Drug screening method and device

Abstract

The invention relates to the field of in-vitro drug screening, in particular to application of an implantation device, cell sorting magnetic beads and primary tumor cells in preparation of a kit for detecting drug sensitivity of the primary tumor cells to antitumor drugs, and the primary tumor cells are from in-vitro tumor tissue samples. The in-vitro tumor tissue sample is obtained from a tumor patient, or is obtained from a PDX model constructed by utilizing tumor cells of the tumor patient, and is separated tumor cells subjected to digestion and cell sorting.

Description

[0001] This application is a divisional application of the patent application with application number 201780003121.3, international application date October 23, 2017, priority application number 201610918458.4, priority date October 21, 2016, entry into the Chinese national phase date March 13, 2018, publication date July 31, 2018, invention name “A drug screening method and device thereof”, and applicant Shanghai Lidi Biotechnology Co., Ltd. Technical Field

[0002] The invention relates to the field of in vitro drug screening, and in particular to a method and a device for rapid drug sensitivity detection of anti-tumor drugs. Background Art

[0003] At present, personalized precision medicine is increasingly being used in clinical practice, especially in the field of tumor treatment. Chemotherapy is currently a major treatment for patients with advanced metastatic cancer, but chemotherapy drugs lack tumor cell specificity. While killing tumor cells, they also kill a large number of bone marrow and other proliferating normal cells, with serious adverse reactions. At the same time, traditional chemotherapy methods are prone to drug resistance after multiple chemotherapy sessions, which will affect the effect of chemotherapy. Therefore, the demand for personalized precision medicine for patients is becoming more and more urgent. On the one hand, personalized precision medicine improves the accuracy of treatment, and on the other hand, it can effectively reduce the randomness and blindness of patients' medication, thereby reducing the harm of patients' medication. In recent years, a patient-derived xenograft model (PDX model) has been widely used in precision medicine. Many large clinical medical centers in North America have established PDX models to conduct preclinical drug development and drug screening and guide personalized precision medication for cancer patients.

[0004] The PDX model better maintains the biological characteristics of the patient's primary tumor. This model is a tumor model established by directly transplanting fresh tumor tissue from the patient into immunodeficient mice (commonly nude mice or SCID mice). This type of model maintains genetic characteristics and tumor heterogeneity similar to those of the patient. At present, the main difficulties in using the PDX model for personalized precision medicine are the long model construction cycle and drug sensitivity testing, as well as the low transplantation tumor rate of the PDX model. For example, primary cell transplantation takes 2 to 3 months, and the subsequent drug sensitivity test takes 3 to 4 months. In addition, the operation technology requirements of the PDX model are very high, and the specimens need to be closely coordinated by surgeons, histologists and researchers after being obtained from the operating room. Therefore, improving the anti-tumor drug sensitivity detection method of the PDX model is an urgent problem to be solved in the field of precision medicine. Summary of the invention

[0005] In order to overcome the technical problems existing in the above-mentioned traditional PDX model drug sensitivity testing, the inventors developed a method for rapidly and accurately detecting the drug sensitivity of anti-tumor drugs.

[0006] In one aspect of the present invention, a method for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs is provided, comprising the following steps:

[0007] (1) Placing primary tumor cells in an implant device;

[0008] (2) implanting an implant device containing the primary tumor cells into a test animal;

[0009] (3) administering the drug to be tested to the test animal;

[0010] (4) Detect the sensitivity of tumor cells to the drug to be tested.

[0011] In one embodiment, the primary tumor cells are from an ex vivo tumor tissue sample. The ex vivo tumor sample can be obtained from a tumor patient, or from a PDX model constructed using tumor cells from a tumor patient.

[0012] In one embodiment, the subject animal is a mouse, preferably a nude mouse.

[0013] In one embodiment, the detection of the sensitivity of tumor cells to the test drug is performed in vitro.

[0014] In one embodiment, the test drug is administered to the test animal orally or parenterally.

[0015] In one embodiment, the primary tumor cells are isolated tumor cells that have been digested and sorted.

[0016] In one embodiment, the detection of the sensitivity of tumor cells to the test drug is performed 5-14 days, preferably 5-7 days, after the test drug is administered to the test animal.

[0017] In one embodiment, the implant device is preferably a tubular device with a molecular weight cutoff of about 500,000 Daltons, preferably a modified polyvinylidene fluoride tube, and its inner diameter is preferably about 1-2 mm.

[0018] Another aspect of the present invention provides a kit for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs, which comprises an implant device, wherein the implant device is a tubular device with a molecular weight cutoff of about 500,000 Daltons, preferably a modified polyvinylidene fluoride tube, and its inner diameter is preferably about 1-2 mm. In one embodiment, the kit is used to implement the method of detecting the drug sensitivity of primary tumor cells to anti-tumor drugs according to the present invention.

[0019] Another aspect of the present invention provides use of the kit of the present invention in detecting the drug sensitivity of primary tumor cells to anti-tumor drugs and in rapidly screening anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0021] Figure 1 It is a schematic diagram of the process of the present invention;

[0022] Figure 2 This is a growth curve of primary gastric cancer tumor cells derived from patients after administration of the anti-tumor drug S-1 in the traditional mouse PDX model GAPF155.

[0023] Figure 3 This is a data diagram of the efficacy of the anti-tumor drug S-1 (S-1) on gastric cancer tumor cells in the ex vivo tumor tissue from the mouse PDX model GAPF155 according to the drug sensitivity detection method of the present invention;

[0024] Figure 4 This is a growth curve of primary gastric cancer tumor cells derived from patients after administration of the anti-tumor drug S-1 in the mouse PDX model GAPF157.

[0025] Figure 5 This is a data diagram of the efficacy of the anti-tumor drug S-1 (S-1) on gastric cancer tumor cells in the ex vivo tumor tissue from the mouse PDX model GAPF157 according to the drug sensitivity detection method of the present invention;

[0026] Figure 6 This is a growth curve of primary gastric cancer tumor cells derived from patients after administration of the anti-tumor drug S-1 in the mouse PDX model GAPF161.

[0027] Figure 7 This is a data diagram of the efficacy of the anti-tumor drug S-1 (S-1) on gastric cancer tumor cells in the ex vivo tumor tissue from the mouse PDX model GAPF161 according to the drug sensitivity detection method of the present invention;

[0028] Figure 8 The results of drug sensitivity testing of lung adenocarcinoma primary tumor cells from patients using the method and kit of the present invention;

[0029] Fig. 9 The present invention is a result of drug sensitivity testing of primary tumor cells of duodenal cancer from patients using the method and kit of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention relates to a method for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs, which comprises the following steps:

[0032] (1) Placing primary tumor cells in an implant device;

[0033] (2) implanting an implant device containing the primary tumor cells into a test animal;

[0034] (3) administering the drug to be tested to the test animal;

[0035] (4) Detect the sensitivity of tumor cells to the drug to be tested.

[0036] Primary tumor cells herein refer to tumor cells isolated from ex vivo tumor tissue. According to one embodiment, the primary tumor cells are isolated from tumor tissue samples of tumor patients, including but not limited to clinically resected tumor tissue, tumor tissue biopsy samples, etc. According to another embodiment, the primary tumor cells are isolated from a PDX mouse model, which contains tumor cells from patient tumor tissue.

[0037] The tumor cells can be derived from tumor tissues of various tumor types, including but not limited to tumors located in the following parts: digestive system (e.g., stomach, intestine, duodenum, colon, pancreas, bile duct, anal canal, etc.), breast, lung, liver, endocrine glands (e.g., adrenal glands, parathyroid glands, pituitary gland, testicles, ovaries, thymus, thyroid gland), urinary and reproductive systems (e.g., kidneys, bladder, ovaries, testicles, prostate, etc.), skeletal muscle system (e.g., bone, smooth muscle, striated muscle, etc.), nervous system (e.g., brain), skin, head and neck, blood system, etc. For example, the tumor cells can be tumor cells derived from gastric cancer, duodenal cancer, and lung cancer. The tumor cells can be any type of tumor cells in the tumor at that part.

[0038] In one embodiment, the primary tumor cells of the present invention are isolated tumor cells that have been digested and sorted. In one embodiment, the digestion is performed by the following method: removing non-tumor tissue and necrotic tissue, cutting the tumor sample into small pieces, washing and collecting tissue pieces with HBSS, and digesting the tumor pieces with 1X collagenase at 37°C for 1-2 hours. In one embodiment, the sorting is performed by the following method: terminating the digestion with a 1:1 dilution of serum culture medium, collecting the cell suspension through a 70μm mesh; centrifuging at 1000rpm for 3 minutes to remove the supernatant, resuspending the cells with 10ml of PBS containing 1% FBS, washing, and adjusting the cell density to 1×10 8 / ml; 20μl / 10 7Add CD45 cell sorting magnetic beads and fibroblast sorting magnetic beads to the cell concentration and incubate at room temperature for 30 minutes; add PBS containing 1% FBS to wash the cells and resuspend them with 2ml PBS containing 1% FBS. Place the magnetic beads on the magnet and rinse them with PBS containing 1% FBS; add the cells to the rinsed magnetic column, wait for the liquid to flow out, wash the column twice with 3ml PBS containing 1% FBS, and collect the flowing liquid; centrifuge the collected liquid at 1000rpm for 3 minutes to remove the supernatant, resuspend the cells in cell culture medium, count, and adjust the cell density to 1-10×10 5 / ml.

[0039] Methods for constructing PDX mouse models are known in the art. For example, see "Melanoma patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions", Oncotarget, Vol.5, No.20, Berglind O. Einarsdottir et al., published on September 8, 2014, and "Personalizing Cancer Treatment in the Age of Global Genomic Analyses: PALB2 Gene Mutations and the Response to DNA Damaging Agents in Pancreatic Cancer", Molecular Cancer Therapies, first published on December 6, 2010; DOI: 10.1158 / 1535-7163.MCT-10-0893, Maria C. Villarroel et al., etc.

[0040] The drug to be tested of the present invention can be a known antitumor drug or a combination thereof, a new antitumor compound or a combination thereof, or a new combination of known antitumor drugs. In the method of the present invention, the drug to be tested can be used in the form of a solid, semisolid or liquid, and can be administered at a desired administration frequency as required.

[0041] In the method of the present invention, the drug to be tested can be administered to the test animal by oral administration, parenteral administration (such as by intravenous, intramuscular, subcutaneous injection or intravenous infusion), topical administration, inhalation administration, transdermal administration such as by skin patch, implant, suppository, etc. Those skilled in the art will select the appropriate administration route according to the needs.

[0042] In one embodiment of the present invention, the sensitivity of tumor cells to the drug to be tested is detected 5-14 days, preferably 5-7 days after the drug to be tested is administered to the test animal. The sensitivity of tumor cells to the drug to be tested in the present invention can be detected by in vitro drug sensitivity detection methods, including but not limited to ATP detection method, MTT method, BrdU labeling method, Ki67 staining method, etc.

[0043] Another aspect of the present invention relates to an implant device for use in the method of the present invention. In one embodiment, the implant device is a tubular implant device, preferably a molecule with a molecular weight cutoff of about 500,000 Daltons. In a preferred embodiment, the tubular implant device is a modified polyvinylidene fluoride tube, and the molecule with a molecular weight cutoff of about 500,000 Daltons, preferably the inner diameter of the modified polyvinylidene fluoride tube is about 1-2mm. In a preferred embodiment of the present invention, the implant device can be implanted subcutaneously in a test animal. Those skilled in the art will appreciate that suitable implantation methods known in the art can be selected as needed.

[0044] Another aspect of the present invention provides a kit comprising the implant device of the present invention. In one embodiment, the kit comprises a modified polyvinylidene fluoride tube having an inner diameter of 1-2 mm and a molecular weight cut-off of 500,000 Dalton molecules. In a preferred embodiment, the kit further comprises an insert sheet comprising instructions for use of the kit.

[0045] The method of the present invention enables primary tumor cells, especially digested and / or sorted separated tumor cells, to survive continuously in an implantation device implanted in an experimental animal (e.g., mouse), and with the aid of the animal as a nutrition provider, the primary tumor cells can grow in the in vivo environment of the test animal, and can quickly and efficiently obtain drug sensitivity test results by detecting the growth state, apoptosis state, and degree of differentiation of cells in vitro as needed after administering the drug to be tested. It has been verified that this method has a very high correlation with the drug sensitivity results obtained by traditional PDX. The present invention can combine in vivo and in vitro experimental techniques to perform rapid and efficient anti-tumor drug evaluation, greatly shortening the time required for the efficacy experiment of the traditional PDX model and saving the cost of clinical experiments. In short, the present invention has the advantages of being fast, easy to operate, low cost, strong repeatability, and easy to promote, especially realizing rapid and accurate detection of the sensitivity of tumor cells to anti-tumor drugs. DETAILED DESCRIPTION

[0046] The present invention discloses a drug screening method and device thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] In order to better illustrate the present invention, it is described below with reference to the accompanying drawings.

[0048] Experimental animals

[0049] Female Nu / Nu mice aged 4-5 weeks were ordered from a supplier (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) and housed in an SPF-level animal room. The animals were adaptively housed for at least three days before the start of the experiment.

[0050] Sample collection

[0051] Take freshly collected clinical tumor surgical samples or mouse PDX samples containing patient tumor cells and place them in shipping tubes, transport them on ice, and transport them to the laboratory as soon as possible.

[0052] Cell digestion and sorting

[0053] After removing non-tumor tissue and necrotic tissue in a biosafety cabinet, the tumor samples were cut into 1-3 cubic millimeter pieces, washed with HBSS and the tissue pieces were collected, centrifuged at 1000 rpm for 3 minutes to remove the supernatant, and the tumor pieces were digested with 1X collagenase (purchased from Gibco) at 37°C for 1-2 hours;

[0054] Digestion was terminated by diluting 1:1 with serum medium (purchased from Life Technologies) and the cell suspension was collected through a 70 μm mesh;

[0055] The supernatant was removed by centrifugation at 1000 rpm for 3 minutes, and the cells were resuspended and washed with 10 ml of PBS containing 1% fetal bovine serum (FBS). The cells were then resuspended in PBS containing 1% FBS, counted, and the cell density was adjusted to 1×10 8 / ml;

[0056] 20 μl / 10 7The cells were added with commercially available CD45 cell sorting magnetic beads (purchased from Miltenyi Biotec) and fibroblast sorting magnetic beads (purchased from Miltenyi Biotec) at the concentration of 10 μg / mL and incubated at room temperature for 30 min;

[0057] Wash the cells with PBS containing 1% FBS and resuspend them with 2 ml of PBS containing 1% FBS. Meanwhile, place the magnetic beads on the magnet and rinse them with PBS containing 1% FBS.

[0058] Add the cells to the rinsed magnetic column, wait until the liquid has drained away, wash the column twice with 3 ml of PBS containing 1% FBS, and collect the drained liquid;

[0059] The collected liquid was centrifuged at 1000 rpm for 3 minutes, the supernatant was removed, and the cell culture medium PC-1 (purchased from Lonza) was resuspended, counted, and the cell density was adjusted to 1-10×10 5 / ml.

[0060] Cell loading, inoculation and viability testing

[0061] Take out the implant device in the biosafety cabinet, i.e., a modified polyvinylidene fluoride tube with a diameter of 1 mm, which has been pre-treated by soaking, rinsing and autoclaving in ethanol. Cut each tube to a length of about 2 cm, rinse it repeatedly with PBS for 3-5 times, and blow off the liquid;

[0062] The tumor cell suspension was added into a modified polyvinylidene fluoride tube and sealed;

[0063] The modified polyvinylidene fluoride tube was inoculated into the subcutaneous tissue of the back of the experimental mice using a tumor puncture needle, and a drug-treated group and a control group were set up. The wounds were sealed with medical glue, and the mice were treated with drugs respectively;

[0064] 5-14 days after administration, the mice were sacrificed, and the modified polyvinylidene fluoride tubes were taken out to detect cell viability by quantitatively measuring ATP using the CellTiter-Glo luminescence method.

[0065] Data analysis

[0066] According to the activity value of the tumor cells in the control group, the activity proliferation percentage (t / c%) of the tumor cells in the drug administration group was calculated, and then the efficacy of the corresponding drug was evaluated.

[0067] Figure 1 The flowchart of the rapid efficacy detection method of anti-tumor drugs of the present invention is shown. First, an in vitro tumor tissue is obtained, then digested and sorted to obtain separated tumor cells, and the tumor cells are loaded into an implant device and inoculated subcutaneously into an animal (e.g., a mouse). After the drug to be tested is administered to the mouse, the implant device is removed 5-7 days later to perform a tumor cell viability test.

[0068] Figure 2-7 A comparison of the traditional PDX drug efficacy assay and the drug efficacy assay of the present invention is shown.

[0069] Example

[0070] Example 1

[0071] The mouse PDX model GAPF155 was obtained by implanting primary gastric cancer tumor cells from patients into mice. After 2 months of feeding, patient-derived gastric cancer cell samples were obtained from GAPF155. The sensitivity of gastric cancer cells in GAPF155 to S-1 was detected using the traditional PDX method and the method of the present invention. Figure 2 and Figure 3 ). The results all showed that the tumor cells were sensitive to S-1.

[0072] Example 2

[0073] The mouse PDX model GAPF157 was obtained by implanting primary gastric cancer tumor cells from patients into mice. After 2 months of feeding, patient-derived gastric cancer cell samples were obtained from GAPF157. The sensitivity of gastric cancer cells in GAPF157 to S-1 was detected using the traditional PDX method and the method of the present invention. Figure 4 and Figure 5 ). The results all showed that the tumor cells were sensitive to S-1.

[0074] Example 3

[0075] The mouse PDX model GAPF161 was obtained by implanting primary gastric cancer tumor cells from patients into mice. After 2 months of feeding, patient-derived gastric cancer cell samples were obtained from GAPF161. The sensitivity of gastric cancer cells in GAPF161 to S-1 was detected using the traditional PDX method and the method of the present invention. Figure 6 and Figure 7 ). The results all showed that the tumor cells were insensitive to S-1.

[0076] The efficacy data obtained by traditional PDX and the method of the present invention showed that S-1 has an inhibitory effect on tumor cells in the GAPF155 model ( Figure 2 and 3 ) and GAPF157 tumor cells in the model ( Figure 4 and 5 ) have good anti-tumor activity, and S-1 has a good anti-tumor activity against tumor cells in the GAPF161 model ( Figure 6 and 7 ) had no significant anti-tumor activity. The results obtained by the traditional PDX method and the method of the present invention were consistent.

[0077] Example 4

[0078] A male patient with lung adenocarcinoma (60 years old) was selected. Based on the clinical experience of lung adenocarcinoma, the doctor selected the medication regimen of Pemetrexe + Cisplatin. After 3 courses of treatment and 9 weeks, the patient's condition relapsed and metastasized. After the patient's informed consent, pleural effusion was collected from the patient with lung adenocarcinoma to obtain primary tumor cells.

[0079] The drug sensitivity detection method of the present invention was used to detect the sensitivity of primary lung adenocarcinoma tumor cells from patients to anti-tumor drugs. The experimental results showed that the paclitaxel + cisplatin regimen showed strong anti-tumor activity against the patient's tumor sample ( Figure 8 ).

[0080] Treatment results after adjustment:

[0081] The doctor changed the medication regimen and chose paclitaxel + cisplatin. After 7 weeks of medication, the patient's pleural effusion disappeared and his condition is now stable and he has been discharged from the hospital.

[0082] Example 5

[0083] A male patient (56 years old) with duodenal ampulla tumor was selected, and primary tumor tissue was obtained from the patient after informed consent.

[0084] Drug screening was performed according to the method of the present invention. The results showed that the patient responded well to gemcitabine for the treatment of pancreatic cancer ( Fig. 9 ).

[0085] Based on the above results, the doctor selected the gemcitabine + cisplatin treatment plan, and the patient's condition was brought under control.

[0086] Table 1 below shows that the inventors performed drug screening on various primary tumor cells from patients using the method of the present invention:

[0087] Table 1

[0088]

[0089]

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An implant device, characterized in that The implant device is a tubular device capable of retaining molecules with a molecular weight of about 500,000 Daltons, preferably a modified polyvinylidene fluoride tube, more preferably a modified polyvinylidene fluoride tube with an inner diameter of about 1-2 mm.

2. Use of the implant device as claimed in claim 1 in preparing a kit for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs and for rapidly screening anti-tumor drugs.

3. The use according to claim 2, characterized in that The method for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs comprises the following steps: Step (1), placing primary tumor cells in the implantation device as claimed in claim 1; Step (2), implanting the implant device containing the primary tumor cells into a test animal; Step (3), administering the drug to be tested to the test animal; Step (4), detecting the sensitivity of the primary tumor cells to the drug to be tested.

4. A kit for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs, characterized in that: It comprises the implant device as claimed in claim 1.

5. Use of the implant device as claimed in claim 1 or the kit as claimed in claim 4 to enable primary tumor cells to survive continuously in the implant device implanted in an experimental animal.

6. The use according to claim 5, characterized in that The implant device uses animals as nutrition providers to enable primary tumor cells to grow in the in vivo environment of the test animal, and after the test drug is administered, the growth state, apoptosis state and differentiation degree of the cells are detected in vitro as needed to quickly and efficiently obtain drug sensitivity test results.

7. The use according to claim 5, characterized in that The primary tumor cells are isolated tumor cells that have been digested and / or sorted.

8. The use according to claim 6, characterized in that The digestion is performed by the following method: removing non-tumor tissue and necrotic tissue, cutting the tumor sample into small pieces, washing with HBSS and collecting the tissue pieces, and digesting the tumor pieces with 1× collagenase at 37° C. for 1-2 hours; The sorting is carried out by the following method: Digestion was terminated by diluting the solution 1:1 with serum medium, and the cell suspension was collected through a 70 μm mesh; Centrifuge at 1000 rpm for 3 minutes, remove the supernatant, resuspend the cells in 10 ml of PBS containing 1% FBS, wash, and adjust the cell density to 1 × 10 8 / m1; 20μ1 / 10 7 The cells were added with CD45 cell sorting magnetic beads and fibroblast sorting magnetic beads at the concentration of 1:1 and incubated at room temperature for 30 min; Wash the cells by adding PBS containing 1% FBS and resuspend them in 2 ml of PBS containing 1% FBS; Place the magnetic beads on the magnet and rinse them with PBS containing 1% FBS; Add the cells to the rinsed magnetic column, wait until the liquid has drained away, wash the column twice with 3 ml of PBS containing 1% FBS, and collect the drained liquid; The collected liquid was centrifuged at 1000 rpm for 3 minutes to remove the supernatant, resuspended in cell culture medium, counted, and the cell density was adjusted to 1-10×10 5 / m1.

9. A method for constructing an animal model for detecting the drug sensitivity of primary tumor cells to anti-tumor drugs, characterized in that: The steps include: Step (1), placing primary tumor cells in the implantation device as claimed in claim 1; Step (2), implanting the implant device containing the primary tumor cells into the body of a test animal.

10. The construction method according to claim 9, characterized in that: The primary tumor cells are tumor cells separated by digestion and sorting; The digestion is performed by the following method: removing non-tumor tissue and necrotic tissue, cutting the tumor sample into small pieces, washing with HBSS and collecting the tissue pieces, and digesting the tumor pieces with 1× collagenase at 37° C. for 1-2 hours; The sorting is carried out by the following method: Digestion was terminated by diluting the solution 1:1 with serum medium, and the cell suspension was collected through a 70 μm mesh; Centrifuge at 1000 rpm for 3 minutes, remove the supernatant, resuspend the cells in 10 ml of PBS containing 1% FBS, wash, and adjust the cell density to 1 × 10 8 / m1; 20μ1 / 10 7 The cells were added with CD45 cell sorting magnetic beads and fibroblast sorting magnetic beads at the concentration of 1:1 and incubated at room temperature for 30 min; Wash the cells by adding PBS containing 1% FBS and resuspend them in 2 ml of PBS containing 1% FBS; Place the magnetic beads on the magnet and rinse them with PBS containing 1% FBS; Add the cells to the rinsed magnetic column, wait until the liquid has drained away, wash the column twice with 3 ml of PBS containing 1% FBS, and collect the drained liquid; The collected liquid was centrifuged at 1000 rpm for 3 minutes to remove the supernatant, resuspended in cell culture medium, counted, and the cell density was adjusted to 1-10×10 5 / m1.

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