidase and heparanase, the mass volume ratio concentration of hyaluronidase is 0.075%-0.15%, and the mass volume ratio concentration of heparanase is 0.01%-0.05%.

[0043] The primary digestion was performed using type IV collagenase solution in a 37°C air bath constant temperature oscillator at a low speed of 100-200 rpm for 45-60 minutes.

[0044] The secondary digestion was performed by oscillating the mixture of hyaluronidase and heparanase at 37°C in an air bath constant temperature oscillator at a low speed of 100-200 rpm for 45-60 minutes.

[0045] After the secondary digestion, the digested mixed solution is filtered using a sieve with a pore size of 70-100 um.

[0046] Preferably, the method comprises the following steps:

[0047] (1) Sample preparation: After the liver tumor tissue was removed during surgery, it was cleaned and cut into several small pieces of 2-5 mm3 using sterile scissors;

[0048] (2) Primary digestion with collagenase: 15 to 30 cut tumor tissue blocks were placed in 2 to 5 mL of type IV collagenase solution and mixed thoroughly; the solution was oscillated at a low speed of 100 to 200 rpm for 45 to 60 minutes in a 37°C air bath constant temperature oscillator; the mass volume ratio of the type IV collagenase solution was 0.075% to 0.15%;

[0049] (3) Cell collection and culture: After digestion, resuspend the cells in 10-20 mL of 1640 medium, balance, and centrifuge at 300 g for 5-8 minutes; discard the supernatant after centrifugation, resuspend the cells in complete lymphocyte medium, inoculate 15-30 mL of each aliquot into a culture flask, and culture in a 37°C, 5% CO2 incubator for 24-48 hours;

[0050] (4) Secondary digestion with hyaluronidase and heparanase: The cell suspensions were recovered and centrifuged at 300 g for 5-8 minutes, the supernatant was discarded, the precipitate was flicked, 2-5 mL of hyaluronidase and heparanase mixture was added to each portion, mixed thoroughly, and oscillated at a low speed of 100-200 rpm for 45-60 minutes in a 37°C air bath constant temperature oscillator;

[0051] (5) Cell collection and re-culture: After digestion, resuspend the cells in 10-20 mL of 1640 medium for each portion. After sufficient pipetting and mixing, filter the digested mixture through a 70-100 um cell sieve, collect the filtrate, and centrifuge at 300g for 5-8 minutes. After centrifugation, discard the supernatant, resuspend the cells in complete lymphocyte medium, inoculate them in a culture flask, and culture them in a directed manner. Cell colonies can be seen in 3-7 days.

[0052] The cells were resuspended in complete lymphocyte medium, wherein the medium was X-VIVO 15 medium containing 5%-15% fetal bovine serum and 500-6000 IU / mL IL-2 in a volume ratio.

[0053] Example 1

[0054] (1) Prepare complete T lymphocyte culture medium: X-VIVO 15 (LONZA / 04-418Q) culture medium containing 10% by volume of fetal bovine serum (Excell / FSP500) and a final concentration of 2500 IU / mL of interleukin 2 (CytoCares / GMP-CD66).

[0057] (2) Prepare type IV collagenase solution: Weigh 0.1 g of type IV collagenase (Worthington / LS004186) powder and dissolve it in 100 mL of 0.9% sodium chloride injection. Mix thoroughly and filter through a 0.2 μm filter membrane into a sterile storage bottle for later use. The concentration of type IV collagenase is 0.1%.

[0060] (3) Prepare a mixed enzyme solution: weigh 0.1 g of hyaluronidase (Sigma-Aldrich / H6254-500MG) powder and 0.05 g of heparanase (MCE / HY-P72235) powder, dissolve them in 100 mL of 0.9% sodium chloride injection, mix thoroughly, and filter through a 0.2 μm filter membrane into a sterile storage bottle for later use. The concentration of hyaluronidase is 0.1%, and the concentration of heparanase is 0.05%.

[0064] (4) Tissue cutting: Place the collection tube containing the surgically removed liver tumor tissue (see Figure 1 ) and the tissue were centrifuged and the supernatant was discarded. 20 mL of 0.9% sodium chloride injection was added to wash once. The tissue was removed with sterile forceps and placed in a sterile culture dish. The size of the tissue was about 1 × 1 × 1 cm. The tissue was cut into about 5 mm pieces with sterile scissors. 3 There are 180 tissue blocks in total.

[0068] (5) Tissue digestion: The sheared tissue blocks were placed into 10 50-mL centrifuge tubes at a rate of 18 blocks per portion. Each tube was supplemented with 10 mL of 0.9% sodium chloride injection for thorough washing and balancing. The tubes were centrifuged at 300 g for 5 min. The supernatant was discarded after centrifugation. Each tube was supplemented with 2 mL of collagenase solution and placed in an air bath constant temperature oscillator at 37°C and 120 rpm for 60 min for digestion.

[0072] (6) Cell collection: After digestion, add 18 mL of 1640 medium (Gibco / C22400500BT) to each tube, balance, and centrifuge at 300 g for 8 minutes.

[0074] (7) Cell inoculation: After centrifugation, discard the supernatant and resuspend the cell pellets from 10 tubes into 2 tubes with complete T lymphocyte culture medium, 30 mL per tube. Mix by pipetting and inoculate into 2 T75 culture flasks. Place in a 37°C, 5% CO 2 Culture in incubator.

[0077] (8) Secondary digestion: Observe the cells under a microscope after 24 hours of culture (see Figure 2 , 3 ), all the cell suspension was recovered into two 50mL centrifuge tubes, balanced, centrifuged at 300g for 8 minutes, the supernatant was discarded, the precipitate was flicked off, 2mL of mixed enzyme solution was added to each tube, and the tubes were placed in an air bath constant temperature oscillator at 37°C and 120rpm for 45 minutes of shaking digestion.

[0081] (9) CD3 / CD28 monoclonal antibody coated culture flask: Take 1.5 mL CD3 / CD28 monoclonal antibody coating solution to coat a T75 culture flask and place it in 37°C, 5% CO 2Incubate in the incubator for 30 minutes. The coating solution is a complete T lymphocyte culture medium containing CD3 / CD28 monoclonal antibodies at a concentration of 1 μg / mL. CD3 monoclonal antibody (CytoCares / GMP-A018), CD28 monoclonal antibody (CytoCares / GMP-A063).

[0082] (10) Cell collection: After digestion, add 18 mL of 1640 medium to each tube, mix well, filter through a 70 μm filter into a new 50 mL centrifuge tube, balance the filtrate, and centrifuge at 300 g for 8 min.

[0083] (11) Cell inoculation: After centrifugation, discard the supernatant, resuspend the cells from two tubes in 12 mL of complete lymphocyte culture medium, and inoculate the cells into the coated T75 culture flask. Add IFN-γ to a final concentration of 1000 IU / mL, mix well, and place in a 37°C, 5% CO 2 Culture in incubator.

[0085] (12) T lymphocyte culture:

[0086] ①Observe the cell status every day. On the 6th day of culture, obvious cell colonies can be seen under the microscope (see Figure 4 , 5 ), pipetting and mixing, take 200 μL and count, result: 0.8×10 9 / L, add 5mL T lymphocyte complete medium and continue culturing.

[0088] ② On the 8th day of culture, samples were taken and counted. The result was: 1.2×10 9 / L, add 4mL of T lymphocyte complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0089] ③ On the 10th day of culture, samples were taken and counted. The result was: 2.2×10 9 / L, transfer all cell suspensions into a T182 culture flask, add CD3 / CD28 monoclonal antibodies at 100 ng / mL, add IFN-γ to a final concentration of 1000 IU / mL, and add 24 mL of T lymphocyte complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0091] ④ On the 12th day of culture, samples were taken and counted. The result was: 2.7×10 9 / L, add 53mL of T lymphocyte complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0093] ⑤ On the 14th day of culture, samples were taken and counted. The result was: 2.8×10 9 / L, transfer all the cell suspension into a cell culture bag, add 257mL T lymphocyte complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0096] ⑥ On the 17th day of culture, samples were taken and counted. The result was: 2.4×10 9 / L, add 560mL of T lymphocyte complete medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0098] ⑦ On the 19th day of culture, samples were taken and counted. The result was: 2.4×10 9 / L, add 640mL of T lymphocyte complete culture medium to a total volume of 1600mL and continue culturing.

[0100] ⑧ On the 21st day of culture, samples were taken and counted. The result was: 2.7×10 9 / L, total number of cells 4.32×10 9 , activity rate 99%, draw the amplification curve (see Figure 6 ) and detected cell surface markers. (Results are shown in Figure 7 )

[0101] Example 2

[0102] (1) Prepare NK cell complete culture medium: X-VIVO 15 (LONZA / 04-418Q) culture medium containing 10% by volume of fetal bovine serum (Excell / FSP500) and 500 IU / mL of interleukin 2 (CytoCares / GMP-CD66).

[0105] (2) Prepare type IV collagenase solution: Weigh 0.1 g of type IV collagenase (Worthington / LS004186) powder and dissolve it in 100 mL of 0.9% sodium chloride injection. Mix thoroughly and filter through a 0.2 μm filter membrane into a sterile storage bottle for later use. The concentration of type IV collagenase is 0.1%.

[0106] (3) Prepare a mixed enzyme solution: weigh 0.1 g of hyaluronidase (Sigma-Aldrich / H6254-500MG) powder and 0.05 g of heparanase (MCE / HY-P72235) powder, dissolve them in 100 mL of 0.9% sodium chloride injection, mix thoroughly, and filter through a 0.2 μm filter membrane into a sterile storage bottle for later use. The concentration of hyaluronidase is 0.1%, and the concentration of heparanase is 0.05%.

[0108] (4) Tissue cutting: Place the collection tube containing the surgically removed liver tumor tissue (see Figure 8) and the tissue were centrifuged and the supernatant was discarded. 20 mL of 0.9% sodium chloride injection was added to wash once. The tissue was removed with sterile forceps and placed in a sterile culture dish. The size of the tissue was about 2 × 0.5 × 1.5 cm. The tissue was cut into about 5 mm pieces with sterile scissors. 3 of tissue blocks, 300 in total.

[0109] (5) Tissue digestion: The sheared tissue blocks were placed into 10 50-mL centrifuge tubes at a rate of 30 blocks per portion. Each tube was supplemented with 30 mL of 0.9% sodium chloride injection for thorough washing and balancing. The tubes were centrifuged at 300 g for 8 min. The supernatant was discarded after centrifugation. Each tube was supplemented with 5 mL of collagenase solution and placed in an air bath constant temperature oscillator at 37°C and 120 rpm for 60 min for digestion.

[0111] (6) Cell collection: After digestion, add 25 mL of 1640 medium (Gibco / C22400500BT) to each tube, balance, and centrifuge at 300 g for 8 minutes.

[0112] (7) Cell inoculation: After centrifugation, discard the supernatant and resuspend the cell pellets from 10 tubes into 2 tubes with NK cell complete medium, 30 mL per tube. Mix well by pipetting and inoculate into 2 T75 culture flasks. Place in a 37°C, 5% CO 2 Culture in incubator.

[0114] (8) Secondary digestion: Observe the cells under a microscope after 24 hours of culture (see Fig. 9 , 10 ), all the cell suspension was recovered into two 50mL centrifuge tubes, balanced, centrifuged at 300g for 8 minutes, the supernatant was discarded, the precipitate was flicked off, 5mL of mixed enzyme solution was added to each tube, and the tubes were placed in an air bath constant temperature oscillator at 37°C and 120rpm for 45 minutes of shaking digestion.

[0116] (9) Cell collection: After digestion, add 25 mL of 1640 medium to each tube, mix well and filter through a 70 μm filter into a new 50 mL centrifuge tube. Balance the filtrate and centrifuge at 300 g for 8 minutes.

[0117] (10) Cell inoculation: After centrifugation, discard the supernatant, resuspend the cells from two tubes with 15 mL of NK cell complete medium, and inoculate the cells into a T75 culture flask. Add one tube of trophoblast cells (Hangzhou Zhongying), mix well, and place in a 37°C, 5% CO 2 Culture in incubator.

[0120] (11) NK cell culture:

[0121] ①Observe the cell status every day. On the 7th day of culture, obvious cell colonies can be seen under the microscope (see Fig.11 , 12), pipetting and mixing, take 200 μL and count, result: 0.6×10 9 / L, add 5mL NK cell complete medium and continue culturing.

[0122] ②On the 9th day of culture, samples were taken and counted. The result was: 0.8×10 9 / L, no fluid replacement is performed and culture is continued.

[0123] ③ On the 11th day of culture, samples were taken and counted. The result was: 1.5×10 9 / L, add 10mL NK cell complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0125] ④ On the 13th day of culture, samples were taken and counted. The result was: 2.0×10 9 / L, add 30mL NK cell complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0127] ⑤ On the 15th day of culture, samples were taken and counted. The result was: 2.4×10 9 / L, transfer all the cell suspension into a T182 culture flask, add 2 tubes of trophoblast cells, and add 84mL of NK cell complete medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0130] ⑥ On the 17th day of culture, samples were taken and counted. The result was: 2.3×10 9 / L, transfer all the cell suspension into a cell culture bag, add 187mL NK cell complete culture medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0131] ⑦ On the 19th day of culture, samples were taken and counted. The result was: 2.6×10 9 / L, add 331mL NK cell complete medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0132] ⑧ On the 21st day of culture, samples were taken and counted. The result was: 2.8×10 9 / L, add 860mL of NK cell complete culture medium to a total volume of 1600mL and continue culturing.

[0133] ⑨On the 23rd day of culture, samples were taken and counted. The result was: 3.2×10 9 / L, total number of cells 5.12×10 9 , activity rate 98%, draw amplification curve (see Fig.11 ) and detected cell surface markers. (Results are shown in Fig.12 )

[0134] Comparative example:

[0135] (1) Prepare complete T lymphocyte culture medium: X-VIVO 15 (LONZA / 04-418Q) culture medium containing 10% by volume of fetal bovine serum (Excell / FSP500) and a final concentration of 2500 IU / mL of interleukin 2 (CytoCares / GMP-CD66).

[0138] (2) Prepare mixed enzyme solution: Weigh 0.1 g of type IV collagenase (Worthington / LS004186) powder, 0.1 g of hyaluronidase (Sigma-Aldrich / H6254-500MG) powder, and 0.01 g of deoxyribonuclease I (ThermoFisher / 18047019) powder and dissolve them in 100 mL of 0.9% sodium chloride injection. Mix thoroughly and filter through a 0.2 μm filter membrane into a sterile storage bottle for later use. The concentration of type IV collagenase is 0.1%, the concentration of hyaluronidase is 0.1%, and the concentration of deoxyribonuclease I is 0.01%.

[0139] (3) Tissue cutting: Place the collection tube containing the surgically removed liver tumor tissue (see Fig.15 ) and the tissue were centrifuged and the supernatant was discarded. 20 mL of 0.9% sodium chloride injection was added to wash once. The tissue was removed with sterile forceps and placed in a sterile culture dish. The size of the tissue was about 1 × 1 × 1 cm. The tissue was cut into about 5 mm pieces with sterile scissors. 3 There are 180 tissue blocks in total.

[0143] (4) Tissue digestion: Tissue digestion: The sheared tissue blocks were placed into 10 50-mL centrifuge tubes at a rate of 18 blocks per portion. Each tube was supplemented with 10 mL of 0.9% sodium chloride injection for thorough washing and balancing. The tubes were centrifuged at 300 g for 5 min. The supernatant was discarded after centrifugation. Each tube was supplemented with 2 mL of the mixed enzyme solution and placed in an air bath constant temperature oscillator at 37°C and 120 rpm for 4 h for digestion.

[0144] (5) Cell collection:

[0145] ① After digestion, add 18 mL of sodium chloride injection to each tube, balance, and centrifuge at 300 g for 8 minutes.

[0146] ②After centrifugation, discard the supernatant, add 20mL of sodium chloride injection to each tube, mix well, filter through a 40μm filter into a new 50mL centrifuge tube, balance, and centrifuge at 300g for 8 minutes.

[0147] ③After centrifugation, discard the supernatant, add 30mL of sodium chloride injection to each tube, mix well and slowly add to a 50mL centrifuge tube containing lymphocyte separation solution (Dayou / 7912011), 15mL of lymphocyte separation solution to each tube.

[0148] ④Balance, 500g, 23 minutes, start slow, brake slow centrifuge.

[0149] ⑤ After centrifugation, slowly aspirate the lymphocytes in the middle of each tube, add them to a new centrifuge tube, add sodium chloride injection to balance, and centrifuge at 300g for 8 minutes.

[0150] ⑥ After centrifugation, discard the supernatant, resuspend the cells with 1 mL of sodium chloride injection, add 5 mL of red blood cell lysis buffer (Beckman / A11895) to lyse for 10 minutes, then add sodium chloride injection again, mix well and balance, and centrifuge at 300g for 8 minutes.

[0152] (6) Cell inoculation: After centrifugation, discard the supernatant, resuspend the cells in 12 mL of complete lymphocyte culture medium, and inoculate them into the coated T75 culture flask. Add IFN-γ to a final concentration of 1000 IU / mL, mix well, and observe and take pictures under a microscope (see Fig.16 , 17 ) and placed in 37°C, 5% CO 2 Culture in incubator.

[0153] (7) T lymphocyte culture:

[0154] ① Observe the cell status every day. On the 7th day of culture, a small number of cell colonies can be seen under the microscope (see Fig.18 , 19 ), pipette and mix thoroughly, take 200 μL and count, result: 0.5×10 9 / L, add 5mL T lymphocyte complete medium and continue culturing.

[0156] ②On the 9th day of culture, samples were taken and counted. The result was: 0.7×10 9 / L, and culture was continued without rehydration.

[0157] ③ On the 11th day of culture, samples were taken and counted. The result was: 1.0×10 9 / L, and culture was continued without rehydration.

[0158] ④ On the 13th day of culture, samples were taken and counted. The result was: 1.4×10 9 / L, add CD3 / CD28 monoclonal antibody at 100ng / mL, add IFN-γ to a final concentration of 1000IU / mL, add 5mL T lymphocyte complete medium, and adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0159] ⑤ On the 15th day of culture, samples were taken and counted. The result was: 1.2×10 9 / L, add 6mL of T lymphocyte complete medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0160] ⑥ On the 17th day of culture, samples were taken and counted. The result was: 1.4×10 9 / L, add 14mL of T lymphocyte complete medium to adjust the cell concentration to 1.0×10 9 / LContinue to culture.

[0162] ⑦ On the 19th day of culture, samples were taken and counted. The result was: 1.5×10 9 / L, transfer all the cell suspension into a T182 culture flask, add 24mL of T lymphocyte complete medium to adjust the cell concentration to 1.0×10 9 / L, continue to cultivate.

[0164] ⑧ On the 21st day of culture, samples were taken and counted. The result was: 1.6×10 9 / L, add 43mL of T lymphocyte complete culture medium to adjust the cell concentration to 1.0×10 9 / L, continue to cultivate.

[0165] ⑨On the 23rd day of culture, samples were taken and counted. The result was: 1.6×10 9 / L, total number of cells 1.84×10 8 , activity rate 92%, draw amplification curve (see Fig. 20 ), detection of cell surface markers (results see Fig.21 ).

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting tumor-infiltrating lymphocytes from surgically removed liver tumor tissue, characterized in that: The liver tumor tissue removed during surgery was cleaned and cut into small pieces, and digested once with type IV collagenase solution at 37°C for 45-60 minutes with shaking. The cells were resuspended with lymphocyte culture medium and inoculated into culture bottles, and cultured in a 37°C, 5% CO2 incubator for 24-48 hours. The cells were then digested again with a mixture of hyaluronidase and heparanase at 37°C for 45-60 minutes. The obtained cells were subjected to directed culture, and obvious cell colonies were visible in 3-7 days, indicating that the cells were successfully isolated and amplified.

2. The method for extracting tumor infiltrating lymphocytes from surgically removed liver tumor tissue according to claim 1, characterized in that: In the mixed solution of hyaluronidase and heparanase, the mass volume ratio concentration of hyaluronidase is 0.075%-0.15%, and the mass volume ratio concentration of heparanase is 0.01%-0.05%.

3. The method for extracting tumor infiltrating lymphocytes from surgically removed liver tumor tissue according to claim 1, characterized in that: The primary digestion was performed using type IV collagenase solution in a 37°C air bath constant temperature oscillator at a low speed of 100-200 rpm for 45-60 minutes.

4. The method for extracting tumor infiltrating lymphocytes from surgically removed liver tumor tissue according to claim 1, characterized in that: The secondary digestion uses a mixture of hyaluronidase and heparanase in an air bath constant temperature oscillator at 37° C., with a low speed of 100-200 rpm for 45-60 minutes.

5. The method for extracting tumor infiltrating lymphocytes from surgically removed liver tumor tissue according to claim 1, characterized in that: After the secondary digestion, the digested mixed solution is filtered using a sieve with a pore size of 70-100 um.

6. The method for extracting tumor infiltrating lymphocytes from surgically removed liver tumor tissue according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Sample preparation: After the liver tumor tissue was removed during surgery, it was cleaned and cut into several small pieces of 2-5 mm3 using sterile scissors; (2) Primary digestion with collagenase: 15 to 30 cut tumor tissue blocks were placed in 2 to 5 mL of type IV collagenase solution and mixed thoroughly; the solution was oscillated at a low speed of 100 to 200 rpm for 45 to 60 minutes in a 37°C air bath constant temperature oscillator; the mass volume ratio of the type IV collagenase solution was 0.075% to 0.15%; (3) Cell collection and culture: After digestion, resuspend the cells in 10-20 mL of 1640 medium, balance, and centrifuge at 300 g for 5-8 minutes; discard the supernatant after centrifugation, resuspend the cells in complete lymphocyte medium, inoculate 15-30 mL of each aliquot into a culture flask, and culture in a 37°C, 5% CO2 incubator for 24-48 hours; (4) Secondary digestion with hyaluronidase and heparanase: The cell suspensions were recovered and centrifuged at 300 g for 5-8 minutes, the supernatant was discarded, the precipitate was flicked, 2-5 mL of hyaluronidase and heparanase mixture was added to each portion, mixed thoroughly, and oscillated at a low speed of 100-200 rpm for 45-60 minutes in a 37°C air bath constant temperature oscillator; (5) Cell collection and re-culture: After digestion, resuspend the cells in 10-20 mL of 1640 medium for each portion. After sufficient pipetting and mixing, filter the digested mixture through a 70-100 um cell sieve, collect the filtrate, and centrifuge at 300g for 5-8 minutes. After centrifugation, discard the supernatant, resuspend the cells in complete lymphocyte medium, inoculate them in a culture flask, and culture them in a directed manner. Cell colonies can be seen in 3-7 days.

7. The method for extracting tumor-infiltrating lymphocytes from surgically removed liver tumor tissue according to claim 6, characterized in that: The cells were resuspended in complete lymphocyte medium, wherein the medium was X-VIVO 15 medium containing 5%-15% fetal bovine serum and 500-6000 IU / mL IL-2 in a volume ratio.

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