A method for extracting and culturing urine-derived cells and its application in preparing drugs for preventing and treating kidney injury

Abstract

The present invention discloses a method for extracting and culturing urine-derived cells and its application in preparing drugs for preventing and treating kidney damage. Through the cell extraction, separation and culture method of the present invention, a group of urine-derived cells with high expression of PAX2, SOX9 and OCT2 genes are obtained from urine, wherein PAX2 and SOX9 genes are stem genes related to kidney development, and OCT2 is a gene related to organic cation and toxin transport in renal tubular epithelial cells. The cells are mainly extracted from suspended cells after urine centrifugation, and feeder layer cells are not required during the culture process, and the cell morphology and growth state are better; moreover, the cells have a good therapeutic effect when used to treat renal ischemia-reperfusion, and can be used to prepare drugs for preventing and treating kidney damage, and have great application value.

Description

Technical Field

[0001] The invention belongs to the field of cell technology, and specifically relates to a method for extracting and culturing urine-derived cells and an application of the method in preparing a drug for preventing and treating kidney injury. Background Art

[0002] The more common cells in human urine include red blood cells, white blood cells (lymphocytes, monocytes and eosinophils, etc.), squamous epithelial cells and renal tubular epithelial cells. The epithelial cells seen in urine are gently shed from the renal tubules, renal pelvis, ureter, bladder, urethra, etc. Recent research reports have pointed out that a type of cell group has been found in urine, which has the biological characteristics and differentiation potential of stem cells, called urine-derived stem cells (USC). Under the action of different induction factors, USC can differentiate into osteoblasts, adipocytes, chondrocytes, urothelial cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes and other tissues. However, the source of human urine-derived stem cells is still unclear. Some studies have reported that they come from the kidneys, while others have reported that they come from the ureters (Zhou Ming, Sun Zhenxiao. New progress in the isolation, culture and application of human urine-derived stem cells [J]. Progress in Biochemistry and Biophysics, 2017, 44(12):7.). In summary, there are many types of cells in urine and their origins are unknown.

[0003] Chinese patent document CN112430567A discloses a method for extracting and culturing urine-derived renal stem cells, wherein urine is collected and centrifuged to obtain a cell precipitate, and then the stem cells with stem properties are screened out through a culture medium; however, the culture process requires the use of trophoblast cells. Chinese patent document CN110305835A discloses a method for preparing urine-derived cells, wherein urine is collected and centrifuged to obtain a cell precipitate, and then the urine-derived cells expressing nephrin and WT1 are screened out through a culture medium; however, relative to the test results of the present invention, it does not indicate whether such cells highly express PAX2, SOX9, and OCT2 genes. Summary of the invention

[0004] The purpose of the present invention is to provide a cell extraction, separation, culture and screening method to obtain a group of urine-derived cells that highly express PAX2, SOX9 and OCT2 genes from healthy human urine. The method does not require feeder cells, has low cost, and the urine-derived cells obtained by the method have higher relative expression levels of PAX2, SOX9 and OCT2 genes than other urine-derived cells, and are better for treating renal ischemia-reperfusion.

[0005] The technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a method for culturing urine-derived cells, comprising the following steps:

[0007] S1: Collect urine, remove the supernatant away from the bottom of the centrifuge tube, and retain the remaining supernatant and cell pellet near the bottom of the centrifuge tube;

[0008] S2: resuspending the cells in the remaining supernatant and cell pellet in step S1, filtering and then adding red blood cell lysis solution for lysis;

[0009] S3: After the lysis is completed, centrifuge and remove the supernatant away from the bottom of the centrifuge tube. Add the culture medium to the remaining supernatant and / or cell pellet, mix well, and then culture to obtain the 0th generation urine-derived cells.

[0010] The present invention finds that the expression level of SOX9 in the precipitated cells after centrifugation is low, and the therapeutic effect of injection in the renal ischemia model is relatively poor, while the expression level of SOX9 in the cells suspended in the supernatant is high, and the therapeutic effect of injection in the renal ischemia model is relatively good. Therefore, the present invention mainly obtains cells in the suspension after centrifugation, and the cell morphology and growth state are better, and the expression levels of PAX2, OCT2, and SOX9 are high, and the therapeutic effect of injection in the renal ischemia model is also good.

[0011] In some embodiments of the present invention, the part of the supernatant away from the bottom of the centrifuge tube removed in step S1 accounts for 75%-85% of the total volume of the supernatant, and the remaining supernatant accounts for 15%-25%.

[0012] In some embodiments of the present invention, the part of the supernatant removed away from the bottom of the centrifuge tube in step S3 accounts for 75%-85% of the total volume of the supernatant, and the remaining supernatant accounts for 15%-25%.

[0013] In some embodiments of the present invention, the centrifugation conditions are: centrifugal force 350×g to 450×g, centrifugal time 8 to 15 min, centrifugal temperature 20 to 25°C; preferably 400×g, 10 min, 20°C.

[0014] In some embodiments of the present invention, the lysis conditions of the lysis solution are: standing at room temperature for 1 to 5 minutes, preferably 5 minutes.

[0015] In some embodiments of the present invention, the pore size of the filter is 30 to 50 μm, preferably 40 μm.

[0016] In some embodiments of the invention, the culture medium comprises REGM and MEF.

[0017] In some embodiments of the present invention, the volume ratio of REGM to MEF is 1:0.5-2, preferably 1:1.

[0018] In some embodiments of the present invention, the culturing environment conditions are 36-37° C., 5% CO 2 ; preferably 37° C., 5% CO 2 .

[0019] In some embodiments of the present invention, the culture time of step S3 is ≥5 days, preferably 5 to 20 days, and the culture medium has a screening function. After 5 days, cells that are not suitable for the culture medium will die and float up; after 13 days, it can be ensured that the fusion degree of the obtained generation 0 cells reaches more than 90%, and then they can be passaged. The present invention has tried culture media such as mTeSR1 and A1640, but none of them can culture urine-derived cells with high expression of PAX2, SOX9, and OCT2 genes. The REGM and MEF culture media of the present invention can ensure the successful culture and expansion of each batch of urine-derived cells with high expression of PAX2, SOX9, and OCT2 genes, so this culture medium is selected for culture.

[0020] In some embodiments of the present invention, passage may be performed when the urine-derived stem cells are cultured to a density of 80% to 90% confluence.

[0021] In some embodiments of the present invention, the passage density is 1.0×(10 6 ~10 7 ) / T75 bottle.

[0022] In some embodiments of the present invention, during the subculture process, the medium is changed every 2 to 3 days; the medium change method is 50% to 100% medium change, preferably 100% medium change.

[0023] In some embodiments of the present invention, the urine is midstream urine of a healthy person.

[0024] The second aspect of the present invention provides the use of urine-derived cells prepared by the culture method described in the first aspect of the present invention in preparing drug screening models, drug toxicity evaluation, cell therapy, cell drug preparation and kidney tissue engineering.

[0025] The third aspect of the present invention provides the use of urine-derived cells prepared by the culture method described in the first aspect of the present invention in the preparation of products for preventing and treating kidney injury.

[0026] In some embodiments of the invention, the renal injury comprises acute renal injury.

[0027] In some embodiments of the present invention, the acute kidney injury comprises renal ischemia-reperfusion injury.

[0028] In some embodiments of the invention, the product comprises a medicament.

[0029] The beneficial effects of the present invention are:

[0030] Through the cell extraction, separation, culture and screening method of the present invention, a group of urine-derived cells with high expression of PAX2, SOX9 and OCT2 genes are obtained from urine, wherein PAX2 and SOX9 genes are stemness genes related to kidney development, and OCT2 is a gene of renal tubular epithelial cells related to organic cation and toxin transport. The cells are mainly extracted from suspended cells after urine centrifugation, and no feeder layer cells are required during the culture process, and the cell morphology and growth state are better; and the cells have good therapeutic effects when used to treat renal ischemia reperfusion, and can be used to prepare drugs for preventing and treating kidney damage, and have great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the P0 cells in Example 1.

[0032] Figure 2 These are the P0 cells in Example 2.

[0033] Figure 3 These are the P0 generation cells in Comparative Example 1.

[0034] Figure 4 These are the P0 generation cells in Comparative Example 2.

[0035] Figure 5 The expression levels of some kidney-related genes in the P3 cells of Example 1.

[0036] Figure 6 The expression levels of PAX2, SOX9 and OCT2 in the P3 generation cells of Example 1, Example 2, Comparative Example 1 and Comparative Example 2.

[0037] Figure 7 It is the BUN expression amount in Example 1, Example 2 and Comparative Example 1.

[0038] Figure 8 It is the Cre expression amount in Example 1, Example 2 and Comparative Example 1.

[0039] Fig. 9 It is the KIM-1 expression amount in Example 1, Example 2 and Comparative Example 1.

[0040] Fig.10 It is the UALB expression amount in Example 1, Example 2 and Comparative Example 1. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0042] The supernatant mentioned in the present application document does not refer to a clear and transparent liquid, but refers to the liquid on the cell pellet after the cell suspension is centrifuged, which is mainly used to distinguish it from the cell pellet.

[0043] Example 1 Extraction of healthy human urine-derived cells

[0044] (1) Collect urine: Disinfect the urethral opening of a healthy person with iodine, and collect 150-200 mL of clean midstream urine in a conical flask; divide the urine into 50 mL centrifuge tubes, with about 50 mL in each tube;

[0045] (2) Centrifugation: centrifugal force 400×g, time 10 min, temperature 20°C; slowly take out the centrifuge tube, use a 10 mL pipette to aspirate the supernatant away from the bottom of the centrifuge tube, and when 15 mL of supernatant remains, use a 1000 μL pipette tip to slowly aspirate the supernatant until 10 mL of supernatant remains (accounting for about 20% of the total volume of the supernatant after centrifugation), and then use a pipette to directly blow the cell pellet and the white substance in the suspension to obtain a suspension; according to multiple extractions, under the conditions of the centrifugal force and centrifugation time, the white substance in the suspension only stays in the supernatant of the 1-5 mL section of the 50 mL centrifuge tube, that is, about 20% of the total volume of the supernatant; therefore, the proportion of the supernatant to be retained is determined based on this result;

[0046] (3) Sieve and add lysis buffer: Take a new 50 mL centrifuge tube, place a 40 μm cell filter at the mouth of the tube, sieve the suspension, and then pour PBS containing 50% ACK Lysis Buffer onto the filter to make the volume of the liquid in the centrifuge tube 25 mL; mix the liquid and let it stand at room temperature for 5 min;

[0047] (4) Centrifugation: 20°C, 400×g for 10 min; discard the supernatant at the bottom of the centrifuge tube until the remaining supernatant is 5 mL; transfer the 5 mL supernatant (containing the cells suspended therein, accounting for about 20% of the total volume of the supernatant after centrifugation) to a new 50 mL centrifuge tube, add 15 mL of urine-derived cell culture medium thereto, and mix well;

[0048] The formula of the urine-derived cell culture medium is as follows: 50% REGM (each 50 mL REGM contains: 49.4 mL REBM + 0.05 mL epidermal growth factor + 0.05 mL transferrin + 0.05 mL insulin + 0.05 mL hydrocortisone + 0.05 mL GA-1000 + 0.05 mL triiodothyronine + 0.05 mL epinephrine + 0.25 mL FBS) + 50% MEF (each 50 mL MEF contains: 44 mL 1×DMEM + 5 mL FBS + 0.5 mL 100×Glutamax + 0.5 mL 100×NEAA);

[0049] (5) Seeding: seed the suspension in a 24-well plate, 1 mL per well, and shake well; place the 24-well plate in an incubator at 37°C and 5% CO2 for 1 day;

[0050] (6) Cultivation: Add 500 μL of urine-derived cell culture medium every day until the amount of urine-derived cell culture medium added to each well reaches 2 mL. No more culture medium is added and the culture is continued for 5 to 13 days to obtain passage 0 cells.

[0051] Example 2

[0052] The difference between Example 2 and Example 1 is step (4), and the other steps are the same. Step (4) of Example 2: centrifuge at 20°C and 400×g for 10 min; discard the supernatant away from the bottom of the centrifuge tube until the remaining supernatant is 5 mL; resuspend, fully mix the 5 mL supernatant (containing the cells suspended therein) and the white precipitate (cell precipitate) at the bottom of the tube, and then add 15 mL of urine-derived cell culture medium thereto and mix well.

[0053] Note: If the centrifugal force is increased from 400×g to 800×g and the time is extended from 10min to 20min, there are still many cells in the suspension of the centrifuge tube after centrifugation, and they have not settled to the bottom of the centrifuge tube. Experimental verification has found that increasing the centrifugal force and centrifugation time will have an adverse effect on the cell state and reduce cell viability, so the centrifugation time and centrifugal force were not increased.

[0054] Comparative Example 1

[0055] The difference between Comparative Example 1 and Example 1 is step (4), and the other steps are the same. Step (4) of Comparative Example 1: centrifuge at 20°C and 400×g for 10 min; discard all the supernatant in the tube (including the cells suspended therein), and then add 20 mL of urine-derived cell culture medium to resuspend the white precipitate (cell precipitate) at the bottom of the tube.

[0056] Comparative Example 2

[0057] The difference between Comparative Example 2 and Example 1 is in step (3) and (4), and the other steps are the same. Step (3) of Comparative Example 2: Pour 1×PBS into the tube until the liquid in the centrifuge tube is fixed to 25 mL; mix the liquid; Step (4): Centrifuge at 20°C and 400×g for 10 min; Discard the supernatant away from the bottom of the centrifuge tube until the remaining supernatant is 5 mL; Resuspend, fully mix the 5 mL supernatant (containing cells) and the white precipitate (cell precipitate) at the bottom of the tube, and then add 15 mL of urine-derived cell culture medium thereto and mix well.

[0058] The results of cell extraction and culture at the 0th generation of Example 1, Example 2, Comparative Example 1 and Comparative Example 2: Figure 1 : The morphology of the cells extracted in Example 1 was spindle-shaped; Figure 2 : Only a part of the cells extracted from Example 2 is the same in morphology as the cells extracted from Example 1, which is spindle-shaped; the morphology of the other part of the cells is different from that of Example 1, which is round; Figure 3 : The morphology of all cells extracted in Comparative Example 1 is different from that in Example 1 and is round; Figure 4 : The cells extracted in Comparative Example 2 had inconsistent morphology and poor growth status.

[0059] Example 3: Amplification Culture

[0060] The cells extracted from Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were cultured and passaged according to the following culture and amplification steps:

[0061] (1) For cells at passage 0, the medium was changed every 2 days. Each change was 100% and the amount of urine-derived cell culture medium replaced was 500 μL per well.

[0062] (2) When the cells of passage 0 are cultured to a confluence of 80-90%, they are subcultured: 1.0×10 6 The cells were inoculated into a T25 flask, and the cells were at the first generation.

[0063] (3) The medium of the first generation cells was changed every 2 days. Each time the medium was changed, 100% of the medium was replaced. The amount of urine-derived cell culture medium replaced each time was 5 mL per bottle.

[0064] (4) When the first generation of cells reaches a confluence of 80-90%, they are subcultured: 3.0×10 6 The cells were inoculated into a T75 flask, and the cells were at the second generation.

[0065] (5) The second generation cells were cultured once every 2 days. Each culture was 100% cultured and the amount of urine-derived cell culture medium replaced was 15 mL per bottle.

[0066] (6) When the second generation cells reach a confluence of 80-90%, they are subcultured: 1.0×10 7 The cells were inoculated into two T75 flasks, and the cells were at the third generation.

[0067] Example 4 Gene Detection

[0068] After the third generation cells of Example 1, Example 2, Comparative Example 1 and Comparative Example 2 were cultured until the cell confluence reached 80-90%, the cells were collected and tested according to the following steps:

[0069] Take 1×10 6 The total RNA was extracted from each cell line using the Takara kit, and the cDNA was obtained by reverse transcription using the Tiangen kit. The expression levels of some kidney-related genes were detected by RT-qPCR using the Tiangen kit.

[0070] Results: Under the same cell culture generation (generation 3), the cells extracted using the extraction scheme in Example 1 had significantly higher expression levels of PAX2, SOX9, and OCT2 genes among the kidney-related genes ( Figure 5 ). The cell population obtained by the scheme of Example 2 also has a good expression level of the above three genes, but because the content of cells with high expression of PAX2, OCT2, and SOX9 in the obtained cell population is lower than that in Example 1, the gene expression levels of PAX2, OCT2, and SOX9 are lower than those in Example 1. The cell population obtained by the scheme of Comparative Example 1 has low gene expression levels of PAX2, OCT2, and SOX9. Figure 1 and Figure 3 The cell shape analysis can determine that the cells obtained in Comparative Example 1 are different from the cells of Example 1. The cells obtained using the Comparative Example 2 scheme have inconsistent morphology and poor growth status. The content of cells with high expression of PAX2, OCT2, and SOX9 in the total cell population is lower than that in Example 2. Step (3) of Comparative Example 2 is also different from that of Example 2. This step difference causes the cells obtained in Comparative Example 2 to have a worse growth status than the cells in Example 2, so the gene expression level is different from that in Example 2 ( Figure 6 ).

[0071] Comprehensive analysis shows that the cell population in the precipitate after centrifugation and the cell population in the suspension contain different cell types, and the cell population in the suspension can obtain cells with high expression of PAX2, SOX9 and OCT2 after culture medium screening. In addition, the insoluble matter in the suspension (Comparative Example 2) affects the cell culture and affects the expression of PAX2, SOX9 and OCT2.

[0072] Example 5 Treatment of renal ischemia-reperfusion injury

[0073] Since the cells of Comparative Example 2 grew poorly and could not be expanded in large quantities for animal experiments, the cells of Comparative Example 2 were not used for animal experiments. Only the third-generation cells of Example 1, Example 2, and Comparative Example 1 were used to treat mice with renal ischemia-reperfusion injury. The mice used were male NOD / SCID mice aged 6 to 8 weeks, weighing 25-30 g. The specific steps are as follows:

[0074] (1) Anesthesia: Hold the syringe filled with chloral hydrate solution and fix the front end of the syringe with your index finger. Insert the needle into the subcutaneous tissue from the lower abdomen to the head in parallel, slightly to the right / left of the linea alba on the lower abdomen of the mouse. After the needle is inserted 3 to 5 mm, insert the needle at a 45° angle to the skin and pass it through the abdominal muscle. When you feel a sense of emptiness, stop inserting the needle and fix the needle. If there is no blood, intestinal fluid, or urine, you can slowly push the drug solution in.

[0075] (2) Skin preparation: When the mouse's toes are pinched or its belly is lightly clamped with forceps, the mouse will be in a deep anesthesia state if there is no obvious pain reaction. Use a razor to prepare the skin of the mouse's abdomen, from the xiphoid process to the pubic bone and from both sides to the anterior axillary line.

[0076] (3) Fixation and disinfection: Place the mouse in a supine position on a warm pad, fix the mouse's limbs and tail with adhesive strips, and disinfect the skin preparation area and 2 cm outward with iodine.

[0077] (4) Opening the abdomen: Use toothed forceps to pinch the skin about 1 cm above the perineum on the midline of the abdomen. Use ophthalmic scissors to cut upward from about 1 cm above the perineum to the xiphoid process. The muscles are cut from the linea alba to expose the liver tissue. Cut the skin and muscle tissue layer by layer.

[0078] (5) Perform renal ischemia-reperfusion: After isolating the renal vein, hold the cerebral aneurysm clip with a hemostatic forceps, and clamp the left and right renal veins in turn without damaging the blood vessels for 30 min each (accurate timing after separating both kidneys). The color of the kidneys gradually changes to purple-black. During this period, cover the abdominal cavity with moistened sterile gauze to reduce the loss of body fluids in the mice. After the timing is over, gently remove the bilateral cerebral aneurysm clips and observe the renal reperfusion. After reperfusion, the kidneys change from purple-black to bright red, indicating that the renal ischemia-reperfusion injury (IRI) model was successfully established.

[0079] (6) Cell injection: According to the grouping (a total of 4 groups: saline negative control group, Example 1 group, Example 2 group, and Comparative Example 1 group (10 mice in each group of Example 1, Example 2, and Comparative Example 1, and 5 mice in the control group), a 34G ultrafine needle (needle length 2.5 mm) was used to perform bilateral renal parenchymal saline or cell injection, with an injection volume of 25 μL per kidney and an injection amount of 4×10 cells per kidney. 5 After the injection, the injection hole can be sealed with tissue glue to prevent cell leakage and bleeding.

[0080] (7) Closing the abdomen: Use a moistened sterile cotton swab to reposition the small intestine, colon, etc., inject 1 mL of normal saline into the abdominal cavity before closing the abdomen, use 5-0 absorbable surgical sutures to suture the peritoneum continuously, and use simple interrupted sutures to suture the skin. Then, disinfect the wound twice with iodine tincture.

[0081] (8) Postoperative warmth preservation and status observation: Move the mice to a cage under an infrared lamp and keep them warm for 15 minutes, waiting for the mice to recover from anesthesia. The postoperative recovery time of mice is generally about five minutes. If the time is too long, the later status of the mice may be affected. Pay attention to the postoperative recovery status of the mice, and give them antibiotics for anti-inflammatory treatment if necessary.

[0082] (9) Animal experiment sample collection:

[0083] (9.1) Urine: Urine was collected from mice 72 hours after surgery using metabolic cages and the urine volume was recorded. The urine was then centrifuged at 2500 rpm / min for 20 min and the supernatant was aliquoted and stored at -80°C for detection of urine albumin using the SDS-Page gel electrophoresis-Coomassie brilliant blue staining method (the urine albumin band is approximately 65 kDa, and each urine sample is added to the electrophoresis system in equal amounts for electrophoresis; after staining, the urine albumin content is calculated by comparing the gray value of the band at 65 kDa). The mouse kidney injury molecule 1 (Kim-1) ELISA kit was used according to the instructions to detect urine kidney injury molecule 1.

[0084] (9.2) Serum: Before sampling, i.e., on the third day after surgery, blood was collected by removing the eyeball. The whole blood was allowed to stand at room temperature for more than 30 min (centrifuged within 2 h). When a small amount of serum was precipitated, the collected blood was centrifuged at 1000×g for 15 min, and the supernatant was collected and packaged, stored at -80℃, and used for the detection of serum urea nitrogen and serum creatinine by the Guangdong Provincial Laboratory Animal Monitoring Institute.

[0085] Results: According to the results of the animal experiment, the various pathological indicators of the mice with renal ischemia-reperfusion injury (IRI) injected with the urine-derived cells extracted in Example 1, namely, blood urea nitrogen (BUN), creatinine (Cre), kidney injury molecule 1 (KIM-1) in urine, and albumin (UALB) in urine, were significantly downregulated compared with those of the IRI control group ( Figure 7-Figure 10); The cells extracted in Example 2 also down-regulated the above-mentioned indicators of IRI mice, but the down-regulation effect was not as obvious as that in Example 1; Although the cells extracted in Comparative Example 1 down-regulated the BUN and Cre indicators of IRI mice, the KIM-1 and UALB indicators were significantly increased compared with the control group, and the therapeutic effect was poor. This shows that the urine-derived cells extracted by the scheme of Example 1 have a significant therapeutic effect on IRI mice, and the urine-derived cells with high expression of PAX2, OCT2, and SOX9 genes have a significant promoting effect on the reduction of IRI pathological indicators.

[0086] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for extracting, culturing and screening urine-derived cells, comprising the following steps: S1: Collect urine; after centrifugation, remove the supernatant far from the bottom of the centrifuge tube, and retain the remaining supernatant and cell pellet near the bottom of the centrifuge tube; S2: resuspending the cells in the remaining supernatant and cell pellet in step S1, filtering and then adding red blood cell lysis solution for lysis; S3: After the lysis is completed, centrifuge and remove the supernatant far from the bottom of the centrifuge tube. Add the culture medium to the remaining supernatant and mix well, then culture to obtain the 0th generation urine-derived cells; The centrifugation conditions in S1 and S3 are: 350×g-450×g, 8-15 min, 20-25°C.

2. The extraction, cultivation and screening method according to claim 1, characterized in that: The part of the supernatant removed away from the bottom of the centrifuge tube described in step S1 and step S3 accounts for 75%-85% of the total volume of the supernatant.

3. The extraction, cultivation and screening method according to claim 1, characterized in that: The lysis conditions for adding red blood cell lysis solution to lyse red blood cells in step S2 are: standing at room temperature for 1 to 5 minutes after adding the lysis solution.

4. The extraction, cultivation and screening method according to claim 1, characterized in that: The volume of the red blood cell lysis solution added in step S2 is 1 to 2 times the volume of the cell suspension.

5. The extraction, cultivation and screening method according to claim 1, characterized in that: The pore size of the filtration in step S2 is 30-50 μm.

6. The extraction, cultivation and screening method according to claim 1, characterized in that: The culture medium comprises REGM culture medium and MEF culture medium; each 50 mL of the MEF culture medium contains 44 mL 1×DMEM, 5 mL FBS, 0.5 mL 100×Glutamax and 0.5 mL 100×NEAA.

7. The extraction, cultivation and screening method according to claim 6, characterized in that: The volume ratio of the REGM culture medium to the MEF culture medium is 1:0.5-2.

8. The extraction, cultivation and screening method according to claim 1, characterized in that: The urine is the midstream urine of a healthy person.

9. The extraction, cultivation and screening method according to claim 1, characterized in that: The culturing time in step S3 is ≥5 days.

10. Use of urine-derived cells obtained by the extraction, culture and screening method according to any one of claims 1 to 9 in the preparation of products for preventing and treating renal injury.

11. Use of urine-derived cells obtained by the extraction, culture and screening method according to any one of claims 1 to 9 in the preparation of drug screening models, drug toxicity evaluation and cell drug preparation.

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