TW200831670A - Methods for the cultivation of tumor cells and application of the same - Google Patents

Abstract

A fast and convenient method is applied to purify tumor cells by removing non-tumour tissue and slough without damaging the tumor cells, followed by extract a cellular activator ''Koyo'', and improving cell growth and proliferation. In addition, the application of the cellular activator ''Koyo '' is performed to activate the DC, CIK, and NK cells collected from the umbilical cord blood or peripheral blood, and the vaccines are made to improve the infection diseases, cancer, cancer metastasis and auto-immune diseases.

Description

200831670 xw \_xj_a\-2 18734twf.doc/0〇6 IX. Description of the invention: [Technical field to which the invention pertains] The present invention is related to - a variety of cell biology, and particularly related to > a tumor biology, Tumor cell molecular genetics, tumor immunity. $ and anti-tumor drugs, and specifically related to the research field of tumor cells and primary culture of tumor cells and their applications. [Prior Art] Since the _* period, how to isolate a large number of highly purified tumor cells from tumor tissues without damaging the tumor cells is straightforward. Due to the difficulty of this technique, the success rate of primary culture of tumor cells in vitro is low (less than 2%). The success of primary culture of tumor cells is often the key to tumor cell research. At the same time, a large number of highly purified tumor cells and primary cultured tumor cells are tumor cell biology, tumor genetics, and tumor molecular biology. An important tool for tumor immunology and anti-tumor drugs. Most experiments show that the main reason for the low success rate of primary culture of tumor cells is that it is difficult to obtain a large number of high-purity tumor cells. 'Tumor tissue is often mixed with many normal cells, including some lymphocytes, fibroblasts, and Qualitative cells and tumor necrotic cells, these cells severely interfere with the growth and growth of tumor cells. NK cells are a group of lymphocyte populations that represent early cell lines that prevent antagonism of viral and tumor cells. The cells involved in the anti-tumor immunity of the non-trio of the antigen and the prevention or transfer of the primary tumor were performed as a function of suppressing or suppressing the stagnation of 200831670 a wl 18734twf.doc/006. In the tumor mouse experiment, LAK cells (lymphocyte-activated NK cells), adhesion NK cells (A-NK), or non-adhesive NK cells were added or not treated with 1L 2 and IL-12/IL_15 ( NA-NK) was observed to have anti-tumor surveillance and was the result of in vitro stimulation of NK cells with high doses of IL-2. However, in the treatment of murine or human tumors or other disorders with NK cells, in vivo or in vitro methods of infection involve the amount of NK cells, which are traditionally based on cytokines and are currently available. It is necessary to administer a high dose of cytokines that are difficult to tolerate by the host, and NK cells have high difficulty in survival in vitro, and it is difficult for cytokines to activate NK cells without nutritional support. Dendritic cells (DCs) are powerful human process cells (APCs) that process the extracted antigen into peptide fragments and then transfer them to secondary lymphoid organs. The major histocompatibility complex (MHC) is bound to T lymphocytes, induces lymphocyte activation, activates the initial immune response, and provides co-stimulatory molecular effects on DC cells, such as CD80, CD86. It can directly activate the initial T lymphocytes in the body, promote the formation of helper T lymphocytes and CTL cells, and also promote B lymphocytes, which produce antibodies. Cytokine-induced killer cells (CIK) are obtained by co-culturing human peripheral blood mononuclear cells in vitro with various cytokines for a period of time. Cytokine-induced killer fine 6 200831670, ~u\-2 18734twf.doc/006 cells can simultaneously express two protein molecules such as CD3+ and CD56+, also known as T lymphocytes of NK cells, and anti-tumor with T lymphocytes Activity and non-MHC restrictive tumoricidal advantages of NK cells. CIK is a new generation of anti-tumor adoptive cell immunotherapy, but clinical reports on malignant solid tumors are still rare. SUMMARY OF THE INVENTION The object of the present invention is to provide a rapid and simple method for removing a large number of non-Φ tumor cells and necrotic tumor cells without any damage to tumor cells, and separating living tumor cells. method. A further object of the present invention is to provide a cell activin (Koyo) derived from the extraction of isolated living tumor cells, which greatly promotes the growth and proliferation of tumor cells, and the accumulation of a large number of purified tumor cells in the tumor cell population effect. It provides good conditions for the growth and proliferation of tumor cells. A further object of the present invention is to provide a method for activating DC, D_CIK, and D-NK cells by using a cell extract activator (Koyo) derived from tumor cells in vitro for long-term culture, passage, and even establishment. Another object of the present invention is to use a cell activin to activate DC, D-CIK, or cells derived from cord blood or peripheral blood of a patient to prepare a vaccine and anti-tumor cells for reinfusion to improve Sense _ infection, cancer, metastatic cancer, autoimmune and other diseases. The present invention provides a rapid and simple no-injury case of removing a large number of non-tumor cells and necrotic tumor cells, and the tumor cells of the live 7 200831670 jlx^/ v_, xx^-2 18734twf.doc/006 are highly purified. The method of isolation improves the success rate of primary culture of tumor cells. Using this efficient purification technique, the success rate of primary culture of tumor cells derived from epithelial cells can be more than 85%. The present invention further provides a method for purifying tumor cell-derived cell activin (Koyo) to activate Dc', D-CIK, D-administered cells obtained from cord blood or peripheral blood of a patient, and the vaccine and anti-tumor cell thereof are prepared. Returning DC, D-CIK and D-NK successfully improved the improvement of infection, cancer # transfer of cancer, autoimmune and other diseases, and even advanced kidney: suffering from illness, providing patients in addition to surgery or chemotherapy -^ Improve and be positive about the quality of life. The advantages of the present invention for separating and purifying tumor cells are rapid, convenient, effective, and low in cost, and there is no sputum in tumor cells, and a large number of purified tumor cells can be obtained, which greatly enhances the swelling of the primary cells. The success rate of cultivation. At the same time, it is also a valuable research tool for tumor biology, swollen=,,, cell molecular genetics, tumor immunology and anti-tumor drugs. The above and other objects, features, and advantages of the present invention will be apparent from the description of the appended claims. [Formula] [Embodiment] Isolation and purification of swollen cells 丨 (1) Treatment of tumor cells before purification. The tumor tissue (1-2 cm3) was surgically removed and placed in a culture flask containing RpMI_164 参 参 ( ( ( 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The visible non-tumor tissue and tumor necrotic tissue were removed with sterile ophthalmic scissors, and the tumor tissue was wetted with a little RPMI-1640 medium (serum free) and the shredded tumor tissue was placed as soon as possible. In a 50 ml centrifuge tube, add 10-15 ml of collagenase 4 solution (Sigma) (collagenase 4 dissolved in D-hanks, final concentration of 1 mg/ml, filter sterilized), and warm bath in a 37 ° C water bath. hour. After 1 hour, the centrifuge tube (50 ml) was taken out, diluted one-fold with a serum-free 1640 solution, repeatedly blown, and the cell suspension was filtered through a sterile 70 mesh filter, centrifuged, and 1200 rpm (8 minutes). The supernatant was removed, and 20 ml of 1640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody was mixed to mix the centrifuged cells to obtain a tumor tissue cell suspension. (2) Separation and purification of tumor cells. Take 20 ml of inactivated fetal bovine serum or human AB serum in a 50 ml centrifuge tube, and mix 20 ml of the cell suspension of the tumor tissue (which contains lymphocytes, fibroblasts, interstitial cells and The tumor necrotic cells were gently added to the A tube from the tube wall, allowed to stand for 6 minutes, and 6 ml of jk clear was taken from the bottom of the A 50 ml centrifuge tube, and placed in a 50 ml centrifuge tube; then the A 50 ml centrifuge tube was allowed to stand. 6 minutes, 6 ml of serum was aspirated from the bottom of a 50 ml centrifuge tube, and placed in a 50 ml centrifuge tube. Then, the A. 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the A 50 ml centrifuge tube, and placed in a No. D 50 ml centrifuge tube. Centrifuge tube No. B 50 ml, centrifuge tube No. C 50 ml and centrifuge tube No. D 50 ml, centrifuge at 1200 rpm (8 min). The cells of the Shen Dian are purified tumor cells. 9 18734twf.doc/006 200831670 V^XX'V"2 Take 5 ml of 10% fetal bovine serum or human AB serum, 1% double-antibody 1640 medium and mix the cells of the B-cell centrifuge tube and place them in a 6-well plate. In one well, many purified tumor cells were visible under the microscope, and the purified tumor cells were cultured for 3-4 days to see tumor cell division. The cells of the C and D centrifuge tubes contain a small amount of non-tumor cells in addition to the tumor cells, and the above process can be repeated to obtain more purified tumor cells. Tumor cell lysate-derived fine-face activin (Koyoj^huangfu: fresh cancer tissue is shredded, sieved to make a single cell suspension, and the tumor is isolated and purified by the present invention. The extract is repeatedly frozen and thawed by liquid nitrogen, and obtained by centrifugation. The clear liquid was filtered and sterilized by 〇·22ιι sieve, and the protein content was determined and stored at -80 ° C. DC ' D_CIK, D-NK cell culture and characterization: The steps related to NK cell culture of autologous peripheral blood and umbilical cord The implementation is in accordance with the Good Manufacturing Practice GMP (Good Manufacturing Practice GMP) guidelines. The suspension step combines Ficoll with sodium metrizoate (eg Hypague) to prepare the standard lymphocyte preparation method. The whole blood is Ficoll-Hypaque density. Gradient centrifugation takes the top layer, and after centrifugation, most of the lymphocytes are found between Ficoll/Hypaque and plasma. Materials: Blood samples (from patient autologous peripheral blood and normal human cord blood) 200831670 y wm -2 18734twf.doc/006 Clean Centrifuge Tube (BD, USA) Disposable Straw (Pasteur Pipettes, USA BD) Various culture bottles Culture flask) 24 well culture dish (24 well culture dish U.S. BD Biosciences beaker T25 flask lymphocyte medium (Ficoll / metrizoate, adjust to 1.077 g / cc)

Aja-V medium, GIBCO; therapeutic level Sterilization dropper Pasteur Pipettes 24 well culture 24 well culture dish

Anti-CD56, Anti-CD 16 ^ Anti-CD3 (OKT-3) and

Single-bead antibody such as Anti-IL-2

Human recombinant IL-2 ( Chiron Company)

Human recombinant IFN-α: ( Immunex company) Human recombinant IL-1 a : ( Ppeprotech company )

Human recombinant CD3 (anti-CD3 MoAb). ( Chiron Company)

Human recombinant GM-CSF: ( Immunex company) Human recombinant IL-4: ( R & D System company)

Human recombinant TNF- a : ( R & D System company)

Human recombinant IL-7: From R & D System company stain Trypan blue 11 20083 1 tZL _tw_a6 Cell counter Hemocytometer or cell counter Saline cross-infection prevention: ^ For the possible interaction of possible infections. Take the following defensive measures. 1. Only sample from well-reputed units. 2. - Only one sample is allowed to be operated. 3. The dropper must never be reused in different samples. 4. The same medium in the container must never be reused in different samples. • 5. The used dropper must never be placed in the container of the medium. Preparation of DC seedlings: Take 50-60ml of peripheral blood of the patient, separate the mononuclear cells into the culture flask with the culture medium, and warm the bath for 1 hour at 37 °C and 5% CO 2 incubator, aspirate and suspend. The cells are cultured with DC cells. The adherent cells were added with fresh DC medium (1640 complete medium + GM-CSF 100 ng/ml + IL_4 30 ng/ml), and placed in a culture environment of 37 ° C and 5% CO 2 . Autologous tumor cell lysate-derived cytokine (Koyo) sensitized DC was added on the third day of culture, and the final concentration of tumor cell lysate protein was 10 mg/ml. On the sixth day, TNFa was added, and on the seventh day, the mature DC vaccine was collected. Preparation of D-CIK cell proliferation: Take suspension cells (according to the isolation and purification of the tumor cells: (1)), add 'INFy-lOOOU/ml D-CIK complete medium, place at 37 ° C and 5% C02 Develop the environment. After 24 hours of culture, a final concentration of 12 '-2 18734 twf.doc/006 200831670 was added to 100 mg/ml mouse anti-human CD3 monoclonal antibody, l〇〇〇U/ml IL-2 and 1000 U/ml IL-Ια, Continue to train. Daily observation, change every 2-3 days, add fresh IL-2 medium, add mature DC cells on the seventh day of culture, and add GM-CSF (Working concentration: 800 ... 1000 iu/ml ), IL-2 ( Working concentration: 20 ... 40 lU/rnl ), IL_7 ( Working concentration: 20ng 40 ng/ml), began to collect D-CIK cells, centrifuged, washed, and 1 在 in culture for about 14 days. 〇ml of physiological saline, 3-5 ml of 20% human albumin weight cells, usually returned to the patient from the vein within one hour. Immunophenotypic assays of mature DC cells and D-CIK cells were stained with FITC-labeled murine anti-human CD80, CD83, CD86, MHC-1, and MHC-II monoclonal antibodies, and these surface molecules were detected by flow cytometry in DC cells. Expression of surface antigen; a small amount of D-CIK cells (1χ1〇6) were taken before reinfusion, using Cy-chromeTM anti-human CD3,

Cy-chromeTM anti-human CD4? FITCanti-human CD8, PE-anti-human CD 19 ? PE-anti-human CD56 (all products of Pharmingen) were used to detect the distribution of lymphocyte subsets. Example 1: Purification of human ovarian cancer cells A, preparation of ovarian cancer cell suspension (1), surgically resected ovarian cancer tissue (l-2 cm3, placed in a culture flask containing RPMI-1640 medium (no gold clearance) Immediately, send it to the laboratory. Use sterile eye scissors to remove the tumor tissue 13 18734twfdoc/006 200831670 and tumor necrotic tissue, moisten the tumor tissue with a little RPMI-1640 medium (serum free) and cut it as soon as possible. Place the shredded tumor tissue in a 50 ml centrifuge tube and add 10-15 ml of collagenase solution (Sigma) (collagenase 4 is dissolved in D-hanks to a final concentration of 1 mg/ml, filtered, sterilized). The bath was incubated in a 37 ° C water bath for 1 hour. After 1 hour, a 50 ml centrifuge tube was taken out, diluted with a serum-free 1640 solution, repeatedly blown, and the cell suspension was filtered through a sterile 70 mesh filter. Centrifuge, 1200 _ turn (8 minutes), remove the supernatant, and take 20 ml of 1640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody. Mix the centrifuged cells to obtain ovarian cancer tissue. Cell suspension. B. Isolation of ovarian cancer cells Purification (2), isolation and purification of tumor cells 20 ml of inactivated fetal bovine serum or human AB serum is placed in a 50 ml centrifuge tube, and 20 ml of mixed cell suspension of ovarian cancer tissue (which contains lymphocytes, Fibroblasts, stromal cells and tumor necrotic cells) (Fig. 1-1) Gently added to the A tube from the tube wall, allowed to stand for 6 minutes, and aspirate 6 ml of serum from the bottom of the A 50 ml centrifuge tube. In a 50 ml centrifuge tube, place the A 50 ml centrifuge tube for 6 minutes, and aspirate 6 ml of serum from the bottom of the A 50 ml centrifuge tube and place it in a 50 ml centrifuge tube. Place the A 50 ml centrifuge tube for 6 minutes. Aspirate 6 ml of serum at the bottom of a 50 ml centrifuge tube, and place it in a 50 ml centrifuge tube of No. D. Centrifuge tube No. B 50 ml, centrifuge tube No. C 50 ml and centrifuge tube No. D 50 ml, centrifuge at 1200 rpm (8 min). That is, the purified egg 14 200831670 i^w^iK-2 18734twf.doc/006 nest cancer cells. Take 5 ml of 10% fetal bovine serum or human AB serum, 1% double-antibody 1640 medium and mix B centrifuge tube sediment Cells are juxtaposed in a well of a 6-well plate, and a series of ovarian cancer cells are visible under the microscope (Figure 1-2). The ovarian cancer cells can be cultured for 3-4 days to see the tumor cell division. The cells of the C and D centrifuge tubes contain a small amount of non-tumor cells in addition to the nest cancer cells, and the above process can be repeated to obtain more purification. Ovarian cancer cells. #Example 2: Purification of human renal cancer cells A. Preparation of renal cancer tissue cell suspensions Surgical resected renal cancer tissues (1-2 cm3) were placed in RPMI-1640-containing medium (no serum) The flask is immediately sent to the laboratory. Exfoliate the tumor tissue and tumor necrotic tissue with sterile ophthalmic scissors, wet the tumor tissue with a little RPMI-1640 medium (serum free) and cut it as quickly as possible. Place the shredded tumor tissue in a 50 ml centrifuge tube. Into, 10-15 ml of collagen I enzyme solution (Sigma) was added (collagenase 4 was dissolved in D-hanks at a final concentration of 1 mg/ml, and sterilized by filtration), and the mixture was incubated in a 37 ° C water bath for 1 hour. After 1 hour, remove the 50 ml centrifuge tube, dilute it twice with serum-free 1640 solution, repeatedly blow, take the cell suspension and filter with a sterile 70 mesh filter, centrifuge, 1200 rpm (8 ^ minutes), go to To remove the 20 ml of 1640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody, the cells after centrifugation are mixed to obtain a cell suspension of renal cancer tissue. 15 18734twfdoc/006 200831670 JL-^ V^/ V-/XX'%."2 Go to the laboratory. Exfoliate the tumor tissue and tumor necrotic tissue with sterile ophthalmic scissors, wet the tumor tissue with a little RPMI-1640 medium (serum free) and cut it as quickly as possible. Place the shredded tumor tissue in a 50 ml centrifuge tube. Into, 10-15 ml of collagenase solution (Sigma) was added (collagenase 4 was dissolved in D-hanks to a final concentration of 1 mg/ml, and sterilized by filtration), and the mixture was incubated in a 37 ° C water bath for 1 hour. After 1 hour, remove 5 〇ml of the centrifuge tube, dilute with serum-free 1640 solution, repeatedly blow, take the cell _ suspension with a sterile 70 mesh filter, centrifuge, 1200 rpm (8 minutes), The supernatant was removed, and 20 ml of the 1640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody was mixed to obtain the cell suspension of the lung cancer tissue. B. Isolation and purification of lung cancer cells Isolation and purification of lung cancer cells 20 ml of inactivated fetal bovine serum or human AB serum is placed in a 50 ml centrifuge tube, and 20 ml of cell suspension of lung cancer tissue is mixed (including Lymphocytes, I fibroblasts, interstitial cells and tumor necrotic cells) (Figure 3-1)

Gently add from the tube wall to the A tube, let stand for 6 minutes, aspirate 6ml serum from the bottom of the A 50ml centrifuge tube, set the B number 50ml centrifuge tube; then let the A number 50ml centrifuge tube stand for 6 minutes, from Aspirate 6 ml of serum at the bottom of a 50 ml centrifuge tube and place it in a 50 ml ml tube. Then, place the A 50 ml centrifuge tube for 6 minutes, and aspirate 6 ml of serum from the bottom of the A 50 ml centrifuge tube, and place the No. D 50 ml • centrifuge tube. Centrifuge tube No. B 50 ml, centrifuge No. C 50 ml and tube No. D 50 ml, centrifuge at 1200 rpm (8 min). Shen Dian 17 200831670 ~m-2 18734twf.doc/006 cells are purified tumor cells. Take 5 ml of 10% fetal bovine jk clear or human AB serum, 1% double-antibody 1640 medium and mix the cells precipitated in the B-cell centrifuge tube and place it in a hole in a 6-well plate. Many strings can be seen under the microscope. Strings of lung cancer cells (Fig. 3-2), the purified tumor cells were cultured for 3-4 days to see lung cancer cell division. The cells of the C_ and D centrifuge tubes contain a small amount of non-lung cancer cells in addition to the renal cancer cells, and the above process can be repeated to obtain more purified lung cancer cells. Example 4: Purification of human breast cancer cells A. Preparation of breast cancer cell suspensions Surgical resected breast cancer tissues (1-2 cm3) were placed in culture flasks containing RPMI-1640 medium (serum free). , immediately sent to the laboratory. Excise the tumor tissue and tumor necrotic tissue with sterile ophthalmic scissors, wet the tumor tissue with a little RPMM640 medium (serum free) and cut it as soon as possible. Place the shredded tumor tissue in a 50 ml centrifuge tube. 10-15 ml of collagenase solution (Sigma) was added (collagenase 4 was dissolved in D-hanks at a final concentration of 1 mg/ml, filter sterilized), and the mixture was incubated in a 37 t: water bath for 1 hour. After 1 hour, remove the 50 ml centrifuge tube and dilute it with the serum-free 1640 solution. Repeat the pipetting, take the cells, filter the suspension with a sterile 70 mesh filter, centrifuge, 1200 rpm (8 minutes), go The supernatant was mixed with 20 ml of 1640 medium containing 10% fetal bovine serum or human 'AB serum and 1% double antibody to obtain a cell suspension of breast cancer tissue. 18 200831670-2 18734twf.doc/006 B. Isolation and purification of breast cancer cells Take 20 ml of inactivated fetal bovine serum or human AB serum in a 50 ml centrifuge tube and mix 20 ml of cells of breast cancer tissue. The suspension (Fig. 4-1) (which contains lymphocytes, fibroblasts, mesenchymal cells and tumor necrotic cells) was gently added from the tube wall to the A tube and allowed to stand for 6 minutes from the bottom of the A 50 ml centrifuge tube. Aspirate 6 ml of serum and place it in a 50 ml centrifuge tube; then place the A-50 ml centrifuge tube for 6 minutes, and aspirate 6 ml of serum from the bottom of the A 50 ml centrifuge tube and place in a 50 ml centrifuge tube. Then, a 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of a 50 ml centrifuge tube, and placed in a No. D 50 ml centrifuge tube. Centrifuge tube No. B 50 ml, centrifuge No. C 50 ml and tube No. D 50 ml, centrifuge at 1200 rpm (8 min). The cells of Shen Dian are purified breast cancer cells. Take 5 ml of 10% fetal bovine serum or human AB serum, 1% double-antibody in 1640 medium and mix the cells precipitated in the B-cell centrifuge tube and place in one well of the 6-well plate. Many purified breast cells can be seen under the microscope ( Figure 4-2), the purified breast cancer cells can be seen for 3 to 4 days to see tumor cell division. The cells of the C and D centrifuge tubes contain a small amount of non-breast cancer cells in addition to the breast cells, and the above process can be repeated to obtain more purified breast cancer cells. Example 5: Purification of human nasopharyngeal carcinoma cells A, preparation of nasopharyngeal carcinoma tissue cell suspension, surgically removed nasopharyngeal carcinoma tissue (l-2 cm3), placed in containing 19 200831670 ~ ΑΑ \-2 18734twf.doc / 006 RPMI-1640 medium (serum-free) in a culture flask and immediately sent to the laboratory. Use sterile ophthalmic scissors to remove tumor tissue and tumor necrotic tissue, moisten the tumor tissue with a little RPMI-1640 medium (serum free) and place it with 砰5 to place the 50% centrifuge tube with the tumor tissue. Into, 10-15 ml of collagenase solution (Sigma) was added (collagenase 4 was dissolved in D-hanks to a final concentration of 1 mg/ml, and sterilized by filtration), and the mixture was incubated in a 37 ° C water bath for 1 hour. After 1 hour, remove the 50 ml centrifuge tube, dilute with _ serum-free 1640 solution, repeatedly blow, take the cell suspension and filter with a sterile 70 mesh filter, centrifuge, 1200 rpm (8 minutes), go to To remove the 20 ml of 1640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody, the cells after centrifugation are mixed to obtain a cell suspension of nasopharyngeal carcinoma tissue. B. Isolation and Purification of Nasopharyngeal Carcinoma Cells Take 20 ml of inactivated fetal bovine serum or human AB serum in a 50 ml centrifuge tube and mix 20 ml of cell suspension of nasopharyngeal carcinoma tissue (Figure 5-1). ) (containing lymphocytes, fibroblasts, mesenchymal cells and tumor necrotic cells) gently added to the tube A from the tube wall, allowed to stand for 6 minutes, and aspirate 6 ml of serum from the bottom of the A 50 ml centrifuge tube, set the B number In a 50 ml centrifuge tube, the A 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the A 50 ml centrifuge tube and placed in a C 50 ml centrifuge tube. Then, a 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of a 50 ml centrifuge tube 20 200831670 a w 18734 twf.cioc/006, and placed in a No. D 50 ml centrifuge tube. Centrifuge tube No. B 50 ml, centrifuge No. C 50 ml and tube No. D 50 ml, centrifuge at 1200 rpm (8 min). The precipitated cells are purified nasopharyngeal carcinoma cells. Take 5 ml of 10% fetal bovine serum or human AB serum, 1% double-antibody in 1640 medium and mix the cells precipitated in the B-cell centrifuge tube into a well of a 6-well plate. Many purified nasopharynx can be seen under the microscope. Cells (Fig. 5-2), the purified nasopharyngeal carcinoma cells were cultured for 3-4 days to see tumor cell division. The cells of the C and D centrifuge tubes contain a small amount of non-nasopharynx cancer cells in addition to the nasopharyngeal cells, and the above process can be repeated to obtain more purified nasopharyngeal carcinoma cells. Example 6: Isolation and purification of human malignant cerebellar blastoma A. Preparation of human malignant sputum blastoma tissue suspension. Human malignant cerebellar medulloblastoma tissue (l-2cm3) was surgically removed and placed in the containing The RPMI-1640 medium (serum-free) was immediately sent to the laboratory. Exclude tumor tissue and tumor necrotic tissue with sterile ophthalmic scissors $, moisten the tumor tissue with a little RPMI-1640 medium (serum free) and cut it as quickly as possible, and place the shredded tumor tissue in 50 ml centrifugation. In the tube, 10-15 ml of collagenase solution (Sigma) was added (collagenase 4 was dissolved in D_hanks, and the final concentration was 1 mg/m b. sterilized), and the bath was incubated in a 37 ° C water bath for 1 hour. After 1 hour, remove the 50 ml centrifuge tube, dilute it with a serum-free 1640 solution, repeatedly beat, and take the cell suspension with a sterile 70 mesh filter, centrifuge, 1200 rpm (8 minutes). Remove the supernatant and take 20 ml 21 18734 tw£doc/006 200831670 JL-^ V_// Mix the centrifuged cells with 1640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody. A cell suspension of malignant cerebellar medulloblastoma tissue. B. Isolation and purification of human malignant cerebellar medulloblastoma cells 20 ml of inactivated fetal bovine serum or human AB serum is placed in a 50 ml centrifuge tube, and 20 ml of mixed malignant cerebellar medullary _ cell tumor tissue The cell suspension (Fig. 6-1) (which contains lymphocytes, fibroblasts, mesenchymal cells and tumor necrotic cells) was gently added from the tube wall to the A tube and allowed to stand for 6 minutes from the A 50 ml centrifuge tube. 6 ml of serum was aspirated at the bottom and placed in a 50 ml centrifuge tube. Then, a 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the A 50 ml centrifuge tube and placed in a C 50 ml centrifuge tube. Then, a 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the 50 ml centrifuge tube of No. A, and placed in a No. D 50 ml centrifuge tube. • Centrifuge tube B 5〇1111, (: No. 5〇1111 centrifuge tube and No. 5 5〇1111 centrifuge tube, centrifuge for 1200 rpm (8 min). The precipitated cells are purified malignant cerebellar medulloblastoma cells. Mix 5 ml of 10% fetal bovine serum or human AB serum, 1% double-antibody in 1640 medium, mix the cells of the B-cell centrifuge tube and place it in a well of a 6-well plate. Many purifications can be seen under the micro-mirror. The cerebellar medulloblastoma cells (Fig. 6-2), the purified malignant cerebellar medulloblastoma cells can be cultured for 3-4 days to see the tumor cell division. The cells of the C and D centrifuge tubes except the malignant cerebellar The cell tumor cells also contain a small amount of non-malignant cerebellum 22 18734twf.doc/006 200831670 blastoma cells, and the above process can be repeated to obtain more purified malignant cerebellar Zhaoblastoma cells. Example 7: Human colon cancer Isolation and purification of cells A, preparation of human colon cancer tissue suspension, surgically resected human colon cancer tissue (1-2 cm3), placed in a culture flask containing RPMI-1640 medium (serum-free), immediately sent to the experiment Room. Use sterile eye scissors to remove tumor tissue and Tumor necrotic tissue, wet the tumor tissue with a little RPMI-1640 medium (serum free) and cut it as soon as possible. Place the shredded tumor tissue in a 50 ml centrifuge tube and add 10-15 ml collagenase solution. (Sigma) (collagenase 4 dissolved in D-hanks, final concentration of lmg/ml, filter sterilization), warm bath for 1 hour in a 37 ° C water bath. After 1 hour, remove 50 ml of centrifuge tube, use no The serum-containing 1640 solution is diluted twice, repeatedly blown, and the cell suspension is filtered with a sterile 70 mesh filter, centrifuged, 1200 rpm (8 minutes), the supernatant is removed, and 20 ml of 10% fetal bovine serum is taken or Human AB serum, 1% double-antibody 1640 medium mixes these centrifuged cells to obtain a cell suspension of colon cancer tissue. B. Separation and purification of human cancer cells Take 20 ml of inactivated fetal bovine serum or Human AB serum was placed in a 50 ml centrifuge tube, and 20 ml of the mixed cell colon tissue suspension (Fig. 7-1) containing lymphocytes, fibroblasts, mesenchymal cells and tumor necrotic cells was light. Light from the tube wall to join 23 200831670? 18734twf.doc/006 into the fistula After standing for 6 minutes, 6 ml of serum was aspirated from the bottom of the 50 ml centrifuge tube, and placed in a 50 ml centrifuge tube. Then, a 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the A 50 ml centrifuge tube, and placed in C. No. 50ml centrifuge tube. Then stand the A 50ml centrifuge tube for 6 minutes, aspirate 6ml serum from the bottom of the A 50ml centrifuge tube, and place it in the No. D 50ml centrifuge tube. Add the B number 50ml centrifuge tube, C No. 50ml centrifuge tube. Centrifuge in a 50 ml centrifuge tube with D, 1200 rpm (8 min). The precipitated cells are purified colon cancer cells. Take 5 ml of 10% fetal bovine jk clear or human AB serum, 1% double-antibody 1640 medium and mix the cells of the B-cell centrifuge tube and place it in one well of the 6-well plate. Many purified colons can be seen under the microscope. Cancer cells (Fig. 7-2) 'The purified colon cancer cells were cultured for 3 days in the stomach for 4 days to see tumor cell division. The cells of the C and D centrifuge tubes contain a small amount of non-colon cancer cells in addition to the colon cancer cells, and the above process can be repeated to obtain a more purified colon cancer. Example 8: Isolation and Purification of Human Hepatoma Cells A. Preparation of Human Hepatocellular Carcinoma Tissue Suspensions Human liver cancer tissues (l-2 cm3) were surgically removed and placed in a culture flask containing RPMI-1640 medium (serum free) immediately. Transfer to the lab. Exfoliate the tumor tissue and tumor necrotic tissue with sterile ophthalmic scissors, wet the tumor tissue with a little RPMM640 medium (serum free) and cut it as quickly as possible. Place the shredded tumor tissue in a 50 ml centrifuge tube. Add 10-15 ml collagenase solution (Sigma) (collagenase 4 dissolved in D-hanks 24 200831670 w w2 18734twf.doc/006, final concentration 1mg/ml, filter sterilized), in a 37 ° C water bath Warm bath for 1 hour. After 1 hour, remove the 50 ml centrifuge tube, dilute with the serum-free 1640 solution, repeatedly blow, take the cell suspension and filter with a sterile 70 mesh filter, centrifuge, 1200 rpm (8 minutes), remove the supernatant. Liquid, 20 ml of 1040 fetal bovine serum or human 'AB serum, 1% double-antibody 1640 medium mixed with these centrifuged cells, you can get the cell suspension of liver cancer tissue _ B, human liver cancer cells isolated and purified Take 20 ml of inactivated fetal bovine serum or human AB serum in a 50 ml centrifuge tube and mix 20 ml of cell suspension of liver cancer tissue (Fig. 8-1) containing lymphocytes, fibroblasts, Mesenchymal cells and tumor necrotic cells were gently added to the A tube from the tube wall, allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the A 50 ml centrifuge tube, placed in a 50 ml centrifuge tube; then centrifuged at 50 ml A. The tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of a 50 ml centrifuge tube, and placed in a C 50 ml centrifuge tube. Then, the No. 50 50 ml centrifuge tube was allowed to stand for 6 minutes, and 6 ml of serum was aspirated from the bottom of the A 50 ml centrifuge tube, and placed in a No. D 50 ml centrifuge tube. Centrifuge tube No. B 50 ml, centrifuge No. C 50 ml and centrifuge tube No. D 50 ml, centrifuge at 1200 rpm (8 min). The precipitated cells are purified liver cancer cells. Take 5 ml of 10% fetal bovine serum or human AB serum, 1% double-antibody 1640 medium and mix the cells of the B-cell centrifuge tube and place it in a hole in a 6-well plate. Many purified liver cancers can be seen under the microscope. Cells (Fig. 8-2), the purified hepatoma cells were cultured for 3-4 days to see swelling 25 2008316709 18734twf.doc/006 tumor cell division. The cells of the C and D centrifuge tubes contain a small amount of non-hepatoma cells in addition to the liver cancer cells, and the above process can be repeated to obtain more purified liver cancer cells.

Example 9: Liver cancer using D-CIK combined with NK A 77-year-old man was diagnosed with highly differentiated hepatocellular carcinoma by liver biopsy on August 18, 2004. After performing CT-guided liver radiofrequency ablation twice, autologous D-CIK cells were returned once, and NK cells were returned 5 times. The patient's life is stable, and the general condition is OK without any discomfort. Table 1: One 77 years old Male diagnosis of high-differentiated hepatocellular carcinoma radiofrequency ablation combined with autologous NK treatment in the treatment of cell count 2004-08-26 2τ guided descending liver radiofrequency ablation 2004-09-01 Autologous CIK cell reinfusion cell count 3 ·2χ109 2004-09-21 CT-guided liver radiofrequency ablation 2004-09-29 NK cell therapy cell count 1.21 X 109 2004-10-12 NK cell therapy cell count 2.0x109 2004-10-22 NK cell therapy Cell count 1.25 X 109 2004-11-1 NK cell therapy cell count 4.35 X 109 2004-11-10 NK cell therapy cell count 4.15 X 109 26 18734 tw£doc/006 200831670 X^K^I WXX^"2 Table 2: - 77-year-old male diagnosed as highly differentiated hepatocellular carcinoma with radiofrequency ablation combined with autologous NK therapy to track tumor markers and HBV-DNA quantification 8/20 11/23 Reference AFP 47.22 ng/ml 99 ng/ml 0 〜 25ng/ml CA199 27.33 u/ml 》0·99 u/ml 0 〜35u/ml CEA 6.65 ng/ml 5.61 Ng/ml 0 〜5ng/ml HBV-DNA 5.37 x 105 copy/ml L.51 x 105 copy/ml 0 〜1·0 x 103 copy/ml Table 3·. — 77-year-old man diagnosed with highly differentiated hepatocellular carcinoma Radiofrequency ablation combined with autologous NK therapy to track T lymphocyte subsets detection 8/25 9/17 10/20 11/23 Reference value CD3+% 58.1 51.5 52.4 54.0 67·0+"7·9 CD3+CD4+% 42.4 42.6 43.6 42.1 33.5+/-6.9 CD3+CD8+% 14.1 13.3 12.7 13.6 29.7+/-6.1 CD4+/CD8+ 3.01 3.20 3.43 3.10 1.31+/-0.5 CD19+% 8.68 7.42 7.74 7.46 9.8+M.3 CD56+% 18.1 27.8 28.6 43.8 18.7 +/-6.3 CD3+CD56+% 3.62 2.58 3.24 2.68 3.0+/-2.0 27 18734twf.doc/006 200831670 Example 10: Liver cancer using D-CIK combined with nk treatment A 43-year-old male, due to HbsAg (+), AFP continued Elevated, diagnosed as primary liver cancer at the time of physical examination in December 2002, performed liver TACE 1 time, liver RFA 2 times and 6 times D-CIK cell reinfusion, performed NK cell reinfusion 3 times, patient Now generally, HbsAg (-), AFP 2.02 ng / ml. Table 4: Radiofrequency ablation performed in a 43-year-old man diagnosed with primary liver cancer _ Combined with D-CIK and autologous NK treatment _ Cycle treatment cell count December 2002 TACE 1 2003-0144 Liver RFA surgery 2003 -0347 Liver RFA surgery 2003-04-01)--CIK cell reinfusion cell count 1·2χ101() 2003-04-15 )-CIK cell reinfusion cell count l.lxlO10 2003-05-01 P-CIK cells Retransmission cell count 1·21χ101() 2003-05-14 )--CIK cell reinfusion cell count 1.22χ101() 2003-06-01 )--CIK cell reinfusion cell count 1.·3χ101() 2003-06 -15)-CIK cell reinfusion cell count 1.21xl01G 2004-09-29 NK cell reinfusion cell count 3. 5χ109 2004-10-12 NK cell reinfusion cell count 1.72χ109 2004-11-01 NK cells Return cell count 4·78χ109 28 200831670 .-2 18734twf.doc/006 Combined D-CIK and autologous NK treatment to track T漱 team, 〆肖融术'~2004-6-30 2004-9-9 2004- 10-11

Table 5: - 43 years old diagnosed as a primary liver cancer male performing _, > Example 11: straight victory cancer liver cancer (Rectum cancer with liver gtastasis) using D_CIK combination # NK treatment 瘙 71-year-old female was diagnosed For patients with rectal cancer liver metastases. In May 2003, the patient was admitted to the hospital for examination and performed a colonoscopy (pathology · tubular adenocarcinoma grade) and abdominal CT examination for rectal cancer liver metastasis. On May 12, 2003, the rectal Dixon, s surgery, radiofrequency ablation of liver metastases were performed twice, D-CIK fine sputum was returned 6 times, and NK cells were returned 4 times. The patient's vital signs are stable. Without any discomfort. 29 v-2 18734twf.doc/006 200831670 Table 6: — 71-year-old woman diagnosed with rectal cancer Liver metastasis after radiofrequency ablation combined with D-CIK and autologous NK treatment. Sputum treatment cell count 2003-05-12 rectal Dixon, s surgery 2003-06-28 Liver metastasis radiofrequency ablation 2003-07-04 Liver metastasis radiofrequency ablation 2003-08-14 D-CIK cells return 6 times 3.2xl09 2003-09-16 Line D -CIK cells were transfused 6 times 5.6xl09 2003-11-18 D-CIK cells were transfused 6 times 5.8xl09 2004-01-28 D-CIK cells were returned 6 times 6.2x10" 2004-04-28 Line D- CIK cells were transfused 6 times 6.6xl09 2004-06-8 D-CIK cells were transfused 6 times 1.2x10" 2004-11-17 NK cell reinfusion cell count 3.27χ109 2004-11-30 NK cell reinfusion Cell count 3.28χ109 2004-12-08 NK cell reinfusion cell count 3.85χ109 2004-12-17 NK cell reinfusion cell count 4·01χ109 30 200831670 v-2 18734twf.doc/006 Table 7: 71-year-old female Diagnosed as rectal cancer liver metastasis after shooting 2004-11-02 melon body N] 2004A2 ^ 〇 T tc treatment after swelling 2004-12-15 6.85ng / ml ginseng a ^ 〇 ~ AFP 6.02ng / ml 6.14ng /ml CEA 4 .52 ng/ml 4.10 ng/ml 3.96 ng/ml 〇~5ng/mi CA199 12.07 u/mi 11.57u/ml 12.24u/mi 〇^35u/ml__ Table 8: — 71-year-old woman diagnosed with rectal cancer liver metastasis Radiofrequency ablation combined with CIK! Changes in tau lymphocyte subsets after autologous NK treatment 2004-11-02 2004-12-01 2004-12-25 Reference value CD3+% 83.0 82.2 82.4 67.0+/-7.y CD3+ CD4+ % 42.4 47.9 46.5 33.5+/-6.y CD3+ CD8+% 27.5 24.1 23.3 29.7+/-6.1 CD4+/CD8+ 1.54 1.98 2.0 1.31+/-0.5 CD 19+% 4.36 2.98 5.18 9.8+/-4.3 CD56+% 8.71 9.3 9.52 18.7+/-6.3 CD3+CD56+% 4.25 7.97 5.74 3.0+/-2.0

Example 12: Clinical case: Colon cancer with liver metastasis using CIK combined with NK - a female patient with liver metastases after colon cancer surgery. The patient underwent "right radical resection of right colon cancer" on 2002-11-8, and the pathological diagnosis was "differentiated adenocarcinoma". After regular chemotherapy, the liver metastasis was found by CT-PET in July 2003. Liver TACE was performed. Times, 31 200831670 ~july-2 18734twf.doc/006 Liver RFA was performed twice, CIK cells were transfused through the hepatic artery for 4 cycles, and NK cells were returned to 9 cycles. Today's patients with gastrointestinal and health conditions are available. Table 9: A case of postoperative liver metastases in women with colon cancer undergoing radiofrequency ablation and CIK combined with autologous NK therapy to record the cell count in the treatment phase 2002-11-08 Radical surgery for semi-colon cancer 2003-11-11 to 2004-09 -16 liver TACE 2 times, liver RFA 2 times, transhepatic arterial CIK cells return 4 times 2004-09-29 NK cell reinfusion cell count 1·2χ109 2004-10-12 NK cell back Transfusion cell count 1.32 X 109 2004-10-22 NK cell reinfusion cell count 1.2xl09 2004-11-01 NK cell reinfusion cell count 4·4χ109 2004-11-10 NK cell reinfusion cell count 4.89 X 109 2004-11-17 NK cell reinfusion cell count 4.78 X 109 2004-11-30 NK cell reinfusion cell count 4.31 X 109 2004-12-08 NK cell reinfusion cell count 4.06 X 109 2004-12-17 NK cell reinfusion cell count 4.32 X 109 32 200831670: 18734twf.doc/006 Table 1 0: - Women with liver metastases after colon cancer undergoing radiofrequency ablation and CIK combined with autologous NK therapy to recover more tumor changes 2004-9-16 2004-10-14 2004-11-11 2〇〇m2>4 Reference value CA199 93.1 u/ml 156.4 u/ml 2 .96 u/ml 321.5 u/ml 0 〜5u/ml CEA 9.15 ng/ml 7.69 ng/ml 5· 19 ng/ml 6.21 ng/ml 0~35u/ml Example: Lung neck shame using D_CIK to treat 68-year-old male Patient 2005.9 because of the bilateral soles of the feet "foot gas, but the side of the side ^ bulging and pain, survey 5 · 9 29 check, the proposed "foot and infection,, = Qi Feng Duo Xin" improved after anti-inflammatory treatment, because of cough during hospitalization嗷 进行 月 月 和 和 和 和 和 和 和 和 和 和 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 月 左 左 左 左 左 左 左Lymphocyte

33 蠡200831670 .-2 18734twf.doc/006 There is a mediastinal and hilar lymphadenopathy, and intrapulmonary tumor biopsy confirmed adenocarcinoma n ~ Π grade. After the discussion of the expert group in 2005·10·13, it was considered that the comprehensive treatment based on radiotherapy should be followed by two-way induction chemotherapy, and whether the recovery of lung function was determined by surgery. Starting from 2005.10.18 Tax〇i (i〇〇mg ivcj dayl and day8) +Carb〇Platin (450mg ivd dayl). Three-dimensional conformal radiotherapy was started on the third day after chemotherapy, and Tax1 (9〇mg) single-agent sensitization was started every week (starting at 11.21). Biotherapy (6 times total D_aK return), lung function improved during the process.

- a ^ _ metastatic lung adenocarcinoma using D-CIK Pod • Dan / 71-year-old male 'patients in the medical examination in Hong Kong in 2004.10 found the right upper Ξ Γ Γ 11 11 treatment, Yu won 11.18 in the tracheal numbness into the rib In addition to the +^ upper segment of the wedge-shaped resection of the knot if the right upper lung lobe can be seen in the adenocarcinoma infiltration, and all the leaching after lying well after gout and pulmonary interstitial pneumonia, after treatment

However, no obvious abnormalities were found with one chest X-ray examination. Section, the squama has a heavy 're- chest ct found that the right upper lung has a knot to seek right for metastatic adenocarcinoma. Patients are reluctant to receive chemotherapy, as long as they are included in the 1251 / particle implantation of the lung car lesion. Thereafter, the continuation of the position is used to expose the above, but it is not the spirit of the second and the second: the enthusiasm of the artist can make some changes and runes without leaving the invention. , , and ########################################################## BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1-1 is a diagram showing human ovarian r cancer cells before purification according to Example 1 of the present invention. < Figure 1-2 is a diagram showing purified ovarian cancer cells according to Example 1 of the present invention. 2-1 is a diagram showing human kidney cancer tissue cells before purification according to Example 2 of the present invention. Fig. 2-2 is a diagram showing the purified renal cell carcinoma of the second embodiment of the present invention. 3-1 is a diagram showing human lung cancer tissue cells before purification according to Example 3 of the present invention. Fig. 3-2 is a view showing the purified lung cancer cells of the third embodiment of the present invention. 4-1 is a diagram showing human breast I cancer tissue cells before purification according to Example 4 of the present invention. 4-2 is a diagram showing purified breast cancer cells according to Example 4 of the present invention. Figure 5-1 is a diagram showing human nasopharyngeal carcinoma tissue cells before purification according to Example 5 of the present invention. Figure 5-2 shows purified nasopharyngeal carcinoma cells according to Example 5 of the present invention. Figure 6-1 is a diagram showing the human cerebellum medulloblastoma tissue cells before purification according to Example 6 of the present invention. 35 18734twfdoc/006 200831670 FIG. 6-2 illustrates purified human cerebellar blastoma cells according to Example 6 of the present invention. 7-1 is a diagram showing human colon cancer cells before purification according to Example 7 of the present invention. Fig. 7-2 is a diagram showing the purified human colon cancer cells of the seventh embodiment of the present invention. Fig. 8-1 is a view showing human liver cancer cells before purification according to Example 8 of the present invention. Fig. 8-2 is a view showing purified human colon cancer cells according to Example 8 of the present invention. Figure 9 is a graph showing the CD3+CD4+% variable curve of a 77-year-old male liver cancer patient using CIK combined with NK treatment. Figure 10 is a graph showing the CD4+/CD8+% variable curve of a 77-year-old male liver cancer patient using a CIK combined with NK. Figure 11 is a diagram showing the ninth embodiment of the present invention. A 77-year-old man with liver cancer is treated with CIK combined with NK: CD56+% variable curve. Fig. 12 is a view showing one of the nineth embodiment of the present invention. A 77-year-old man with liver cancer. A man with CIK combined with NK treatment performed a liver CT scan on 8/19 and found several wounds on the right side. Figure 13 is a view showing a ninety-seven-year-old man with liver cancer using hepatocellular carcinoma using CIK combined with NK treatment according to Example 9 of the present invention. (a) 8A/26 RFA liver damage (b) 9/21 RFA liver recovery The phenomenon. Figure 14 is a diagram showing the use of CIK combined with NK in a 77-year-old man with liver cancer in a patient of 77 years old in the present invention: no liver damage detected in the liver at 11/23 to 36 18734 twf.doc/006 200831670 CT/PET scan of the liver . Figure 15 is a view showing a nine-year-old male with a liver cancer using ακ combined with NK treatment according to the embodiment of the present invention. In the two liver cancer treatments and five autologous NK cell reinfusions, the CT/PET smelting debt is not detected. damage. Figure 16 is a graph showing the variation of CD3+CD4+% in a 43-year-old male with hepatocellular carcinoma undergoing radiofrequency ablation combined with ακ and self-existing therapy. K rule Figure 17 shows a tenth embodiment of the present invention: a primary liver cancer hybrid radiofrequency ablation combined with CIΚ and self = for tracking CD4+/CD8+ variable curve after treatment. Fig. 18 is a diagram showing the tenth of the present invention: a 43-year-old male with primary liver cancer undergoing a radiation (four) fusion combined with ακ static as a curve for tracking CD56+%. Figure 19 is a diagram showing the results of a radiofrequency ablation combined with cik and primary therapy for a 43-year-old man with hepatocellular carcinoma in the present invention. The results of CT tracing were used after liver TACE treatment. Fig. 20 is a view showing the results of a radiofrequency ablation combined with ακ and autologous body = original therapy in a 43-year-old male with hepatocellular carcinoma after heal FRA treatment. Figure 21 shows that one of the tenth embodiment of the present invention is 杞年玲=hepatocarcinoma male _,; Xiao Rong _+ ακ map... prepared for dry two-two field inspection results without recurrence. Radiofrequency ablation combined with CIK in men with hepatocellular carcinoma and == 37 200831670 -2 18734twf.doc/006

After treatment: No results were observed from CT/PET scans on 11/18/04. Fig. 23 is a view showing that the liver cancer has not recurred after the TACE combined with liver RFA is performed in a 43-year-old male patient with primary liver cancer who has been diagnosed as a primary liver cancer. The patient was negative for AFp and negative for HBsAg. Figure 24 is a diagram showing that a 71-year-old female is a rectal cancer transplanting liver cancer (Rectum cancer with liver* _ metastasis) performing rectal Dixon, 3 surgery, and liver metastases radiofrequency ablation twice. The CIK cells were transfused 6 times and the NK cells were retrieved 4 times and the CD3+CD4+% variable curve was followed. 25 is a diagram showing a 71-year-old woman diagnosed with rectal cancer with liver metastasis performing rectal Dixon, s surgery, liver metastases radiofrequency ablation twice, CIK cells are returned. The CD4+/CD8+ variable curve was followed after 6 transfusions and 4 rounds of NK cell reincarnation. Lutu 26 depicts a 71-year-old woman diagnosed with rectal cancer with liver metastasis performing rectal Dixon, s surgery, liver metastases radiofrequency ablation twice, CIK cells. The CD56+% variable curve was traced after 6 times of return and 4 times of NK cell reinfusion. Figure 27 is a diagram showing that a 71-year-old female is diagnosed with rectal cancer with liver metastasis and performs rectal Dixon, s surgery, liver metastases radiofrequency ablation twice, CIK cells. 6 times of reinfusion and 4 times of NK cell reinfusion 38 20083 1670.2 18734twf.doc/006 The results were tracked by liver CT at 2003-5-30. 28 is a diagram showing that a 71-year-old female is diagnosed with rectal cancer with liver metastasis performing rectal Dixon, s surgery, and liver metastases radiofrequency ablation twice 'CIK cell gyrus. After 6 transfusions and 4 NK cell retransmissions, the results were followed by liver RFA at 2003-6-28. 29 shows a 71-year-old female diagnosed with rectal cancer with liver (metastasis) performing rectal Dixon, s surgery, liver metastases radiofrequency ablation twice, CIK cells, according to an eleventh embodiment of the present invention. After 6 times of reinfusion and 4 times of NK cell reinfusion, the liver RFA was followed at 2003-74. Figure 3 is not a case in which a 71-year-old woman is diagnosed with rectal cancer with liver metastasis (RectUm cancer with liver metastasis) performing rectal Dixon, s surgery, liver metastases radiofrequency ablation twice, CIK After the cells were returned 6 times and the NK cells were reinfused 4 times, the liver CT scan showed no recurrence of the cancer. The cancer marker has returned to the normal index. Figure 31 is a graph showing the tracking of CD3+CD4+% in a female patient with liver metastasis after colon cancer after receiving radiofrequency ablation and CIK combined with autologous NK therapy. 32 is a graph showing the tracking of CD4+/CD8+% in a female patient with liver metastases after undergoing radiofrequency ablation and CIK combined with autologous NK treatment. 33 is a graph showing the tracking of CD45+% of a female patient with liver metastasis after colon cancer after radiofrequency ablation and CIK combined with NK treatment. Figure 34 is a view showing the twelfth to the sequel to the liver of the female patient with liver metastasis after radiofrequency ablation and Clg 1 intestinal cancer NK treatment in 2003-12-20.

RFA Fig. 35 is a view showing the CT scan of the liver in 2004-1_17 after the insertion of the sputum sputum node and the postoperative liver metastasis in a female patient undergoing radiofrequency ablation and Ci=j intestinal cancer NK treatment. Figure 36 is a diagram showing the embodiment of the present invention. The human liver MRI of the u/04 showed no recurrence after radiofrequency ablation and combined with autologous NK treatment in women with liver metastasis after sputum colon cancer operation. 0 [Main component symbol description benefits

Claims (1)

200831670 n, w2 18734twf.doc/006 X. Patent scope: 1. A method for isolation and purification of human tumor cells, including (a) pre-purification treatment of tumor cells; (b) separation and purification of tumor cells. 2. The separation and purification technique of human tumor cells according to claim 1, wherein the pre-purification treatment process of the tumor cells further comprises (1) removing visible non-tumor tissue and tumor necrotic tissue; (ii) using a little RPMI-containing 1640 medium (serum free) wets the tumor tissue and cuts it as quickly as possible; (iii) places the cut tumor tissue in the first centrifuge tube and adds the collagenase solution; (iv) at 37° C bath in the water bath for 1 hour; (v) 1 hour after the removal of the centrifuge tube is diluted with serum-free RPMI-1640 solution, repeated pipetting to take the cell suspension; (vi) Centrifugal speed between 1000-1600 After 5-16 minutes of transfer; (vii) Remove the supernatant and mix the centrifuged cells with an appropriate amount of RPMM640 medium containing 10% fetal bovine serum or human AB serum and 1% double antibody. 3. The separation and purification technology of human tumor cells as described in claim 1 of the patent scope, wherein the step of separating and purifying the tumor cells further comprises: (1) taking an appropriate amount of inactivated fetal bovine serum or human AB serum, and the centrifugation speed is between 2500 and 3600 18-30 minutes; (ii) taking the supernatant in a second centrifuge tube; (iii) taking the human serum or fetal bovine serum of the step (1) in a third centrifuge tube; (iv) the appropriate amount in the first a mixed tumor group of the centrifuge tube. The woven cell suspension is added to the third centrifuge tube through the tube wall, allowed to stand for a suitable time, and then 6 ml of serum is aspirated from the bottom of the third centrifuge tube to be placed in the 'fourth centrifuge tube. (v) the third centrifuge tube is allowed to stand for a suitable time, and then 6 ml of serum is aspirated from the bottom of the third tube and placed in a fifth centrifuge tube; (vi) 41 18734 twf.doc/006 200831670 v^jljl^-2 The bottom of the blanket multi-tube is aspirated 6ml and then the third tube is allowed to stand properly _, from the # μ, the fifth serum is placed in the sixth centrifuge tube; (10) the 诵-precision to the centrifuge tube, and the sixth centrifuge tube is transferred in the centrifuge ' ^ ^ ^ , . I purified tumor cells, for 5-16 minutes, the resulting precipitated cells are ▲ Q Qing and i/G double machine (viii) take appropriate amount of 10% fetal bovine serum or human A3 π ^: the cells of the temple sub-set 6 well of 1640 medium mix the fourth centrifuge tube ice aw a 1 work such as ^ + cut The cell culture meal is in a hole in the plate for 3_4 days; (ix) the purified _%, \, , and the six cells of the centrifuge can be seen to divide the tumor cells; (X) the fifth centrifugation @a to *; A 0# tumor cells, repeated sputum cells in addition to tumor cells also contain a small amount of non-reporting process, can obtain more purified tumor fine glands.. 7 ^ Λ, yV ^ ^ to describe human tumor cells 4 · as patent application The first range of the range ^ @ & μ & μ _ ^ is therefore applied as epithelial swelling and purification technology, of which the best tumors are sputum, lung cancer cells, ceremonial adenoma, selected from ovarian cancer cells, kidney cancer cells Tumor cells, cancer cells, nasopharyngeal carcinoma cells, cerebellar medulla, sarcophagus, guangyi, tl 挢 cancer, chyme cancer, lung cells, liver cancer cells, gliomas, sputum cancer cells, etc. The group of people formed. ,, 5, such as the scope of the patent application, the third 顼 斤 述 : : : : 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤 瘤, the ratio of sputum clear is the cell suspension of the tumor tissue, the phase #人0·8-1·5. r , φ ^ ^ & 沭 human tumor cell fraction 6 · 申 26 patent scope 3 顼 51,, from the purification technology, where the appropriate time of steps (iv), (v), (vi) is between 4 -8 minutes. 7. The application of an isolation and purification technique for human tumor cells, which is more suitable for vaccines, anti-tumor drugs, living cell therapy, gene therapy 42 18734 tt/doc/006 200831670 8 · The application of the separation and purification technology of human tumor cells as described in the 7th patent scope of Shenqing, wherein the isolation and purification technology of human tumor cells further includes (a) pre-purification treatment of tumor cells; (b) separation of tumor cells. purification. 9. The application of the separation and purification technique of human tumor cells as described in claim 8 of the patent application, wherein the pre-purification treatment steps of the tumor cells further comprise (1) removing visible non-tumor tissue and tumor necrotic tissue; Wetting the tumor tissue with RJPMI-1640 medium (no ash clear) and cutting it as soon as possible; (iii) placing the cut tumor tissue in the first centrifuge tube and adding the collagenase solution; (iv) The bath was incubated in a 37 ° C water bath for 1 hour; (v) the centrifuge tube taken out after 1 hour was diluted twice with serum-free RPMI-1640 solution, and the cell suspension was taken after repeated blows; (vi) Centrifugal speed was between 1000-1600 after 5-16 minutes; (vii) Removal of supernatant 'The appropriate amount of PFMI-1640 medium containing 10% fetal bovine serum or human AB clear and 1% double antibody was mixed to mix the centrifuged cells. 10. The application of the separation and purification technology of human tumor cells according to item 8 of the patent application scope, wherein the step of separating and purifying the tumor cells further comprises: (1) taking an appropriate amount of inactivated fetal bovine serum or human AB serum at a centrifugal speed of 25 〇. 0_3600 for 18-30 minutes: (9) take the supernatant into the second centrifuge tube; (Hi) take the step (丨) of human serum or fetal bovine serum in a suitable amount in the third centrifuge tube; (iv) the appropriate amount The cell suspension of the mixed tumor tissue of the first centrifuge tube is added to the second miscellaneous tube through the tube wall, and is allowed to stand for a suitable time, and then placed from the third centrifuge tube: 2:2, 43,78034 twfdoc/006 200831670 (4) the third centrifuge tube is allowed to stand for a suitable time, and then 6 ml of serum is aspirated from the bottom of the third tube and placed in the fifth centrifuge tube; (vi) the third tube is allowed to stand still. Time, 6ml of serum is aspirated from the bottom of the third tube and placed in the sixth centrifuge tube; (vii) the fourth centrifuge tube, the fifth centrifuge tube, and the sixth centrifuge tube are rotated at a centrifugal speed of 1000-1600 to 5 - After 16 minutes, the resulting precipitated cells were purified tumor cells; (viii) Mix 10% fetal bovine serum or human AB serum and 1% double-antibody in 1640 medium to mix the cells precipitated in the fourth centrifuge tube and place in one well of the 6-well plate; (ix) culture the purified tumor cells 3-4 The tumor cell division can be seen in the day; (X) the cells of the fifth centrifuge tube and the sixth centrifuge tube contain a small amount of non-tumor cells in addition to the tumor cells, and the above process is repeated to obtain more purified tumor cells. 11. The application of the separation and purification technology of human tumor cells according to claim 8 of the patent application, wherein the preferred tumor cells are epithelial tumors selected from the group consisting of ovarian cancer cells, renal cancer cells, lung cancer cells, breast cancer cells, and nose. A group consisting of pharyngeal cancer cells, cerebellar medulloblastoma cells, colon cancer cells, liver cancer cells, gliomas, colon cancer, esophageal cancer, lung cancer cells, and the like. 12. The use of the separation and purification technique of human tumor cells according to claim 10, wherein the proportion of step (iv) to the cell suspension of the mixed tumor tissue of the third centrifuge tube is proportional to the ratio of human serum Between 0.8-1.5. 13. The use of the separation and purification technique of human tumor cells as described in claim 10, wherein the appropriate time of steps (iv), (v), (vi) is between 4 and 8 minutes. 44 200831670~2 18734twf.doc/006 1 4. A cell activin (Koyo) derived from the isolation and purification technique of human tumor cells, wherein the cell activin (Koyo) activates cells such as DC, CIK, NK and the like. 15. The cell activin (Koyo) derived from the isolation and purification technique of human tumor cells according to claim 14, wherein the cells of the initial DC, CIK, NK, etc. are selected from cord blood or peripheral blood of the subject. . 16. The cell activin (Koyo) derived from the isolation and purification technique of human tumor cells as described in claim 15, wherein activated DC, CIK, NK and the like are further prepared into a vaccine to improve infection and cancer. , transferred cancer, autoimmune and other diseases. 17. The cytokine (Koyo) derived from the isolation and purification technique of human tumor cells according to claim 16, wherein the prepared vaccine is preferably an epithelial tumor selected from the group consisting of ovarian cancer cells and renal cancer cells. , lung cancer cells, breast cancer cells, nasopharyngeal carcinoma cells, cerebellar Zhao cell tumor cells, colon cancer cells, liver cancer cells, nerve glioblastoma, colorectal cancer, esophageal cancer, lung cancer cells and the like. 45