Method for extracting and identifying human urine stem cells

Abstract

The invention discloses a method for extracting mesenchymal stem cells from urine. The method comprises the following steps: (1) sampling and inoculating urine; (2) amplification and passage of mesenchymal stem cells; (3) identifying the urine stem cells; and (4) a cryopreservation and resuscitation method of the urine stem cells. And performing biological phenotype identification according to the standard formulated by the International Cell Therapy Association to confirm the mesenchymal stem cells. The method is low in cost, simple and rapid, and the used culture solutions are all commercialized and are very convenient to purchase and use. The culture solution special for the stem cells is used in the first two weeks of the extraction period, after passage, when enough stem cells are obtained, the culture medium can be replaced with a common DMEM culture medium, and the manufacturing cost is greatly reduced. And secondly, the materials are simple, rapid and non-invasive, and the sample of the method for extracting the mesenchymal stem cells from urine comes from a patient, so that the problem of heterogeneity of cell sources is reduced. And a foundation is laid for stem cell treatment and gene editing treatment of genetic diseases and major difficult diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for extracting epithelial cells and mesenchymal stem cells from urine. Background Art

[0002] Human urine-derived stem cells (hUSCs) are a type of stem cells derived from urine. Compared with other stem cells, they have the advantages of non-invasive sampling, simple extraction and separation steps, relatively high telomerase activity and long telomere sequences, good multi-directional differentiation potential, high safety, and no tumorigenicity. Many experiments have shown that they are more easily induced to differentiate into various cells of the body, so they have greater potential for clinical treatment and a larger market.

[0003] The transplantation of human mesenchymal stem cells (MSCs) has been widely studied in the treatment of various diseases, including degenerative neuropathy, premature ovarian failure, repair of genitourinary system injury, spinal cord injury, brain injury, myocardial defect, vascular endothelial defect, cartilage defect, lung injury, etc., as well as cancer treatment, and has been clinically applied in multiple fields, and no tumor occurrence has been seen in these studies.

[0004] Through retrieval, the following several patent disclosure documents related to the present patent application are found: 1. A method for preparing primitive mesenchymal stem cells (CN101629165B). Disclosed is a method for preparing primitive mesenchymal stem cells, which uses fresh human umbilical cord, extracts Wharton's jelly, and is separated and cultured after collagenase digestion, and is suitable for cryopreservation. The invention also relates to the primitive mesenchymal stem cells prepared by the above method, which are identified as mesenchymal stem cells according to the standards formulated by the International Society for Cellular Therapy.

[0005] The steps of the above invention method are relatively numerous, the materials are not easily obtained and require strict ethical restrictions, and the clinical application is limited.

[0006] A method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placental amniotic tissue (CN105420179A). The invention relates to a method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placental amniotic tissue, and the steps are as follows: umbilical cord and placental amniotic tissue sampling and inoculation, mesenchymal stem cells and amniotic epithelial cells are amplified and passaged in a culture flask, the mesenchymal stem cells and amniotic epithelial cells are identified, when the cell culture reaches the third generation, after trypsin digestion, the purity of the cultured cells is identified, and it is verified that the cell purity reaches more than 90%, that is, it meets the standard, and a mixture of epithelial cells and mesenchymal stem cells can be obtained.

[0007] The above-mentioned inventive method has limitations such as the same large number of steps, relatively difficult sampling, and high costs, and the source determines the limitations in application.

[0008] Through technical comparison, this patent application is quite different from the above two patent disclosure documents. Summary of the Invention

[0009] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for extracting mesenchymal stem cells from urine at low cost, simply and quickly, and non-invasively. This method can be derived from the patient himself, reducing the problem of cell source heterogeneity. It lays a foundation for stem cell therapy, gene editing therapy for genetic inherited diseases and major intractable diseases.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for extracting mesenchymal stem cells from urine, the steps are as follows: (1) Urine collection and inoculation: ① High-temperature sterilize surgical instruments and large glassware, and at the same time disinfect the ultra-clean workbench with ultraviolet light; ② Prepare the culture medium: The composition of the stem cell basal medium is: DMEM / F12 / KSFM / REGM Bullet kit / REGM SingleQuot kit plus 10% fetal bovine serum of the total volume of the complete culture medium, 1% penicillin-streptomycin, and store at 4°C.

[0011] In the DMEM / F12 / KSFM / REGM Bullet kit / REGM SingleQuot kit, the volume ratio of DMEM:F12:KSFM:REGM Bullet kit:REGM SingleQuot kit is 1:1:1:1:1.

[0012] Strictly screen donors, excluding urinary system diseases, infectious diseases, metabolic diseases and malignant tumors; Collect about 200 ml of fresh midstream urine from healthy adults. The operation process strictly follows aseptic operation to avoid urine contamination. Volunteers wash their hands (seven-step handwashing method) before collecting urine, and thoroughly clean and disinfect the external urethral orifice and the surrounding area with iodophor and 70% alcohol (1-3 times). Pre-place 2.5 ml of double antibody and 5 ml of FBS in a sterile container (500 ml glass bottle, used after being dried and cooled by high-temperature high-pressure), immediately transfer it to a 500 ml culture bottle (dried and cooled by high-temperature high-pressure), and take it to the ultra-clean workbench for operation.

[0013] Immediately aliquot the urine sample in the culture flask into 4 sterile 50-ml centrifuge tubes. After centrifuging at 1500 r / min at room temperature for 10 min, discard the supernatant. Approximately 0.3 - 0.5 ml of liquid remains in each tube. Add 3 ml of stem cell basal medium to each tube to resuspend the precipitate. Gently pipette the mixed cell suspension into a 6-well plate or a 24-well plate, adding approximately 0.5 ml of the suspension and 1 ml of stem cell culture medium to each well. Incubate statically in an incubator. Observe the cell attachment after 6 h. Without any abnormalities, replace the hUSCs complete medium (half-medium change) after 24 h, and discard the non-attached cells to complete the preliminary screening.

[0014] Continue the culture, change the medium every 2 days, adding 1 ml and 0.5 ml of culture medium to the 6-well and 24-well plates respectively, for continuous screening and purification. At the same time, observe the cell morphology, growth status, and bacterial contamination through an ordinary optical microscope every day. If bacterial contamination occurs, clean it in a timely manner. Passage the cells by digestion when the confluence rate of the attached cells reaches 90%. These are the P0 generation of hUSCs.

[0015] Expansion and passage of mesenchymal stem cells: At the first passage, discard the original medium, wash 1 - 2 times with PBS buffer, and then add 0.5 ml of 0.25% trypsin. Incubate at 37°C for 2 - 3 min and transfer to a 6-well plate at a 1:1 ratio.

[0016] Incubate statically in an incubator and change the medium once every 2 - 3 days. Passage the cells when the cell confluence reaches 90%.

[0017] Trypsin digestion and passage: At the next passage, passage the cells to a 6-well plate at a 1:3 ratio. Change the medium once every 2 - 3 days and passage the cells again when the cell confluence reaches 90%.

[0018] Identification of urine stem cells: Growth morphology of stem cells: The cells attach and grow in a fibroblast-like manner.

[0019] Expression of stem cell surface antigens: High expression of CD44 and CD73, low expression or non-expression of CD45 and HLA-DR.

[0020] Verify by flow cytometry that the cell purity reaches over 90%. Conduct tests to rule out common infectious diseases, and exclude bacterial and mycoplasma contamination. If it meets the standards, it is a mixture of epithelial cells and mesenchymal stem cells.

[0021] Inductive differentiation ability of stem cells into adipocytes.

[0022] Preparation of adipogenic induction medium: Stem cell basal medium, insulin, indomethacin, dexamethasone, 3-isobutylmethylxanthine.

[0023] When the stem cells of the 3rd passage in the 6-well plate are fused to 80-90%, add 2 ml of osteogenic / adipogenic induction medium to each well and label.

[0024] After 21 days, perform Oil Red O staining and Alizarin Red staining to determine the induction results.

[0025] The method for cryopreservation and resuscitation of urine stem cells is as follows: ① When the cells are passaged to the 3rd passage, digest them with trypsin and centrifuge. Wash the medium with PBS, digest with 0.25% trypsin, 0.5 ml per well, at 37 °C for 2-3 min. After the cells are suspended, add 0.5 ml of DMEM to each well to neutralize the trypsin reaction. Centrifuge at 1500 r / min for 3 min; ② Then suspend them in the cell cryopreservation solution at a cell density of (1-3)×10 6 / ml, aliquot into 2 ml cryotubes, and cool down to liquid nitrogen step by step.

[0026] The step-by-step cooling sequence: -20 °C for 2 h; -40 °C for 2 h; -80 °C overnight; liquid nitrogen.

[0027] The composition of the cell cryopreservation solution: 10%-20% DMSO, 70%-80% human fetal bovine serum, and stem cell basal culture medium.

[0028] Before using the cells, quickly thaw the cells in the cryotube in a 37 °C water bath within 1 min, add them to a 15 ml centrifuge tube containing 6 ml of culture medium, centrifuge at 1500 r / min for 3 min, and then suspend them in PBS to prepare a cell suspension with a cell density of (1-3)×10 6 / ml for experimental and clinical applications including transplantation, and keep it at 4 °C until use.

[0029] The advantages and positive effects achieved by the present invention are: This method has low cost, is simple and fast, and all the culture media used are commercial, which is very convenient to purchase and use. In the first two weeks of the extraction cycle, use the special culture medium for stem cells. When enough stem cells are obtained after passage, it can be replaced with ordinary DMEM medium, which greatly reduces the production cost. Secondly, the material collection is simple, fast, and non-invasive. The method of extracting mesenchymal stem cells from urine uses samples from the patients themselves, reducing the problem of cell source heterogeneity. It lays a foundation for stem cell therapy, gene editing therapy for genetic diseases, and major and intractable diseases. Description of the Drawings Figure 1 Observation of the morphology of hUSC adherent culture cells under a light microscope. Figure 2Detected by flow cytometer (Becman, DxFLEX, USA), with fluorescein FITC and analyzed by CytExpert. Figure 3 Observed the morphology of adherent hUSC cells under light microscope. Specific implementation mode

[0031] The present invention will be further described in detail below through specific embodiments. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0032] Based on the content listed in the following table, according to the method of the present invention, the reagents used in the present invention are all common reagents in the art without special regulations; the methods used in the present invention are all conventional methods in the art without special regulations.

[0033] Reagent Name Composition REGMSingleQuotkit (LONZA, CC-3191) 1×Bottle Basal Medium (F-12) 500 mL REGM Bulletkit (LONZA, CC-3190) 1×Green Cap Vial with hEGF, 0.50 mL1×Lilac Cap Vial with Insulin, 0.50 mL1×Natural Cap Vial with Hydrocortisone, 0.50 mL1×Red Cap Vial with GA-1000, 0.50 mL1×Bottle with FBS, 2.50 mL1×Natural Cap Vial with Transferrin, 0.50 mL1×Amber Vialwith Triiodothyronine, 0.50 mL1×Amber Vial with Epinephrine, 0.50 mL Stem Cell Basal Medium KSFM (Giboco, 17005042), 100 mLHG-DMEM (Solarbio, 12100), 100mLREGM Bullet kit (LONZA, CC-3190), 100 mLREGM SingleQuot kit (LONZA, CC-3191), 100 mL10% Fetal Bovine Serum (FBS, Sangon Biotech, E6000010100), 40 mL1% Penicillin-Streptomycin (Sangon Biotech, E6070110100), 4.4 mL Adipogenic Induction Medium Stem Cell Basal Medium 50 mLInsulin (10 ug / mL; Aladdin, I302196), 130 uLIndomethacin (100 uM; Aladdin, I106885), 70 uLDexamethasone (1 uM; Sangon Biotech, E6011870005), 70 uL3-Isobutylmethylxanthine (500 uM; Sangon Biotech, E6066300100), 70 uL Stem Cell Cryopreservation Medium Cryopreservation Medium (Solarbio, 24800): hUSCs Medium = 1:1 A method for extracting mesenchymal stem cells from urine, the steps are as follows: Urine sampling and inoculation: Sterilize surgical instruments and large glass dishes at high temperature, and sterilize the ultra-clean workbench with ultraviolet light at the same time.

[0034] Prepare the culture medium: Prepare the stem cell basal medium according to the above table and store it at 4°C.

[0035] Strictly screen donors to exclude urinary system diseases, infectious diseases, metabolic diseases and malignant tumors; Collect about 200 ml of fresh midstream urine from healthy adults. The operation process strictly follows aseptic operation to avoid urine contamination. Volunteers wash their hands (seven-step handwashing method) before collecting urine, and thoroughly clean and disinfect the external urethral orifice and its surrounding area with iodophor and 70% alcohol (1-3 times). Pre-place 2.5 ml of double antibody and 5 ml of FBS in a sterile container (500 ml glass bottle, used after drying and cooling at high temperature and high pressure), immediately transfer it to a 500 ml culture bottle (dried and cooled at high temperature and high pressure), and take it to the ultra-clean workbench for operation.

[0036] Immediately sub-pack the urine sample in the culture bottle into 4 sterile 50 ml centrifuge tubes, centrifuge at 1500 r / min at room temperature for 10 min, then discard the supernatant. Each tube remains about 0.3 - 0.5 ml of liquid. Add 3 ml of stem cell basal culture medium to each tube to resuspend the precipitate. Gently pipette and mix the cell suspension and add it to a 6-well plate or 24-well plate. Add about 0.5 ml of suspension and 1 ml of stem cell culture medium to each well, and let it stand and culture in an incubator. Observe the cell adhesion situation after 6 h. If there is no abnormality, replace the hUSCs complete medium (half-medium change) after 24 h, and discard the non-adherent cells to complete the preliminary screening.

[0037] Continue the culture, change the medium every 2 days, add 1 ml and 0.5 ml of culture medium to 6-well and 24-well plates respectively, and continuously screen and purify. At the same time, observe the cell morphology, growth status and bacterial contamination through an ordinary optical microscope every day. If bacterial contamination occurs, clean it in time. When the fusion rate of adherent cells reaches 90%, digest and passage the cells. This is the P0 generation of hUSCs.

[0038] Expansion and passage of mesenchymal stem cells: At the first passage, discard the original medium, wash it 1 - 2 times with PBS buffer, and then add 0.5 ml of 0.25% trypsin. Incubate at 37 °C for 2 - 3 minutes, and transfer it to a 6-well plate at a ratio of 1:1.

[0039] Static culture in an incubator, change the medium once every 2 - 3 days, and passage the cells when the cell fusion reaches 90%.

[0040] Trypsin digestion and passage: At the next passage, passage it to a 6-well plate at a ratio of 1:3. Change the medium once every 2 - 3 days, and passage the cells again when the cell fusion reaches 90%.

[0041] Identification of urine stem cells: ① Growth morphology of stem cells: The cells adhere and grow in a fibroblast-like shape. (As Figure 1 shown) ② Expression of stem cell surface antigens: High expression of CD44 and CD73, low expression or non-expression of CD45 and HLA-DR. (As Figure 2 shown)

[0042] Inductive differentiation ability of stem cells into adipogenic direction. (As Figure 3 shown) Preparation of adipogenic induction medium: See the above table for details.

[0043] When the stem cells of the 3rd generation in a 6-well plate are fused to 80 - 90%, add 2 ml of osteogenic / adipogenic induction medium to each well and label it.

[0044] After 21 days, perform Oil Red O staining and Alizarin Red staining to determine the induction results.

[0045] Freezing preservation and resuscitation method of urine stem cells, the steps are as follows: ① When the cells are passaged to the 3rd generation, digest them with trypsin, centrifuge, wash the medium with PBS, digest with 0.25% trypsin, 0.5 ml per well, at 37 °C for 2 - 3 minutes. After the cells are suspended, add 0.5 ml of DMEM to each well to neutralize the trypsin reaction. Centrifuge at 1500 r / min for 3 minutes; ② Then, according to the cell density (1 - 3) × 10 6Suspended in the cell cryopreservation solution at / ml, aliquoted into 2-ml cryotubes, and cooled stepwise to liquid nitrogen (-20°C for 2 h; -40°C for 2 h; -80°C overnight; liquid nitrogen).

[0046] The composition of the cell cryopreservation solution: See the above table for details.

[0047] Before using the cells, quickly thaw the cells in the cryotube in a 37°C water bath within 1 min, add them to a 15-ml centrifuge tube containing 6 ml of culture medium, centrifuge at 1500 r / min for 3 min, and then resuspend in PBS to prepare a cell suspension with a cell density of (1-3)×10 6 / ml for experimental and clinical applications including transplantation, and keep it at 4°C until use.

[0048] The above identification results indicate that the stem cells isolated from urine by the method of the present invention belong to mesenchymal stem cells.

Claims

1. Claims: A method for extracting mesenchymal stem cells from urine, characterized in that: The steps are as follows: (1) Urine collection and inoculation: ① Sterilize surgical instruments and large glassware at high temperature, and simultaneously disinfect the laminar flow hood with ultraviolet light; ② Prepare the culture medium: The composition of the stem cell basal medium is: DMEM / F12 / KSFM / REGM Bullet kit / REGMSingleQuot kit plus 10% fetal bovine serum, 1% penicillin-streptomycin of the total volume of the complete medium, and store at 4°C. In the DMEM / F12 / KSFM / REGM Bullet kit / REGM SingleQuot kit, the volume ratio of DMEM:F12:KSFM:REGM Bullet kit:REGM SingleQuot kit is 1:1:1:1:

1.

2. Strictly screen donors to exclude urinary system diseases, infectious diseases, metabolic diseases and malignant tumors; Collect about 200 ml of fresh midstream urine from healthy adults. The operation process strictly follows aseptic operation to avoid urine contamination. Volunteers wash their hands (seven-step handwashing method) before collecting urine, and thoroughly clean and disinfect the external urethral orifice and its surrounding area with iodophor and 70% alcohol (1 - 3 times). Pre-place 2.5 ml of double antibiotics and 5 ml of FBS in a sterile container (500 ml glass bottle, used after drying and cooling by high-temperature high-pressure), immediately transfer it to a 500 ml culture flask (dried and cooled by high-temperature high-pressure), and take it to the laminar flow hood for operation.

3. Immediately sub-divide the urine sample in the culture flask into 4 sterile 50 ml centrifuge tubes, centrifuge at 1500 r / min at room temperature for 10 min, then discard the supernatant. Each tube remains about 0.3 - 0.5 ml of liquid, and add 3 ml of stem cell basal culture medium to each tube to resuspend the precipitate. Gently pipette and mix the cell suspension and add it to a 6-well plate or a 24-well plate. Add about 0.5 ml of the suspension and 1 ml of stem cell culture medium to each well, and let it stand and culture in the incubator. Observe the cell attachment situation after 6 h. If there is no abnormality, replace the hUSCs complete medium (half-medium change) after 24 h, and discard the non-attached cells to complete the preliminary screening.

4. Continue culturing, change the medium every 2 days, add 1 ml and 0.5 ml of the culture medium to the 6-well plate and 24-well plate respectively, and continuously screen and purify. At the same time, observe the cell morphology, growth situation and bacterial contamination situation through an ordinary optical microscope every day. If bacterial contamination occurs, clean it in time. When the confluence rate of the attached cells reaches 90%, digest and passage the cells. This is the P0 generation of hUSCs.

5. Amplification and passage of mesenchymal stem cells: At the first passage, discard the original medium, wash it 1 - 2 times with PBS buffer, and then add 0.5 ml of 0.25% trypsin. Incubate at 37°C for 2 - 3 min, and transfer it to a 6-well plate at a ratio of 1:

1.

6. Let it stand and culture in the incubator, change the medium once every 2 - 3 days, and passage the cells when the cell confluence reaches 90%.

7. Trypsin digestion and passage: At the next passage, passage the cells to a 6-well plate at a ratio of 1:

3. Change the medium once every 2 - 3 days, and passage the cells again when the cell confluence reaches 90%.

8. Identification of urine stem cells: Growth morphology of stem cells: Cells adhere to the wall and grow in a fibroblast-like manner.

9. Expression of stem cell surface antigens: High expression of CD16, CD44, CD90, CD73, CD105, and low expression or non-expression of CD19, CD34, CD45, CD11B, HLA-DR.

10. Flow cytometry verifies that the cell purity reaches over 90%. Laboratory tests exclude common infectious diseases, bacteria, and mycoplasma contamination. If it meets the standards, it is a mixture of epithelial cells and mesenchymal stem cells.

11. Inductive differentiation ability of stem cells into osteoblasts and adipocytes.

12. Preparation of osteogenic induction medium: Stem cell basal medium, β-glycerophosphate, vitamin C, dexamethasone.

13. Preparation of adipogenic induction medium: Stem cell basal medium, insulin, indomethacin, dexamethasone, 3-isobutylmethylxanthine.

14. When the stem cells of the 3rd passage in the 6-well plate are fused to 80-90%, add 2 ml of osteogenic / adipogenic induction medium to each well and label. After 21 days, perform Oil Red O staining and Alizarin Red staining to determine the induction results.

15. Freezing preservation and resuscitation method of urine stem cells, the steps are as follows: ① When the cells are passaged to the 3rd passage, digest with trypsin and centrifuge. Wash the culture medium with PBS, digest with 0.25% trypsin, 0.5 ml per well, at 37°C for 2-3 min. After the cells are suspended, add 0.5 ml of DMEM to each well to neutralize the trypsin reaction. Centrifuge at 1500 r / min for 3 min; ②Then, according to the cell density of (1-3)×10 6 / ml, suspend it in the cell cryopreservation solution, aliquot it into 2-ml cryopreservation tubes, and cool it down to liquid nitrogen by gradient cooling. The gradient cooling sequence: -20°C for 2 h; -40°C for 2 h; -80°C overnight; liquid nitrogen. The composition of the cell cryopreservation solution is: 10% - 20% DMSO, 70% - 80% fetal bovine serum, and stem cell basal culture medium.

16. Before using the cells, quickly thaw the cells in the cryopreservation tube in a 37°C water bath within 1 minute, add them to a 15 ml centrifuge tube containing 6 ml of culture medium, centrifuge at 1500 r / min for 3 minutes, and then resuspend in PBS to prepare a cell suspension with a cell density of (1-3)×10 6 / ml for experimental and clinical applications including transplantation, and keep it at 4°C until use.

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