A complete culture medium for renal tubular epithelial cells and its application

Abstract

The invention discloses a complete culture medium for renal tubular epithelial cells and its application, wherein the complete culture medium for renal tubular epithelial cells includes a serum-free basal medium, fetal bovine serum accounting for 5-10% (v / v) of the serum-free basal medium, 1-10 μg / mL growth hormone, 1-2 μg / mL chitosan, 10-20 ng / mL epidermal growth factor, 10-20 ng / mL interleukin-17 cytokine and 10-20 ng / mL cholera toxin. The complete culture medium for renal tubular epithelial cells of the present invention can significantly improve the proliferation ability of renal tubular epithelial cells, and effectively maintain the morphology and properties of the cells, and when cultured to the P3 generation, the cells can still grow vigorously and the cell purity can reach more than 90%, and at the same time, each component of the complete culture medium is easily obtained and convenient to prepare, so it can be developed into a commercial product.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a complete culture medium for renal tubular epithelial cells and application thereof in in vitro culture of renal tubular epithelial cells. Background Art

[0002] Renal tubular epithelial cells are important carriers and tool cells for studying acute renal injury, chronic kidney disease, diabetic nephropathy, renal interstitial fibrosis, kidney stones and other renal diseases. They are one of the main cells that constitute the tubular interstitium and are also another important component of the nephron. Together with the glomerulus, they form a complete functional nephron with the following functions: reabsorption; secretion, excretion; regulation of water and electrolyte balance; regulation of acid-base balance, etc. Renal tubular epithelial cells are not only related to the pathological and physiological processes of the renal tubules, but are also the main site of injury in many congenital diseases, metabolic diseases and inflammation.

[0003] The in vitro culture of renal tubular epithelial cells has high requirements for the culture medium nutrition, and increasing the growth rate, increasing the number of passages and maintaining the cell characteristics after passage are particularly important for using renal tubular epithelial cells to study various diseases in vitro. Patent application CN 111593016A discloses a method for separating and culturing human renal epithelial cells, and its culture medium components are: 5mL 100× double antibody, 10-20ng / ml human recombinant EGF, 5-10ng / ml interleukin-6, 15-20% fetal bovine serum, and the balance is DMEM / F12. Patent US4456687A discloses that cholera toxin, methyl isobutyl xanthine, and isoproterenol effectively promote growth and can increase cell cyclic AMP and cell proliferation. However, no commercial products developed based on the above patents are seen on the market, and the actual culture effect is still uncertain.

[0004] At present, the more mature epithelial cell culture medium on the market is mainly Sciencell's EPICM culture medium, which has a relatively good effect in culturing epithelial cells in vitro, but its formula is confidential and expensive, and the inventors found that the culture medium is not suitable for the culture of renal tubular epithelial cells. When the cells are subcultured to the P3 generation, the cell morphology has changed significantly and the cell purity has been significantly reduced. Therefore, the development of a special culture medium for renal tubular epithelial cells with clear effects and low costs has become a necessary prerequisite for in-depth research and application of renal tubular epithelial cells, and is particularly important for using renal tubular epithelial cells to study various diseases in vitro. Summary of the invention

[0005] In view of this, the present invention aims to provide a culture medium suitable for renal tubular epithelial cells, which, when used to culture renal tubular epithelial cells, can effectively improve the proliferation and passage ability of renal tubular epithelial cells.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a complete culture medium for renal tubular epithelial cells, which complete culture medium comprises: a serum-free basal culture medium, fetal bovine serum (FBS) accounting for 5-10 v / v% of the serum-free basal culture medium, 1-10 μg / mL growth factor, 1-2 μg / mL chitosan, 10-20 ng / mL epidermal growth factor, 10-20 ng / mL interleukin-17 cytokine and 10-20 ng / mL cholera toxin.

[0008] In the complete culture medium for renal tubular epithelial cells of the present invention, the serum-free culture medium can be selected from DMEM / F12, DMEM, 1640, etc. In some specific embodiments of the present invention, the serum-free culture medium is DMEM / F12.

[0009] In one embodiment of the present invention, the complete culture medium for renal tubular epithelial cells includes: serum-free basal medium, fetal bovine serum accounting for 5% volume of the serum-free basal medium, 5 μg / mL growth factor, 1.5 μg / mL chitosan, 13 ng / mL epidermal growth factor, 15 ng / mL interleukin-17 cytokine and 13 ng / mL cholera toxin.

[0010] The experimental data of the present invention show that there is a synergistic effect between the components such as auxin, chitosan, epidermal growth factor, interleukin-17 cytokine and cholera toxin in the complete culture medium. Under reasonable concentration and ratio conditions, the proliferation ability of renal tubular epithelial cells can be effectively improved, the morphology and properties of the cells can be effectively maintained, and the growth of foreign cells can be inhibited; when the renal tubular epithelial cells are cultured to the P3 generation using the complete culture medium, the cells still grow vigorously and the cell purity can reach more than 90%.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned complete culture medium for renal tubular epithelial cells, comprising the following steps:

[0012] Weigh 0.25 g of auxin and dissolve it in 5 mL of anhydrous ethanol to obtain reagent 1; weigh 0.01 g of chitosan and dissolve it in 10 mL of 1% acetic acid to obtain reagent 2; use a sterile syringe and a sterile 0.22 μm filter membrane to filter and sterilize reagent 1 and reagent 2;

[0013] Dissolve 50 μg of epidermal growth factor in 1 mL of sterile purified water to obtain reagent 3; dissolve 50 μg of interleukin-17 cytokine in 1 mL of sterile purified water to obtain reagent 4; dissolve 50 μg of cholera toxin in 1 mL of sterile purified water to obtain reagent 5;

[0014] According to the composition of the complete medium for renal tubular epithelial cells, take appropriate amounts of reagent 1, reagent 2, reagent 3, reagent 4, reagent 5, FBS and serum-free basal medium and mix them evenly to obtain the complete medium.

[0015] The third aspect of the present invention provides the use of the above-mentioned complete culture medium for renal tubular epithelial cells, which is at least any of the following:

[0016] a) Application in in vitro culture of renal tubular epithelial cells;

[0017] b) Application in improving the proliferation and passage ability of renal tubular epithelial cells.

[0018] A fourth aspect of the present invention provides a method for culturing renal tubular epithelial cells in vitro, wherein the method uses the complete renal tubular epithelial cell culture medium of the present invention to culture renal tubular epithelial cells in vitro.

[0019] Furthermore, the method for culturing renal tubular epithelial cells in vitro specifically comprises the following steps:

[0020] S1, adding renal tubular epithelial cells into complete medium for renal tubular epithelial cells to obtain P1 generation cells;

[0021] S2. When the cell confluence reaches 70% to 90%, the digested cells are processed and inoculated into a complete culture medium for renal tubular epithelial cells to obtain P2 generation cells;

[0022] S3. Repeat step S2 to subculture and expand the renal tubular epithelial cells.

[0023] Furthermore, in the method for culturing renal tubular epithelial cells in vitro, the process of the treatment described in step S2 is: after washing with a washing solution, adding a digestion reagent for digestion, and then terminating the digestion with a complete medium for renal tubular epithelial cells. In some specific embodiments of the present invention, the washing solution is PBS buffer, and the digestion reagent is 0.25% (w / v) trypsin solution containing 0.04% (w / v) EDTA.

[0024] Furthermore, in the method for culturing renal tubular epithelial cells in vitro, the culture conditions include: a culture temperature of 37° C. and a CO 2 concentration of 5% (v / v).

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] Based on the characteristics of renal tubular epithelial cells, the present invention specifically develops a complete culture medium suitable for renal tubular epithelial cells, which can effectively improve the proliferation ability of renal tubular epithelial cells, effectively maintain the morphology and properties of the cells, and inhibit the growth of foreign cells; when the renal tubular epithelial cells are cultured to the P3 generation using the complete culture medium, the cells still grow vigorously, and the cell purity can reach more than 90%. Moreover, the various components of the complete culture medium are easy to obtain, and the preparation is convenient, which greatly reduces the cost of culturing renal tubular epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The cell morphology diagram of renal tubular epithelial cells under different culture medium conditions of the present invention.

[0028] Figure 2 The figure is a cell morphology diagram of the renal tubular epithelial cells after multiple subcultures of the renal tubular epithelial cells using the complete culture medium for renal tubular epithelial cells of the present invention and the EPICM culture medium.

[0029] Figure 3 It is a growth curve diagram of renal tubular epithelial cells under different culture medium conditions of the present invention.

[0030] Figure 4 The figure is a fluorescence staining diagram of cells after multiple subculture of renal tubular epithelial cells with the complete culture medium for renal tubular epithelial cells of the present invention and the EPICM culture medium. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these embodiments are exemplary and are only used to explain the present invention, rather than to limit the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art; the term "including" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0033] The FBS used in the following examples was purchased from Wuhan Prosai Life Science Co., Ltd. (Cat. No. 164210), the primary rat renal tubular epithelial cells used were purchased from Wuhan Prosai Life Science Co., Ltd. (Cat. No. CP-R062), and the EPICM (Epithelial Cell Medium) culture medium used was purchased from Sciencell (Cat. No. 4101).

[0034] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0035] Example 1

[0036] The complete culture medium for renal tubular epithelial cells provided in this example includes the following components and contents:

[0037] 2.5 mg of auxin, 0.75 mg of chitosan, 6.5 μg of epidermal growth factor, 7.5 μg of interleukin-17 cytokine, 6.5 μg of cholera toxin, 25 mL of fetal bovine serum and 475 mL of DMEM / F12 basal medium.

[0038] Weigh 0.25 g of auxin and dissolve it in 5 mL of anhydrous ethanol to obtain reagent 1; weigh 0.01 g of chitosan and dissolve it in 10 mL of 1% acetic acid to obtain reagent 2; use a sterile syringe and a sterile 0.22 μm filter membrane to filter and sterilize reagent 1 and reagent 2; dissolve 50 μg of epidermal growth factor in 1 mL of sterile purified water to obtain reagent 3; dissolve 50 μg of interleukin-17 cytokine in 1 mL of sterile purified water to obtain reagent 4; dissolve 50 μg of cholera toxin in 1 mL of sterile purified water to obtain reagent 5; take 50 μL of reagent 1, 0.75 mL of reagent 2, 130 μL of reagent 3, 150 μL of reagent 4, 130 μL of reagent 5, 25 mL of FBS and 475 mL of serum-free basal culture medium, mix them evenly, filter and sterilize them with a 0.2 μm PVDF (polyvinylidene fluoride) filter membrane to obtain a complete culture medium for renal tubular epithelial cells.

[0039] Example 2

[0040] The complete culture medium for renal tubular epithelial cells provided in this example includes the following components and contents:

[0041] 5 mg of auxin, 0.5 mg of chitosan, 10 μg of epidermal growth factor, 10 μg of interleukin-17 cytokine, 10 μg of cholera toxin, 45 mL of fetal bovine serum and 455 mL of DMEM basal medium.

[0042] The preparation method of complete culture medium for renal tubular epithelial cells is as described in Example 1.

[0043] Example 3

[0044] The complete culture medium for renal tubular epithelial cells provided in this example includes the following components and contents:

[0045] 0.5 mg auxin, 1 mg chitosan, 5 μg epidermal growth factor, 5 μg interleukin-17 cytokine, 5 μg cholera toxin, 25 mL fetal bovine serum and 475 mL 1640 basal medium.

[0046] The preparation method of complete culture medium for renal tubular epithelial cells is as described in Example 1.

[0047] Example 4

[0048] The complete culture medium for renal tubular epithelial cells provided in this example includes the following components and contents:

[0049] 3 mg of growth factor, 0.55 mg of chitosan, 7.5 μg of epidermal growth factor, 7.5 μg of interleukin-17 cytokine, 7.5 μg of cholera toxin, 25 mL of fetal bovine serum and 475 mL of DMEM basal medium.

[0050] The preparation method of complete culture medium for renal tubular epithelial cells is as described in Example 1.

[0051] Example 5

[0052] The complete culture medium for renal tubular epithelial cells provided in Examples 1-4 was used to culture renal tubular epithelial cells in vitro, and the effects of each complete culture medium on the cell morphology and proliferation of renal tubular epithelial cells were detected.

[0053] 1. Detection of cell morphology, including the following steps:

[0054] (1) Primary rat renal tubular epithelial cells were centrifuged and added to 5 mL of complete renal tubular epithelial cell culture medium and suspended evenly. The cells were then inoculated into a 6 cm culture dish and cultured in an incubator at 37° C. containing 5% (v / v) CO 2 . The resulting cells were designated as the P1 generation.

[0055] (2) When the cell confluence reaches 90%, the supernatant is discarded, the cells are washed twice with PBS buffer, and 0.25% (w / v) trypsin containing 0.04% (w / v) EDTA is added for digestion. The amount of trypsin added is 1 mL / 6 cm culture dish. After the cells fall off, complete culture medium for renal tubular epithelial cells is added to terminate digestion. The volume of complete culture medium for renal tubular epithelial cells is 5 times the volume of trypsin.

[0056] (3) The digested cells were centrifuged at 1000 prm for 5 min, the supernatant was discarded, and the cells were centrifuged at 3 × 10 5 The cells were resuspended in complete tubular epithelial cell culture medium at a density of 1 cell / mL, and 1 mL of the cell suspension was inoculated into a 6-well plate at each well and cultured in an incubator at 37°C containing 5% (v / v) CO2. After 48 h of culture, the cell morphology was observed under an inverted microscope.

[0057] (4) Repeat steps (2) and (3) to perform conventional subculture on the cells.

[0058] 2. The detection of proliferation effect includes the following steps:

[0059] (1) Primary rat renal tubular epithelial cells were centrifuged and added to 5 mL of complete renal tubular epithelial cell culture medium and suspended evenly. The cells were then inoculated into a 6 cm culture dish and cultured in an incubator at 37° C. containing 5% (v / v) CO 2 . The resulting cells were designated as the P1 generation.

[0060] (2) When the P1 cell confluence reaches 90%, the supernatant is discarded, the cells are washed twice with PBS buffer, and 0.25% (w / v) trypsin containing 0.04% (w / v) EDTA is added for digestion. The amount of trypsin added is 1 mL / 6 cm culture dish. After the cells fall off, complete culture medium for renal tubular epithelial cells is added to terminate digestion. The volume of complete culture medium for renal tubular epithelial cells is 5 times the volume of trypsin.

[0061] (3) The digested cells were centrifuged at 1000 prm for 5 min, the supernatant was discarded, and the cells were centrifuged at 3 × 10 5 The cells were resuspended in complete culture medium for renal tubular epithelial cells at a density of 10 / mL, and 0.1 mL of the cell suspension was inoculated into a 96-well plate per well and cultured in an incubator at 37°C containing 5% (v / v) CO2. The medium was changed every 2 days.

[0062] (4) On the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day of culture, the proliferation effect of renal tubular cells was measured using a CCK8 kit according to the instructions of the kit.

[0063] Comparative Example 1

[0064] Different from Example 5, this example uses EPICM culture medium to culture renal tubular epithelial cells in vitro.

[0065] Comparative Example 2

[0066] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 0.1 mg of auxin, 0.1 mg of chitosan, 4 μg of epidermal growth factor, 15 μg of interleukin-17 cytokine, 4 μg of cholera toxin, 25 mL of fetal bovine serum and 475 mL of DMEM / F12 basal culture medium. The preparation method of the culture medium is referred to Example 1.

[0067] Compared with the complete culture medium of Example 1, the contents of auxin, chitosan, epidermal growth factor, interleukin-17 cytokine and cholera toxin in the culture medium of this example are not within the scope defined by the present invention.

[0068] Comparative Example 3

[0069] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 2.5 mg of growth hormone, 25 mL of fetal bovine serum and 475 mL of DMEM / F12 basal medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium in Example 1, the culture medium in this example contains only FBS and growth hormone in addition to the basal medium.

[0070] Comparative Example 4

[0071] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 0.75 mg chitosan, 25 mL fetal bovine serum and 475 mL DMEM / F12 basal medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium in Example 1, the culture medium in this example contains only FBS and chitosan in addition to the basal medium.

[0072] Comparative Example 5

[0073] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 6.5 μg epidermal growth factor, 25 mL fetal bovine serum and 475 mL DMEM / F12 basal medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium in Example 1, the culture medium in this example contains only FBS and epidermal growth factor in addition to the basal medium.

[0074] Comparative Example 6

[0075] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 7.5 μg interleukin-17 cytokine, 25 mL fetal bovine serum and 475 mL DMEM / F12 basal medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium in Example 1, the culture medium in this example contains only FBS and interleukin-17 cytokine in addition to the basal medium.

[0076] Comparative Example 7

[0077] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 6.5 μg cholera toxin, 25 mL fetal bovine serum and 475 mL DMEM / F12 basal medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium in Example 1, the culture medium in this example contains only FBS and cholera toxin in addition to the basal medium.

[0078] Comparative Example 8

[0079] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 2.5 mg of auxin, 0.75 mg of chitosan, 25 mL of fetal bovine serum and 475 mL of DMEM / F12 basal medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium of Example 1, the culture medium in this example does not contain epidermal growth factor, interleukin-17 cytokine, and cholera toxin.

[0080] Comparative Example 9

[0081] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 2.5 mg of auxin, 0.75 mg of chitosan, 6.5 μg of epidermal growth factor, 25 mL of fetal bovine serum and 475 mL of DMEM / F12 basal culture medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium of Example 1, the culture medium in this example does not contain interleukin-17 cytokine and cholera toxin.

[0082] Comparative Example 10

[0083] Different from Example 5, the culture medium used for in vitro culture of renal tubular epithelial cells in this example includes the following components: 2.5 mg of auxin, 0.75 mg of chitosan, 6.5 μg of epidermal growth factor, 7.5 μg of interleukin-17 cytokine, 25 mL of fetal bovine serum and 475 mL of DMEM / F12 basal culture medium. The preparation method of the culture medium is referred to Example 1. Compared with the complete culture medium of Example 1, the culture medium in this example does not contain cholera toxin.

[0084] According to the culture method of Example 5, after culturing renal tubular epithelial cells for 48 hours using the complete culture medium in Examples 1 to 4 and the culture medium in Comparative Examples 1 to 10, the cell morphology is as follows: Figure 1 As shown (a to n correspond to Example 1, Example 2, Example 3, Example 4 and Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, Comparative Example 8, Comparative Example 9, Comparative Example 10, respectively). Figure 1It can be seen that the cell morphology in Comparative Example 1 and Examples 1 to 4 is oval and paving stone-like, which is a typical epithelial cell morphology; although the cell morphology of Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, Comparative Example 8, Comparative Example 9, and Comparative Example 10 is normal, the cell amount is small; the cell morphology in Comparative Example 2 has changed significantly. The above results show that when the complete medium for renal tubular epithelial cells provided by the present invention is used to culture renal tubular epithelial cells, it can well maintain the morphology and growth rate of renal tubular epithelial cells. Compared with the EPICM medium of Comparative Example 1, there is no significant difference. However, if the main component composition and ratio of the complete medium for renal tubular epithelial cells provided by the present invention are changed, the renal tubular epithelial cells will not be able to maintain the normal morphology and growth rate.

[0085] Figure 2 The cell morphology of renal tubular epithelial cells after multiple wound culture using the complete medium of Example 1 and the EPICM medium of Comparative Example 1, respectively. Figure 2 It can be seen that when the complete medium of the present invention is used to culture renal tubular epithelial cells, the cells still maintain normal morphology and growth rate at the P3 generation, while when the EPICM medium is used, the cell morphology has changed significantly at the P3 generation, and the number of miscellaneous cells has increased.

[0086] According to the culture method of Example 5, after the complete culture medium in Examples 1 to 4 and the culture medium in Comparative Examples 1 to 10 were used to culture the renal tubular epithelial cells, the cell growth curves were as follows: Figure 3 As shown. Figure 3 It can be seen that when the complete culture medium of the present invention is used to culture renal tubular epithelial cells, the cells grow vigorously and proliferate rapidly. After reaching the plateau phase, a large number of cells can be maintained alive, and there is no significant difference among the embodiments; when the culture medium in comparative examples 2 to 10 is used to culture renal tubular epithelial cells, not only is the cell proliferation rate lower than that of the embodiments, but the total amount of cells after reaching the plateau phase is also less than that of the embodiments; in the comparative examples, the propagation rate of comparative examples 3 to 12 is also significantly lower than that of comparative example 2, and the total amount of cells after reaching the plateau phase is also less; when the renal tubular epithelial cells are cultured using the EPICM culture medium in comparative example 1, the cell proliferation rate in the first 2 days of the growth curve is not significantly different from that of the embodiments, but the cell proliferation rate and the maximum cell number are significantly lower than those of the embodiments as time goes on. The above results show that when the complete medium for renal tubular epithelial cells provided by the present invention is used to culture renal tubular epithelial cells, it can effectively increase the proliferation rate of renal tubular epithelial cells and maintain the survival of a large number of cells. However, changing the composition and ratio of the main components in the complete medium for renal tubular epithelial cells provided by the present invention will lead to a decrease in the proliferation rate of renal tubular epithelial cells and a decrease in the total amount of cells obtained.

[0087] In addition, using EPICM medium as a control, the purity of the renal tubular epithelial cells obtained by culturing the complete medium for renal tubular epithelial cells of the present invention (Example 1) was identified by immunofluorescence method, and the specific steps were as follows:

[0088] ① Place the poly-lysine-coated slides in a 6-well plate, then inoculate renal tubular epithelial cells at a volume of 300,000 cells / well. After culturing for 2 days, aspirate the culture medium, add 1 mL of PBS, slowly add from the edge of the well plate, and aspirate the PBS after about 10 seconds; add 500 μL of 4% (v / v) paraformaldehyde, fix at room temperature for 15 minutes, and avoid light. Aspirate the paraformaldehyde, add 1 mL of PBS to wash once, and then add 1 mL of PBS to soak the slides again.

[0089] ② Add 1 mL of 0.5% (v / v) Triton X-100 to the 6-well plate to cover the cells and fix for 20 minutes. Aspirate the Triton X-100 and add 1 mL of pre-cooled PBS to wash three times, 3 minutes each time.

[0090] ③ Transfer the slide to a glass slide and store the slide in a black box away from light. Add 50 μL of 10% (v / v) goat serum (purchased from Wuhan Boster Biotechnology Co., Ltd., item number AR1009) to each slide for blocking and incubate at room temperature for 30 minutes. After blocking, use tweezers to remove the slide, absorb the serum from the side, and place it back on the slide.

[0091] ④ Add 50 μL CK18 primary antibody (purchased from Proteintech, catalog number 10830-1-AP), incubate overnight (about 16 hours) in a 4°C refrigerator, and store in a black box away from light. After incubation, clamp the slide to a 6-well plate, wash it 3 times with PBS, soak it for 3 minutes, and pay attention to avoid light during the process. Then transfer the slide back to the slide in the black box.

[0092] ⑤ Add 50 μL of secondary antibody (purchased from Wuhan Boster Biotechnology Co., Ltd., catalog number BA1032) and incubate at 37°C for 1 hour in the dark. After the secondary antibody incubation, clamp the slide to a 6-well plate, wash it 3 times with PBS, soak it for 3 minutes, and keep it away from light. Then transfer the slide back to the slide in the black box.

[0093] ⑥ Add 50 μL DAPI drops (purchased from Elabscience, catalog number E-IR-R103) and stain at room temperature in the dark for 5 minutes. After DAPI staining, clamp the slide to a 6-well plate, wash it 3 times with PBS, soak it for 3 minutes, and then transfer the slide back to the slide in the black box.

[0094] ⑦ Add 10ul of anti-fluorescence quenching sealing agent (purchased from Southern Biotech, product number 0100-01) on the slide for sealing.

[0095] ⑧ Take photos and observe under a fluorescence microscope, calculate the number of immunofluorescence positive cells (shown in red), and its percentage in the total nucleated cells (shown in blue) is the purity of renal tubular epithelial cells.

[0096] The results of the purity test of renal tubular epithelial cells are as follows Figure 4 As shown. Figure 4 It can be seen that when the complete medium of the present invention is used to culture renal tubular epithelial cells, the purity of each generation of cells is high, and the cell purity is still maintained at more than 90% at the P3 generation; when the EPICM medium is used, the cell purity at the P3 generation decreases significantly. This shows that the complete medium for renal tubular epithelial cells provided by the present invention can better maintain the purity of renal tubular epithelial cells in multiple subcultures of renal tubular epithelial cells.

[0097] In summary, the complete culture medium for renal tubular epithelial cells provided by the present invention can significantly enhance the proliferation ability of renal tubular epithelial cells, effectively maintain the morphology and properties of the cells, and inhibit the growth of foreign cells. It is of great significance for the in vitro culture and research of renal tubular epithelial cells. Moreover, the components are simple and easy to obtain, and the preparation is convenient, which is conducive to the development into commercial products.

[0098] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A complete culture medium for renal tubular epithelial cells, characterized in that The invention comprises a DMEM / F12 culture medium, fetal bovine serum accounting for 5-10% (v / v) of the DMEM / F12 culture medium, 1-10 μg / mL growth factor, 1-2 μg / mL chitosan, 10-20 ng / mL epidermal growth factor, 10-20 ng / mL interleukin-17 cytokine and 10-20 ng / mL cholera toxin.

2. Use of the complete renal tubular epithelial cell culture medium as claimed in claim 1 in the in vitro culture of primary rat renal tubular epithelial cells.

3. Use of the complete renal tubular epithelial cell culture medium as claimed in claim 1 in improving the proliferation and passage ability of primary rat renal tubular epithelial cells.

4. A method for culturing renal tubular epithelial cells in vitro, characterized in that: Primary rat renal tubular epithelial cells are cultured using the complete renal tubular epithelial cell culture medium according to claim 1.

5. The method according to claim 4, characterized in that The following steps are involved: S1, adding primary rat renal tubular epithelial cells into complete renal tubular epithelial cell culture medium to obtain P1 cells; S2. When the cell confluence reaches 70% to 90%, the digested cells are processed and inoculated into complete culture medium for renal tubular epithelial cells to obtain P2 generation cells; S3. Repeat step S2 to subculture and expand primary rat renal tubular epithelial cells.

6. The method according to claim 5, characterized in that The treatment process in step S2 is: after washing with a washing solution, adding a digestion reagent for digestion, and then terminating the digestion with a complete culture medium for renal tubular epithelial cells.

7. The method according to claim 6, characterized in that The washing solution is PBS buffer.

8. The method according to claim 6, characterized in that The digestion reagent is 0.25% (w / v) trypsin solution containing 0.04% (w / v) EDTA.

9. The method according to claim 4, characterized in that The culture conditions include: a culture temperature of 37° C. and a CO 2 concentration of 5% (v / v).

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