Application of Small Molecule Compounds in Renal Cell Culture

Abstract

The present invention discloses the application of small molecule compounds in the culture of kidney-derived cells. The kidney-derived cells include renal tubular epithelial cells and urine-derived cells from the kidney. The present invention discovers that Corydaline, ARN2966, PKG drug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, Buparvaquone can maintain the long-term culture of primary human renal tubular epithelial cells and improve cell viability. It is also found that Corydaline, ARN2966, and PKG drug G1 have an obvious effect of improving cell viability on urine-derived cells from the kidney, and even a proliferation-promoting effect, which can ensure that a larger number of urine-derived cells can be obtained at an earlier passage.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture, and particularly relates to the application of a small molecule compound in the culture of primary human kidney-derived cells. Background Art

[0002] It is reported that more than 3,000 chemical substances (some are metabolites) can be detected in human urine. In addition, there are also some cells shed from the blood or tissues (mainly the kidneys) in urine. The common cells in human urine include red blood cells, white blood cells (lymphocytes, monocytes, eosinophils, etc.), transitional epithelial cells, squamous epithelial cells, and renal tubular epithelial cells, etc. The epithelial cells seen in urine are shed from the renal tubules, renal pelvis, ureters, bladder, urethra, etc.

[0003] Research reports in recent years have pointed out that a cell population with the biological characteristics and differentiation potential of stem cells has also been found in urine, which is called urine-derived stem cells (USC). Under the action of different inducing factors, USC can differentiate into various tissues such as osteoblasts, adipocytes, chondrocytes, urothelial cells, smooth muscle cells, skeletal muscle cells, and cardiomyocytes.

[0004] Because the acquisition of urine-derived cells is non-invasive and the number of cells is relatively large, they have been widely studied for regenerative medicine. For example, urine cells are directly reprogrammed into neural stem cells (Nature Methods, doi:10.1038 / nmeth.2283); for example, induced pluripotent stem cells derived from urine cells are differentiated into epithelial-like membranous structures to replace the epithelial tissue required for the construction of regenerated teeth (Cell Regeneration, doi:10.1186 / 2045-9769-2-3).

[0005] Primary cells are closest to the originating tissue. They are directly taken from the tissue and processed to be established under optimal culture conditions. Since they are derived from the tissue and unmodified, they are more similar to the in vivo state and exhibit normal physiological characteristics. Therefore, they provide an excellent model system for studying the normal physiology and biochemical properties of cells (such as metabolic research, aging, signal transduction research) and the effects of drugs and toxic compounds on cells. However, it should be noted that primary cells have a limited lifespan and will stop dividing (or senesce) after a certain number of cell divisions, and are more difficult to culture and maintain than continuous cell lines.

[0006] The most commonly used primary cell types in research are epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells. The initial cultures are heterogeneous (i.e., a mixture of multiple cell types present in the tissue) and can only be cultured in vitro for a limited time range. Renal tubular epithelial cells refer to a layer of cells outside the renal tubules. It is not recommended to passage primary renal tubular epithelial cells because as the number of passages increases or the culture time is too long, cell viability will severely decline.

[0007] Primary cells are used to prepare cell drugs (cell therapy), and the number of cells must meet the treatment requirements. However, the number of passages of primary cells generally does not exceed 10 generations. It is difficult to amplify cells in large quantities under a limited number of passages. Renal-derived cells (cells derived from the kidney) do not have a relatively stable growth rate. After amplifying cells in large quantities, the number of passages of their primary cells is already relatively high, and cell viability decreases, which is also not conducive to cell therapy. Therefore, it is necessary to treat urothelial cells with small molecule drugs that can improve the proliferative ability of renal-derived cells to ensure that a larger number of urothelial cells can be obtained at an earlier passage. Summary of the Invention

[0008] The purpose of the present invention is to provide small molecule compounds that are beneficial to maintaining the long-term in vitro culture of renal-derived cells (cells derived from the kidney), improving their cell viability, and even promoting their cell proliferation. These small molecule compounds include at least one of Corydaline, ARN2966, PKG drug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, Buparvaquone.

[0009] The technical solution adopted by the present invention is:

[0010] In the first aspect of the present invention, a culture medium for culturing renal-derived cells is provided. The culture medium contains at least one of Corydaline, ARN2966, PKG drug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, Buparvaquone, preferably at least one of Corydaline, ARN2966, PKG drug G1.

[0011] In some embodiments of the present invention, the renal-derived cells include renal tubular epithelial cells and / or urothelial cells derived from the kidney.

[0012] Compounds Corydaline, ARN2966, PKG drug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, and Buparvaquone have significant effects on improving the cell viability and promoting the cell proliferation of renal tubular epithelial cells, which is beneficial to the long-term culture of renal tubular epithelial cells.

[0013] In some embodiments of the present invention, the concentration of Corydaline is 0.003 - 10 μM; the concentration of ARN2966 is 0.003 - 10 μM; the concentration of PKG drug G1 is 0.003 - 10 μM; the concentration of Succinobucol is 0.003 - 10 μM; the concentration of Meptyldinocap is 0.003 - 10 μM; the concentration of Rifamycin sodium salt is 0.003 - 10 μM; the concentration of Carnosic acid is 0.003 - 10 μM; the concentration of TBHQ is 0.003 - 10 μM; the concentration of C188-9 is 0.003 - 10 μM; the concentration of Buparvaquone is 0.003 - 10 μM.

[0014] In some embodiments of the present invention, the culture medium further comprises a basal medium, and the basal medium contains fetal bovine serum with a volume fraction of 5% - 20%.

[0015] In some embodiments of the present invention, the basal medium is DMEM / F12 medium.

[0016] Compounds Corydaline, ARN2966, and PKG drug G1 have obvious effects on improving the cell viability and promoting the cell proliferation of urine-derived cells from the kidney, which is beneficial to the long-term culture of renal tubular epithelial cells derived from the kidney.

[0017] When the renal cells are urine-derived cells, the concentration of Corydaline is 1 - 5 μM; the concentration of ARN2966 is 1 - 5 μM; the concentration of PKG drug G1 is 3 - 5 μM; the concentration of Buparvaquone is 0.003 - 10 μM.

[0018] In some embodiments of the present invention, the culture medium further contains fetal bovine serum with a volume fraction of 5% - 20%.

[0019] In some embodiments of the present invention, the basal medium is a mixture of REGM medium and DMEM-high Glucose medium at a volume ratio of 1:(0.5 - 1.5).

[0020] Among them, every 50 mL of REGM contains: 49.4 mL of REBM + 0.05 mL of epidermal growth factor + 0.05 mL of transferrin + 0.05 mL of insulin + 0.05 mL of hydrocortisone + 0.05 mL of GA-1000 + 0.05 mL of triiodothyronine + 0.05 mL of adrenaline + 0.25 mL of FBS.

[0021] Every 50 mL of MEF contains: 44 mL of 1×DMEM + 5 mL of FBS + 0.5 mL of 100×Glutamax + 0.5 mL of 100×NEAA.

[0022] In the second aspect of the present invention, there is provided the use of a small molecule compound or a pharmaceutically acceptable salt thereof in at least one of (I) - (VI);

[0023] (I) Improving the viability of kidney-derived cells;

[0024] (II) Preparing a product for improving the viability of kidney-derived cells;

[0025] (III) Promoting the proliferation of kidney-derived cells;

[0026] (IV) Preparing a product for promoting the proliferation of kidney-derived cells;

[0027] (V) Culturing kidney-derived cells;

[0028] (VI) Preparing a product for culturing kidney-derived cells; the small molecule compound comprises at least one of Corydaline, ARN2966, PKGdrug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, Buparvaquone.

[0029] In some embodiments of the present invention, the kidney-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

[0030] In some embodiments of the present invention, when the renal-derived cells are renal tubular epithelial cells, the concentrations of the small molecule compounds Corydaline, ARN2966, PKG drug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, and Buparvaquone are all 0.003 - 10 μM.

[0031] In some embodiments of the present invention, when the renal-derived cells are urinary-derived cells, the concentration of Corydaline is 1 - 5 μM; the concentration of ARN2966 is 1 - 5 μM; the concentration of PKG drug G1 is 3 - 5 μM.

[0032] In a third aspect of the present invention, a method for improving the viability of renal-derived cells is provided, which comprises culturing renal-derived cells using the culture medium described in the first aspect of the present invention.

[0033] In some embodiments of the present invention, the renal-derived cells include renal tubular epithelial cells and / or urinary-derived cells from the kidney.

[0034] In some embodiments of the present invention, the culture time is 24 - 72 h.

[0035] In some preferred embodiments of the present invention, the culture time is 24 - 48 h.

[0036] The beneficial effects of the present invention are as follows:

[0037] The present invention screens small molecule compounds capable of maintaining the long-term culture of primary human renal tubular epithelial cells through a high-throughput drug screening method, including Corydaline, Succinobucol, Asunaprevir, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, PKG drug G1, Buparvaquone, and ARN2966. Except for Asunaprevir, the other 10 compounds have a dose-effect dependence; the above small molecule compounds can be used for the long-term culture of primary human renal tubular epithelial cells, improve cell viability, and even promote cell proliferation.

[0038] On this basis, the present invention further discovers that Corydaline, ARN2966, and PKG drug G1 among the above 10 compounds have obvious effects of improving the viability and promoting the proliferation of urinary-derived cells from the kidney, which can ensure that a larger number of urinary-derived cells can be obtained at an earlier passage. Brief Description of the Drawings

[0039] Figure 1 It is a high-throughput screening flow chart.

[0040] Figure 2 It is the result of primary screening.

[0041] Figure 3 It is the result of secondary screening.

[0042] Figure 4 They are the dose-effect curves of each compound.

[0043] Figure 5 They are the effects of each compound on cell proliferation.

[0044] Figure 6 They are pictures of the morphology of urinary-derived cells.

[0045] Figure 7 They are the gene detection results of urinary-derived cells.

[0046] Figure 8 They are the experimental design diagrams for primary screening of urinary-derived cells with each compound added alone.

[0047] Figure 9 They are the results of primary screening of urinary-derived cells with each compound added alone.

[0048] Figure 10 They are the experimental design diagrams for secondary screening of urinary-derived cells with each compound added alone.

[0049] Figure 11 They are the results of secondary screening of urinary-derived cells with Corydaline, ARN2966, and PKG drug G1 added alone.

[0050] Figure 12 They are the results of secondary screening of urinary-derived cells with Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, and Buparvaquone added alone.

[0051] Figure 13 They are the experimental design diagrams for combined use of Corydaline, ARN2966, and PKG drug G1.

[0052] Figure 14 They are the results of combined use of Corydaline, ARN2966, and PKG drug G1. Detailed Implementation Manner

[0053] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0054] Materials: Cells: Primary human proximal tubular cells (HPTC), human urinary-derived cells (UPC), cell counting kit (cck-8) (GLPBIO, GK10001), Calcein-AM / PI Double Staining Kit (DOJINDO, C542), EdU detection kit (Beyotime, C0081SL), Ki67 (abcam, ab16667), DMEM / F12 (ThermoFisher, C11330500BT), FBS (Gibco, 10270-106). Among them, HPTC was extracted, isolated, and purified according to the protocols described in the references <Isolation of two distinct populations of cells from rat kidney cortex and their use in the study of chemical-induced toxicity> and <Isolation and characterisation of human proximal tubular cells derived from kidney cortical segment>.

[0055] Example 1

[0056] 1. High-throughput preliminary screening

[0057] The fifth-generation (P5) HPTC were seeded into 384-well plates at a density of 2000 cells per well and cultured in DMEM / F12 medium (containing 10% fetal bovine serum) for 24 hours. Then, the small molecule compounds to be screened (working concentration 5 μM) were added to each well using an automatic liquid workstation, and the HPTC were further cultured for 48 hours. Then, 10 μl of CCK-8 was added to each well, and after incubation at 37 °C for 3 hours, the absorbance at 450 nm was measured using a microplate reader ( Figure 1 ). Each compound had 2 replicates.

[0058] A total of 8400 compounds were screened and detected, and the relative cell viability of each compound in two 384-well plates was plotted ( Figure 2) Considering the compound's effect on promoting cell survival and repeatability, 11 compounds were initially screened (Table 1) for subsequent verification.

[0059] Table 1. 11 candidate compounds for subsequent verification

[0060]

[0061]

[0062] 2. Re-screening

[0063] HPTC at passage 5 was seeded into a 384-well plate at a density of 2000 cells per well and cultured in DMEM / F12 medium (containing 10% fetal bovine serum) for 24 hours. Then, in the experimental groups, 11 small molecule compounds shown in Table 1 at different concentrations were added (each compound was dissolved and diluted with DMSO, and the working concentrations after adding to the cells were 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003 μM respectively). The blank control group was added with the same volume of DMSO, and the positive control group was added with the corresponding concentration of CHIR99021 (GSK-3 inhibitor). HPTC was continuously cultured for 48 hours, then 10 μl of CCK-8 was added to each well, and after incubating at 37 °C for 3 hours, the absorbance at 490 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader. Each compound was replicated 4 times.

[0064] The verification results of CCK-8 detection are as Figure 3 . Except for Asunaprevir, the other 10 compounds all had the effect of increasing the viability of renal tubular epithelial cells, and all had a dose-effect dependence ( Figure 3 and Figure 4 )

[0065] 3. Ki-67 detection

[0066] Ki-67 is often used in pathological immunohistochemistry to indicate the degree of cell proliferation activity. Cell division is divided into four cycles, namely G1, S, G2, and M phases. After the mitotic phase (M phase) ends, the cell enters the quiescent phase (G0) phase, during which the cell does not divide and proliferate. Ki-67 is expressed in all phases of cell proliferation, but not in the G0 phase. The level of the Ki-67 index in the pathological report indicates the rate of tumor cell proliferation.

[0067] EdU (5-Ethynyl-2'-deoxyuridine) is a thymidine analogue, and the alkynyl group it carries is rarely seen in natural compounds. It can replace thymine (T) and infiltrate into the DNA molecule being synthesized during the DNA replication period. Based on The specific reaction of fluorescent dyes with EdU can directly and accurately detect DNA replication activity, which is widely used in cell proliferation, cell differentiation, growth and development, and DNA damage repair.

[0068] For each compound, two concentrations were selected for verification according to the dose-effect response curve (Table 2), and the cells were of two passages, P4 and P6. The HPTC of P4 and P6 were seeded into 384-well plates at 2000 and 2500 per well respectively, cultured overnight, then treated with drugs for 48 h. The control group was treated with DMSO for 48 h. Then, EdU with a final concentration of 10 μM was added and incubated at 37 °C for 3 h, followed by fixation with 4% PFA. The DNA synthesis was detected using an EdU detection kit, the cell proliferation marker Ki67 was detected by immunofluorescence staining, and images were obtained and data were analyzed using a high-content image analysis system.

[0069] Table 2 Concentrations of small molecule compounds

[0070]

[0071] The results showed that whether it was P4 or P6, these compounds basically did not affect the expression of Ki-67 in human primary renal tubular epithelial cells, and 10 μM meptyldinocap had a significant inhibitory effect on the expression of cell Ki-67 ( Figure 5 ), and these drugs were not carcinogenic.

[0072] Based on the comprehensive analysis of the CCK8 experiment results, cell counting results, and Ki-67 results, the principle of the CCK8 experiment is that CCK8 indirectly reflects the number of viable cells by reacting with dehydrogenases in cells. From the CCK8 results, it can be seen that these compounds at least increased the viability of primary renal tubular epithelial cells, indirectly indicating an increase in the number of renal tubular epithelial cells. Ki-67 is an indicator reflecting cell proliferation, but there was little difference between the groups with and without the above-mentioned compounds, probably because the cells were just in the G0 phase during detection, so there was little difference in Ki-67 between the groups. Therefore, after the end of the M phase, Ki-67 will be quickly degraded, and cells in the G0 phase do not express Ki-67. It should be noted that as Figure 5 shown, compared with the DMSO control group, for P6 generation renal tubular epithelial cells, after using the drugs shown in Table 2, the cell number increased; for P4 generation renal tubular epithelial cells after using the drugs shown in Table 2 (except corydaline), the cell number also increased, indicating that the above-mentioned drugs promoted cell proliferation.

[0073] Example 2 Effects of small molecule compounds on urinary-derived cells

[0074] 1. Extraction and isolation of urinary-derived cells

[0075] (1) Urine collection: Disinfect the urethral opening of a normal person with iodine and collect 150-200 mL of clean midstream urine in a conical flask. Dispense the urine into 50 mL centrifuge tubes, approximately 50 mL per tube.

[0076] (2) Centrifugation: 400 × g, 10 min, 20°C. Discard the supernatant and retain the cell pellet, then resuspend in PBS.

[0077] (3) Sieve: Take a new 50 mL centrifuge tube, place a 40 μm cell strainer on the tube mouth, sieve the suspension, and then pour 1× PBS onto the strainer to make the volume of the centrifuge tube 25 mL; mix the liquid;

[0078] (4) Resuspend after centrifugation: Centrifuge at 25°C for 10 min, 400 × g; discard the supernatant and retain the cell pellet; then add 15 mL of UPC medium to resuspend the white pellet in the tube;

[0079] The UPC culture medium formula is: 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).

[0080] (5) Seeding: Divide the suspension into 24-well plates, 1 mL per well, and shake well. Place the 24-well plates in an incubator at 37°C and 5% CO2 for 1 day.

[0081] (6) Culture: Add 500 μL of UPC culture medium every day until the amount of UPC culture medium added to each well reaches 2 mL. No more culture medium is added and culture is continued for 5 days. After cell clones appear, replace the UPC culture medium every 2 days.

[0082] Results: As Figure 6 As shown, urine-derived cells were obtained.

[0083] 2. RT-qPCR Gene Detection of Cells

[0084] After the urine-derived cells in step 1 are cultured to a confluence of 80-90%, the cells are collected and tested according to the following steps:

[0085] Take some cells and extract total RNA using a Takara kit. Reverse transcribe to obtain cDNA using a Tiangen kit, and detect the expression levels of urinary cell gene markers using a Tiangen RT-qPCR kit.

[0086] Results: The results of gene marker detection are as Figure 7 shown. The extracted urinary cells express genes related to kidney function, kidney development, or renal tubule development, indicating that the urinary cells are derived from the kidney.

[0087] 3. Preliminary screening of small molecule drugs

[0088] (1) Seeding plates: Experimental group and control group: Seed P2-generation urinary cells into 96-well plates at an inoculation density of 2000 cells per well, with 100 μL of UPC medium in each well; Blank group: Add 100 μL of UPC medium to each well; Incubate at 37 °C and 5% CO2 for 24 hours.

[0089] (2) Detection at 0 hour: Add 10 μL of CCK-8 to each well, incubate at 37 °C for 3 hours, and then measure the absorbance at 450 nm using a microplate reader.

[0090] (3) Changing the medium: Add 100 μL of UPC medium to the experimental group (the UPC medium contains the corresponding small molecule compounds shown in Table 2, and their working concentrations are all 5 μM. Each compound is dissolved and diluted with DMSO). Add 100 μL of UPC medium containing the same concentration of DMSO (0.05%) as the experimental group to the control group; Add 100 μL of medium to each well in the blank group. The sample addition diagram for the 96-well plate is shown in Figure ( Figure 8 ). Incubate at 37 °C and 5% CO2 for 48 hours.

[0091] (4) Detection at 48 hours: Add 10 μL of CCK-8 to each well, incubate at 37 °C for 3 hours, and then measure the absorbance at 450 nm using a microplate reader.

[0092] Results: A total of 10 compounds were detected, with 3 replicates for each compound (Table 2). The results of CCK8 are as Figure 9 shown. Corydaline, ARN2966, and PKG drug G1 have a significant effect on improving the cell viability of urinary cells under the conditions of a working concentration of 5 μM and an incubation time of 48 h, indicating their promotion of cell proliferation; The remaining compounds did not show an effect on improving cell viability under the same conditions and had an inhibitory effect on the cells.

[0093] 4. Rescreening

[0094] Experimental purpose:

[0095] Determine the optimal incubation concentration and time of drugs that can promote the cell viability of urinary-derived cells.

[0096] Experimental operations:

[0097] (1) Seeding plates: For the experimental group and the control group, passage 2 urinary-derived cells were seeded into 96-well plates at an inoculation density of 2000 cells per well, with 100 μL of UPC medium in each well; Blank group: 100 μL of UPC medium was added to each well; Incubate at 37 °C and 5% CO2 for 24 hours.

[0098] (2) Detection at 0 hour: Add 10 μl of CCK-8 to each well, incubate at 37 °C for 3 hours, and then measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0099] (3) Adding drugs alone: Discard the medium, and the experimental group was added with UPC medium containing the small molecule compounds shown in Table 2 (for Corydaline, ARN2966, or PKG drug G1, there were two working concentration groups, 1 μM and 5 μM; the working concentration of the remaining drugs in Table 2 was 1 μM), 100 μL of medium per well (each well in the same group was added with the corresponding same compound). The control group was added with 100 μL of medium containing 0.05% DMSO per well; The blank group was added with 100 μL of medium per well. The sample addition diagram of the 96-well plate is shown in Figure ( Figure 10 ). Incubate at 37 °C and 5% CO2 for 48 hours.

[0100] (4) Detection at 48 hours: Add 10 μL of CCK-8 to each well in the 1 μM experimental group of Corydaline, ARN2966, and PKG drug G1, each well in the experimental group of the remaining drugs, each well in the control group, and each well in the blank group. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using an ELISA reader.

[0101] (5) Repeating adding drugs alone: Discard the medium from all wells, repeat the operation of the sample addition part in step (3), and then continue to incubate at 37 °C and 5% CO2 for 24 hours.

[0102] (6) Detection at 72 hours: Add 10 μl of CCK-8 to each well in the 5 μM experimental group of Corydaline, ARN2966, and PKG drug G1, each well in the experimental group of the remaining drugs, each well in the control group, and each well in the blank group. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using an ELISA reader.

[0103] Results: A total of 10 compounds were detected, with 3 replicates for each compound. Using the CCK-8 assay results of each compound, i.e., plotting the relative cell viability, and comprehensively considering the effect of the compound on improving cell viability and repeatability, the conclusion of the rescreening was that Corydaline, ARN2966, and PKG drug G1 significantly increased the viability of urinary-derived cells under the conditions of a concentration of 5 μM and an incubation time of 48 h, suggesting their promotion of cell proliferation. On this basis, when the concentration was reduced to 1 μM or the incubation time was increased to 72 h, this effect would be significantly reduced, and even inhibitory effects were observed( Figure 11 ); for the remaining compounds under the conditions of a concentration of 1 μM and an incubation time of 48 h or 72 h, only Meptyldinocap, Rifamycin sodium salt, Buparvaquone, and Carnosic acid showed a weak effect of increasing cell viability. After increasing the concentration to 5 μM on the basis of an incubation time of 48 h, none of them showed the effect of increasing cell viability, and they had inhibitory effects on cell viability( Figure 12 ).

[0104] Therefore, it was finally determined that Corydaline, ARN2966, and PKG drug G1 had a significant effect of increasing the viability of urinary-derived cells, suggesting their promotion of cell proliferation, and the corresponding optimal incubation conditions were a concentration of 5 μM and an incubation time of 48 h.

[0105] Example 3: Combination drug use

[0106] Experimental purpose: To determine whether the combination of Corydaline, ARN2966, and PKG drug G1, which can increase the viability of urinary-derived cells, also has an effect of increasing cell viability, and to determine its optimal incubation time and concentration.

[0107] Experimental method:

[0108] The specific method for grouping the combination drugs is shown in Table 3; the incubation times for each group of drugs are set at 24 h, 48 h, and 72 h respectively, and the concentrations of various drugs in each group of drugs are the same, with 20 μM, 10 μM, 5 μM, and 2.5 μM set.

[0109] Table 3: Combination drug use

[0110] Group Additives in the culture medium Experimental group 1 corydaline + ARN2966 Experimental group 2 corydaline + PKG drug G1 Experimental group 3 ARN2966 + PKG drug G1 Experimental group 4 corydaline + ARN2966 + PKG drug G1 Control group 0.2% DMSO Blank group None

[0111] (1) Seeding plates: Urinary-derived cells at passage 2 were taken for the experimental group and the control group and seeded into 96-well plates at an inoculation density of 2000 cells per well, with 100 μL of UPC medium in each well; blank group: 100 μL of UPC medium was added to each well; cultured at 37 °C and 5% CO2 for 24 hours.

[0112] (2) 0-hour detection: Add 10 μl of CCK-8 to each well. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using a microplate reader.

[0113] (3) Combination drug treatment: Discard the culture medium. Add UPC medium containing the small molecule compound combinations shown in Table 3 to the experimental groups (each compound combination in each experimental group has 4 working concentrations (the working concentrations of each drug in the same group are the same), namely 20 μM, 10 μM, 5 μM, and 2.5 μM), 100 μL of medium per well (each well in the same group is added with the corresponding same compound combination). Add 100 μL of medium containing 0.2% DMSO to each well in the control group; add 100 μL of medium to each well in the blank group. The sample addition diagram for the 96-well plate is shown in Figure ( Figure 13 ). Incubate at 37 °C and 5% CO2 for 24 hours.

[0114] (4) 24-hour detection: Add 10 μl of CCK-8 to each well. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using a microplate reader.

[0115] (5) Repeat combination drug treatment: Discard the culture medium from all wells. After repeating the operation of the sample addition part in step (3), continue to culture at 37 °C and 5% CO2 for 24 hours.

[0116] (6) 48-hour detection: Add 10 μl of CCK-8 to each well. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using a microplate reader.

[0117] (7) Repeat combination drug treatment: Discard the culture medium from all wells. After repeating the operation of the sample addition part in step (3), continue to culture at 37 °C and 5% CO2 for 24 hours.

[0118] (8) 72-hour detection: Add 10 μl of CCK-8 to each well. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using a microplate reader.

[0119] Results: A total of 4 combinations of combination drug treatments were detected, with 3 replicates for each combination. Using the CCK-8 detection verification results of each compound, that is, plotting the relative cell viability, and comprehensively considering the effect of the compound on improving cell viability and repeatability, the re-screening conclusion is: The compound combination ARN2966 + PKG drug G1 has a relatively obvious effect on improving the viability of urinary-derived cells under the conditions of a concentration of 20 μM and an incubation time of 24 h, and this effect is equivalent to the individual drug administration effect of these 2 drugs under the conditions of 5 μM and an incubation time of 48 h ( Figure 14 ).

[0120] Therefore, the compound combination ARN2966 + PKG drug G1 should have the effect of enhancing the viability of urinary-derived cells, suggesting its proliferative promoting effect. The corresponding optimal incubation conditions are a concentration of 20 μM and an incubation time of 24 h.

[0121] The above specific embodiments have described the present invention in detail. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. A culture medium for culturing kidney-derived cells, characterized in that, The culture medium contains small molecule compounds, and the small molecule compounds include Corydaline; the concentration of Corydaline is 0.003 - 10 μM; The renal-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

2. The culture medium according to claim 1, wherein The culture medium further contains a basal medium.

3. The culture medium according to claim 2, wherein: The basal medium is DMEM / F12 medium.

4. The culture medium according to claim 1, characterized in that, When the renal-derived cells are urine-derived cells, the concentration of Corydaline is 1 - 5 μM.

5. The culture medium according to claim 4, wherein: The culture medium further contains a basal medium.

6. The culture medium according to claim 5, wherein: The basal medium is a mixture of REGM medium and DMEM-high Glucose medium in a volume ratio of 1:(0.5 - 1.5).

7. The use of Corydaline or a pharmaceutically acceptable salt thereof in at least one of (I) - (VI); (I) Enhancing the viability of renal-derived cells; (II) Preparing a product for enhancing the viability of renal-derived cells; (III) Promoting the proliferation of renal-derived cells; (IV) Preparing a product for promoting the proliferation of renal-derived cells; (V) Culturing renal-derived cells; (VI) Preparing a product for culturing renal-derived cells; The renal-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

8. The application according to claim 7, wherein, When the renal-derived cells are renal tubular epithelial cells, the concentration of the small molecule compound Corydaline is 0.003 - 10 μM.

9. The application according to claim 7, wherein When the renal-derived cells are urine-derived cells, the concentration of Corydaline is 1 - 5 μM.

10. A method for improving the viability of renal cells, characterized in that, Culturing renal-derived cells using the culture medium according to any one of claims 1 - 6; The renal-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

11. The method according to claim 10, wherein: The culture time is 24 - 72 h.

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