Application of ARN2966 in renal cell culture

Abstract

The invention discloses an application of ARN2966 in culture of renal cells. The kidney-derived cells comprise renal tubular epithelial cells and urine-derived cells from kidneys. According to the present invention, it is found that the Corydaline, the ARN2966, the PKG drg G1, the Succinobcol, the Meptyldinocap, the Rifamycin sodium salt, the Carnosic acid, the TBHQ, the C188-9 and the Buparvaquone can maintain the long-term culture of the primary human renal tubular epithelial cells, and the cell activity can be improved; the application also finds that the Corydaline, the ARN2966 and the PKG drg G1 have an obvious cell viability improving effect and even a proliferation promoting effect on the urine-derived cells from the kidney, and can ensure that more urine-derived cells can be obtained at an early generation.

Description

[0001] Divisional Application Information

[0002] This invention is a divisional application. The invention title of the original application is: Application of Small Molecule Compounds in Renal Cell Culture. The application date is: September 20, 2022. The application number is: 202211143348.7. Technical Field

[0003] This invention belongs to the field of cell culture and specifically relates to the application of ARN2966 in renal cell culture. Background Art

[0004] 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 the 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.

[0005] 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.

[0006] Because the acquisition of urine-derived cells is non-invasive and the number of cells is relatively large, it has been widely studied for regenerative medicine. For example, directly reprogramming urine cells into neural stem cells (Nature Methods, doi:10.1038 / nmeth.2283); for example, inducing pluripotent stem cells derived from urine cells to differentiate 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).

[0007] 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 (e.g., metabolic studies, aging, signal transduction studies) as well as 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.

[0008] 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, the cell viability will severely decline.

[0009] 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 a large number of cells under a limited number of passages. Renal source cells (cells derived from the kidney) do not have a relatively stable growth rate. After a large number of cells are amplified, the generation number of their primary cells is already relatively high, and the cell viability decreases, which is also not conducive to cell therapy. Therefore, it is necessary to treat urinary-derived cells with small molecule drugs that can improve the proliferation ability of renal source cells to ensure that a larger number of urinary-derived cells can be obtained at an earlier generation. Summary of the Invention

[0010] The object of the present invention is to provide small molecule compounds that are beneficial to maintaining the long-term in vitro culture of renal source 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 sodiumsalt, Carnosic acid, TBHQ, C188-9, Buparvaquone.

[0011] The technical solution adopted by the present invention is:

[0012] In a first aspect of the present invention, there is provided a culture medium for culturing kidney-derived cells, the culture medium containing 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; more preferably, the small molecule compound is ARN2966.

[0013] In some embodiments of the present invention, the kidney-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

[0014] Compounds Corydaline, ARN2966, PKG drug G1, Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, Buparvaquone have a significant effect of improving cell viability and promoting cell proliferation on renal tubular epithelial cells, which is beneficial to the long-term culture of renal tubular epithelial cells.

[0015] When the kidney-derived cells are renal tubular epithelial cells, 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.

[0016] In some embodiments of the present invention, the culture medium further contains a basal medium, and the basal medium contains fetal bovine serum with a volume fraction of 5% - 20%.

[0017] In some embodiments of the present invention, the basal medium is DMEM / F12 medium.

[0018] Compound Corydaline, ARN2966, and PKG drug G1 have a significant effect of enhancing cell viability and promoting cell proliferation on renal-derived urinary cells, which is beneficial to the long-term culture of renal-derived renal tubular epithelial cells.

[0019] When the renal cells are urinary 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.

[0020] In some embodiments of the present invention, the culture medium further contains fetal bovine serum with a volume fraction of 5% - 20%.

[0021] 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).

[0022] 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.

[0023] 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.

[0024] In the second aspect of the present invention, there is provided the use of ARN2966 or a pharmaceutically acceptable salt thereof in at least one of (I) - (VI);

[0025] (I) Enhancing the viability of renal cells;

[0026] (II) Preparing a product for enhancing the viability of renal cells;

[0027] (III) Promoting the proliferation of renal cells;

[0028] (IV) Preparing a product for promoting the proliferation of renal cells;

[0029] (V) Culturing renal cells;

[0030] (VI) Preparing a product for culturing renal cells.

[0031] In some embodiments of the present invention, the renal-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

[0032] In some embodiments of the present invention, when the renal-derived cells are renal tubular epithelial cells, the concentration of the small molecule compound ARN2966 is 0.003 - 10 μM.

[0033] In some embodiments of the present invention, when the renal-derived cells are urine-derived cells, the concentration of ARN2966 is 1 - 5 μM.

[0034] In a third aspect of the present invention, a method for improving the viability of renal-derived cells is provided, which is to culture renal-derived cells using the culture medium described in the first aspect of the present invention.

[0035] In some embodiments of the present invention, the renal-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

[0036] In some embodiments of the present invention, the culture time is 24 - 72 h.

[0037] In some preferred embodiments of the present invention, the culture time is 24 - 48 h.

[0038] The beneficial effects of the present invention are as follows:

[0039] 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.

[0040] 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 renal-derived urine cells, which can ensure that a larger number of urine-derived cells can be obtained at an earlier passage. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a high-throughput screening flow chart.

[0042] Figure 2It is the primary screening result.

[0043] Figure 3 It is the rescreening result.

[0044] Figure 4 It is the dose-effect curve of each compound.

[0045] Figure 5 It is the influence of each compound on cell proliferation.

[0046] Figure 6 It is the picture of the morphology of urinary-derived cells.

[0047] Figure 7 It is the gene detection result of urinary-derived cells.

[0048] Figure 8 It is the experimental design diagram of the primary screening of urinary-derived cells with each compound added alone.

[0049] Figure 9 It is the result of the primary screening of urinary-derived cells with each compound added alone.

[0050] Figure 10 It is the experimental design diagram of the rescreening of urinary-derived cells with each compound added alone.

[0051] Figure 11 It is the result of the rescreening of urinary-derived cells with Corydaline, ARN2966, and PKG drug G1 added alone.

[0052] Figure 12 It is the result of the rescreening of urinary-derived cells with Succinobucol, Meptyldinocap, Rifamycin sodium salt, Carnosic acid, TBHQ, C188-9, and Buparvaquone added alone.

[0053] Figure 13 It is the experimental design diagram of the combined use of Corydaline, ARN2966, and PKG drug G1.

[0054] Figure 14 It is the result of the combined use of Corydaline, ARN2966, and PKG drug G1. Specific implementation manners

[0055] 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 all fall within the scope of protection of the present invention.

[0056] 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>.

[0057] Example 1 Screening of small molecule compounds

[0058] 1. High-throughput preliminary screening

[0059] The fifth-generation (P5) HPTC 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, the small molecule compounds to be screened (working concentration 5 μM) were added to each well of the plate using an automated liquid workstation, and the 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 450 nm was measured using a microplate reader ( Figure 1 ). Each compound had 2 replicates.

[0060] 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 cell survival promoting effect and repeatability of the compounds comprehensively, 11 compounds were initially screened (Table 1) for subsequent verification.

[0061] Table 1. 11 candidate compounds for subsequent verification

[0062]

[0063]

[0064] 2. Re-screening

[0065] HPTC at passage 5 was seeded into a 384-well plate at a seeding 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 respectively (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). 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. 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 made with 4 replicate wells.

[0066] 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 )

[0067] 3. Ki-67 detection

[0068] 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 this period, the cell does not divide and proliferate. Ki-67 is expressed in all phases of cell proliferation, but not in the G0 phase. The index level of Ki-67 in the pathological report indicates the rate of tumor cell proliferation.

[0069] 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.

[0070] For each compound, two concentrations were selected for verification according to the dose-response curve (Table 2). Cells of two passages, P4 and P6, were used. HPTCs 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. 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, and the cell proliferation marker Ki67 was detected by immunofluorescence staining. Images were acquired and data were analyzed using a high-content image analysis system.

[0071] Table 2 Concentrations of small molecule compounds

[0072]

[0073]

[0074] 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. Meptyldinocap at 10 μM had a significant inhibitory effect on the expression of cell Ki-67 ( Figure 5 ), and these drugs were not carcinogenic.

[0075] 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, the number of cells in P6 renal tubular epithelial cells increased after treatment with the drugs shown in Table 2; the number of cells in P4 renal tubular epithelial cells also increased after treatment with the drugs shown in Table 2 (except corydaline), suggesting that the above-mentioned drugs promote cell proliferation.

[0076] Example 2 Effects of small molecule compounds on urinary-derived cells

[0077] 1. Extraction and isolation of urinary-derived cells

[0078] (1) Urine collection: Disinfect the urethral orifice of normal individuals with iodophor, and collect 150 - 200 mL of clean midstream urine in a Erlenmeyer flask. Aliquot the urine into 50 mL centrifuge tubes, approximately 50 mL per tube;

[0079] (2) Centrifugation: Centrifugal force 400×g, time 10 min, temperature 20 °C. Discard the supernatant and retain the cell pellet, resuspend with PBS;

[0080] (3) Filtration through a sieve: Take a new 50 mL centrifuge tube, place a 40 μm cell filter mesh at the tube orifice, filter the suspension, and then pour 1×PBS onto the filter mesh until the liquid in the centrifuge tube is made up to 25 mL; Mix the liquid evenly;

[0081] (4) Resuspension 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;

[0082] The formula of the UPC medium: 50% REGM (per 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) + 50% MEF (per 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).

[0083] (5) Seeding plates: Seed the suspension into 24 - well plates, 1 mL per well, and shake well; Place the 24 - well plates in an incubator at 37 °C, 5% CO 2 and incubate statically for 1 day.

[0084] (6) Culture: Add 500 μL of UPC medium every day. After the addition amount of UPC medium in each well reaches 2 mL, stop adding medium and continue culturing for 5 days; After cell clones appear, change the UPC medium every 2 days.

[0085] Results: As Figure 6 shown, the urine - derived cells were obtained.

[0086] 2. RT - qPCR gene detection of cells

[0087] After culturing the urine - derived cells in step 1 until the cell confluence reaches 80 - 90%, collect and detect the cells according to the following steps:

[0088] Take some cells and extract total RNA using the Takara kit. Reverse transcribe to obtain cDNA using the Tiangen kit, and detect the expression levels of urinary cell gene markers using the Tiangen kit for RT-qPCR.

[0089] Results: The results of the 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.

[0090] 3. Preliminary screening of small molecule drugs

[0091] (1) Seeding plates: Experimental group, control group: Take P2-generation urinary cells and inoculate them 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% CO 2 for 24 hours.

[0092] (2) Detection at 0 hour: Add 10 μL of CCK-8 to each well, incubate at 37 °C for 3 hours, and then detect the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0093] (3) Change 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% CO 2 for 48 hours.

[0094] (4) Detection at 48 hours: Add 10 μL of CCK-8 to each well, incubate at 37 °C for 3 hours, and then detect the absorbance at 450 nm using an ELISA reader.

[0095] 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 of increasing cell viability on 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 of increasing cell viability under the same conditions and had an inhibitory effect on the cells.

[0096] 4. Re-screening

[0097] Experimental purpose:

[0098] Determine the optimal incubation concentration and time of drugs that promote the cell viability of urinary-derived cells.

[0099] Experimental operation:

[0100] (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% CO 2 for 24 hours.

[0101] (2) Detection at 0 hour: 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.

[0102] (3) Adding drugs alone: Discard the medium, 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% CO 2 for 48 hours.

[0103] (4) Detection at 48 hours: Add 10 μL of CCK-8 to each well of the 1 μM experimental group of Corydaline, ARN2966, PKG drug G1, each well of the experimental group of the remaining drugs, each well of the control group, and each well of the blank group. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using a microplate reader.

[0104] (5) Repeating adding drugs alone: Discard the medium from all wells, after repeating the operation of the sample addition part in step (3), continue to incubate at 37 °C and 5% CO 2 for 24 hours.

[0105] (6) Detection at 72 hours: Add 10 μL of CCK-8 to each well of the 5 μM experimental group of Corydaline, ARN2966, PKG drug G1, each well of the experimental group of the remaining drugs, each well of the control group, and each well of the blank group. After incubating at 37 °C for 3 hours, measure the absorbance at 450 nm using a microplate reader.

[0106] 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 enhancing cell viability and repeatability, the rescreening conclusion was that Corydaline, ARN2966, and PKG drug G1 significantly increased the cell viability of urinary-derived cells under the conditions of a concentration of 5 μM and an incubation time of 48 h, indicating 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 or even inhibitory ( 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 an effect of increasing cell viability, and they had an inhibitory effect on cell viability ( Figure 12 ).

[0107] Therefore, it was finally determined that Corydaline, ARN2966, and PKG drug G1 significantly increased the cell viability of urinary-derived cells, indicating their promotion of cell proliferation, and the corresponding optimal incubation conditions were a concentration of 5 μM and an incubation time of 48 h.

[0108] Example 3: Combination drug

[0109] 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 on increasing cell viability, and to determine its optimal incubation time and concentration.

[0110] Experimental method:

[0111] The specific method for grouping the combination drugs is shown in Table 3; the incubation times for each group of drugs were set at 24 h, 48 h, and 72 h respectively, and the concentrations of various drugs in each group of drugs were the same, with 20 μM, 10 μM, 5 μM, and 2.5 μM.

[0112] Table 3 Combination drugs

[0113] 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

[0114] (1) Seeding: Urinary-derived cells of passage 2 were taken for the experimental group and the control group and seeded into a 96-well plate 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; incubated at 37 °C and 5% CO 2 for 24 hours.

[0115] (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.

[0116] (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% CO 2 for 24 hours.

[0117] (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.

[0118] (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 incubate at 37 °C and 5% CO 2 for 24 hours.

[0119] (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.

[0120] (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 incubate at 37 °C and 5% CO 2 for 24 hours.

[0121] (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.

[0122] Results: A total of 4 combinations of combined 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 in enhancing cell viability and repeatability, the re-screening conclusion is: The compound combination ARN2966 + PKG drug G1 has a relatively obvious effect in enhancing 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 administration effect of these two drugs under the conditions of 5 μM and an incubation time of 48 h ( Figure 14 ).

[0123] 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.

[0124] 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 relevant technical field, various changes can be made without departing from the spirit 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 culture medium for culturing kidney-derived cells, characterized in that: the culture medium contains small molecule compounds, and the small molecule compounds include ARN2966.

2. The culture medium according to claim 1, characterized in that: the kidney-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney; preferably, the concentration of ARN2966 is 0.003 - 10 μM.

3. The culture medium according to claim 1, characterized in that: the culture medium further includes small molecule compound Corydaline and / or PKG drug G1.

4. The culture medium according to claim 3, characterized in that: when the kidney-derived cells are renal tubular epithelial cells, the concentrations of the small molecule compounds ARN2966, Corydaline, and / or PKG drug G1 are all 0.003 - 10 μM; preferably, the culture medium further contains a basal medium, and the basal medium is preferably DMEM / F12 medium; when the kidney-derived 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; preferably, the culture medium further contains a basal medium, and the basal medium is a mixture of REGM medium and DMEM-high Glucose medium in a volume ratio of 1:(0.5 - 1.5).

5. Use of ARN2966 or a pharmaceutically acceptable salt thereof in at least one of (I) - (VI); (I) Enhancing the viability of kidney-derived cells; (II) Preparing a product for enhancing the viability of kidney-derived cells; (III) Promoting the proliferation of kidney-derived cells; (IV) Preparing a product for promoting the proliferation of kidney-derived cells; (V) Culturing kidney-derived cells; (VI) Preparing a product for culturing kidney-derived cells.

6. The use according to claim 5, characterized in that, the kidney-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

7. The use according to claim 5, characterized in that, when the kidney-derived cells are renal tubular epithelial cells, the concentration of the small molecule compound ARN2966 is 0.003 - 10 μM.

8. The use according to claim 5, characterized in that, when the kidney-derived cells are urine-derived cells, the concentration of ARN2966 is 1 - 5 μM.

9. A method for enhancing the viability of kidney-derived cells, characterized in that, culturing kidney-derived cells using the culture medium according to any one of claims 1 - 4; preferably, the culturing time is 24 - 72 h.

10. The method according to claim 9, characterized in that, the kidney-derived cells include renal tubular epithelial cells and / or urine-derived cells from the kidney.

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