Construction method and application of kidney cell co-culture microsphere model

Abstract

The invention discloses a construction method and application of a kidney cell co-culture microsphere model, and belongs to the technical field of cell culture. According to the method, an in-vitro model closer to in-vivo physiological characteristics is successfully constructed by optimizing the proportion and the initial balling amount of three cells with different physiological functions, and the method comprises the following steps: human renal cortex proximal tubule epithelial cells HK-2, human embryo kidney cells HEK293T, human kidney cells HEK293T, human kidney cells HEK293T, human kidney cells HEK293T, human kidney cells HEK293T and human kidney cells HEK293T are preparing a mixed cell suspension from human umbilical vein fusion cells EA.hy926 according to a ratio of 4: 1: 1 and an initial balling amount of 1500 cells; inoculating to a 96-hole ultra-low adsorption culture plate, and carrying out three-dimensional self-assembly culture for 48 hours to finally form a kidney microsphere model with a space structure; the model disclosed by the invention is closer to a cell environment in a human body, and has important application value in the aspects of toxin safety evaluation and kidney disease mechanism research.

Description

Technical Field

[0001] The present invention relates to a method for constructing a renal cell co-culture microsphere model and application thereof, belonging to the technical field of cell culture. Background Art

[0002] The toxicological evaluation system has undergone a transformation from traditional animal experiments to in vitro models with stronger physiological relevance. Based on the 3R principle (replacement, reduction, and optimization), traditional animal models are limited in extrapolation due to species differences and metabolic heterogeneity, and have bottlenecks such as long cycles and low throughput. The two-dimensional monolayer cell model is widely used in in vitro toxicity assessment due to its standardized operation advantages and has certain advantages in early drug research. However, its static culture system has limitations such as the lack of complex structures necessary for cell-cell and cell-matrix interactions, loss of polarity and attenuation of metabolic function. It is unable to reconstruct the three-dimensional topological structure of the tissue microenvironment, and in vitro cells are prone to phenotypic drift, resulting in a significant reduction in physiological relevance, which significantly affects the accuracy of toxicity prediction.

[0003] The kidney is a vital organ for metabolism and excretion, responsible for numerous key physiological functions, including filtering blood, regulating electrolyte balance and blood pressure, and secreting hormones. It is also a target organ for a variety of toxins and drug toxicity. Compared to other organs, the kidney has a highly complex microstructure and cellular composition, making in vitro simulation of its function particularly difficult but also necessary. Currently, three-dimensional cell models are often formed with the assistance of scaffolds or materials. Traditional scaffold materials and photoinitiation processes can cause immune responses, chronic inflammation, or toxicity issues from degradation products, and are complex to operate. Studies have shown that when the concentration of the photoinitiator Irgacure 651 is 0.01% (w / w), the relative cell survival rate is 54%; when the concentration increases to 0.02% (w / w), the relative cell survival rate drops significantly to 2%. Therefore, while ensuring evaluation accuracy, it is crucial to simplify the process and reduce potential biotoxicity.

[0004] Therefore, it is particularly important to construct an evaluation model that can better simulate the real physiological environment and meet the advantages of high throughput and accurate screening. The scaffold-free three-dimensional renal cell spheres reduce unnecessary animal consumption, can simulate in vivo cell responses and interactions, and have the advantages of high throughput screening and easy operation, which helps to improve the accuracy of fungal toxin evaluation. Summary of the Invention

[0005] To address the above problems, the present invention provides a method for constructing a scaffold-free three-dimensional renal cell microsphere model and its application in nephrotoxicity assessment. Human renal cortical proximal tubule epithelial cells HK-2 with metabolic function, human embryonic kidney cells HEK293T that provide matrix support, and human umbilical vein fusion cells EA.hy926 that form the prototype of a microvascular network are co-cultured, the cell ratio and the number of spheres are optimized, and the cell self-aggregation tendency is utilized to realize the construction of a renal microsphere model under scaffold-free conditions. This provides a new in vitro research platform for studying the mechanism of action of nephrotoxic substances and improves the accuracy of nephrotoxicity toxicological evaluation of fungal toxins.

[0006] The first object of the present invention is to provide a method for constructing a scaffold-free three-dimensional cell microsphere model for nephrotoxicity evaluation, comprising the steps of:

[0007] (1) Human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T, and human umbilical vein fusion cells EA.hy926 were mixed to obtain a mixed cell suspension;

[0008] (2) culturing the mixed cell suspension to obtain a scaffold-free three-dimensional cell microsphere model;

[0009] Wherein, in step (1), the ratio of the number of human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T and human umbilical vein fusion cells EA.hy926 is 1-8:1:1;

[0010] The initial cell amount of the scaffold-free three-dimensional cell microsphere model in step (2) is 500 to 3000 cells.

[0011] In one embodiment, in step (1), the quantitative ratio of human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T and human umbilical vein fusion cells EA.hy926 is 4:1:1.

[0012] In one embodiment, the initial cell amount of the scaffold-free three-dimensional cell microsphere model in step (2) is 1500 cells.

[0013] In one embodiment, the culturing in step (2) is for 3 to 18 days.

[0014] In one embodiment, the culture in step (2) is for 6 to 8 days.

[0015] In one embodiment, the culture temperature in step (2) is 37°C.

[0016] In one embodiment, in step (2), the culture medium is replaced by half every 48 hours.

[0017] The second object of the present invention is to provide a scaffold-free three-dimensional cell microsphere model constructed by any of the above methods.

[0018] The third object of the present invention is to provide the application of the above-mentioned scaffold-free three-dimensional cell microsphere model in the evaluation of nephrotoxicity.

[0019] In one embodiment, the nephrotoxicity is assessed as nephrotoxicity induced by OTA toxin or CIT toxin.

[0020] Beneficial effects of the present invention

[0021] (1) The present invention co-cultures HK-2 cells, HEK293T cells, and EA.hy926 cells to simulate the metabolic interaction network of key functional cells in renal tissue in vitro. Compared with the traditional monolayer culture system, this model dynamically reconstructs the renal tissue-specific microenvironment by enhancing the synergistic effects of cell-matrix-cell three-dimensional interactions and paracrine signaling pathways, significantly improving the physiological relevance of the in vitro nephrotoxicity assessment model.

[0022] (2) Compared with animal experiments, the cell evaluation model used in the present invention is a species homologous system constructed based on human cells, which can effectively avoid the metabolic pathway deviation caused by species differences in rodent experiments. In addition, the experimental process and operation are simpler, the experimental cycle is significantly shortened, and the cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Bright field images of microsphere morphology at different initial sphere-forming cell amounts and after 3, 6, 12, and 18 days of culture;

[0024] Figure 2 Changes in microsphere diameters at different initial sphere-forming cell amounts and after 3, 6, 12, and 18 days of culture;

[0025] Figure 3 When the initial spheroid cell amount was 1500, the proportion of live cells changed after microspheres were formed at different cell ratios;

[0026] Figure 4 The initial cell number for spheroid formation was 1500, and the ratio of HK-2:HEK293T:EA.hy926 was 4:1:1. After 3 days of culture, the survival of cells in the spheroids was observed. (A) is a 3D image, and (B) is a plan view.

[0027] Figure 5 After OTA stimulation for 12h, 24h, 48h, and 72h, the cell viability changes with the drug concentration; (A) is the adherent cells, and (B) is the renal microspheres;

[0028] Figure 6After CIT stimulation for 12h, 24h, 48h, and 72h, the changes in cell viability with drug concentration were observed. (A) shows adherent cells, and (B) shows renal microspheres.

[0029] Figure 7 The effects of OTA+CIT on cell viability under isotoxic exposure and exposure at different toxicity ratios; (A) is isotoxic exposure of adherent cells, (B) is isotoxic exposure of renal microspheres, (C) is exposure of adherent cells at different toxicity ratios, and (D) is exposure of renal microspheres at different toxicity ratios.

[0030] Figure 8 The effect of OTA+CIT at the concentrations of 1 / 16IC50, 1 / 8IC50, and 1 / 4IC50 on the levels of inflammatory factors in adherent cells;

[0031] Figure 9 This is the effect of OTA+CIT at concentrations of 1 / 16IC50, 1 / 8IC50, and 1 / 4IC50 on the levels of glomerular inflammatory factors. DETAILED DESCRIPTION

[0032] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0033] The raw materials used in the embodiment are:

[0034] HK-2 cells, HEK293T cells, and EA.hy926 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0035] OTA toxin and CIT toxin were purchased from Beijing Puhuashi Technology Development Co., Ltd.

[0036] Cell culture methods:

[0037] Prepare complete culture medium, basal culture medium DMEM / F12 + 1% double antibody + 10% fetal bovine serum + 0.05% human epidermal growth factor (EGF); remove cryovials containing human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T, and human umbilical vein fusion cells EA.hy926 from liquid nitrogen, thaw in a 37°C water bath, centrifuge at 800 rpm for 5 minutes, resuspend in 1 mL of complete culture medium, and transfer to a culture dish pre-filled with fresh complete culture medium for culture; change the medium every 48 hours, and when the cells cover 70-80% of the bottom of the culture dish, digest with trypsin digestion solution. When gaps between cells are observed under a microscope, cells become rounded, and some cells are about to detach from the wall, complete culture medium is immediately added to terminate digestion; collect the digested cells in a 5 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add the corresponding amount of complete culture medium to resuspend in a ratio of one to two or one to three, and distribute to multiple new culture dishes to complete the subculture after cell recovery.

[0038] Example 1: Construction of a scaffold-free three-dimensional cell microsphere model

[0039] A method for constructing a scaffold-free three-dimensional cell microsphere model comprises the following steps:

[0040] (1) Human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T, and human umbilical vein fusion cells EA.hy926 were prepared into a mixed cell suspension; wherein the concentration of human renal cortical proximal tubule epithelial cells HK-2 was 5000 cells / mL, the concentration of human embryonic kidney cells HEK293T was 1250 cells / mL, and the concentration of human umbilical vein fusion cells EA.hy926 was 1250 cells / mL;

[0041] (2) The mixed cell suspension was added to a 96-well plate with ultra-low adsorption at 200 μL per well and cultured for 72 h. Half of the medium was replaced every 48 h to obtain a scaffold-free three-dimensional cell microsphere model with an initial cell volume of 1500 cells / sphere.

[0042] Example 2: Cell number optimization

[0043] On the basis of Example 1, the initial cell amount in step (2) was changed to 500 cells / sphere and 3000 cells / sphere, and the remaining steps were consistent with Example 1; using 1500 cells / sphere in Example 1 as a control, three scaffold-free three-dimensional renal microsphere models with different diameters were obtained.

[0044] The microsphere model was observed for spheroid morphology and diameter measurement using a wide-field imaging high-content system on days 3, 6, 12, and 18. Figure 1 、 Figure 2The results showed that within 3 days, the cell spheroids were uniformly sized (200-400 μm) with relatively stable morphology, and there were significant differences in spheroid size between the three initial cell numbers. Thereafter, the spheroid diameter continued to increase with culture time, with significant size changes at 6 days. After 12 days, all spheroids exceeded 500 μm in size, with a blackening of the center and relatively slower growth compared to 6 days. The size differences between the three different initial cell numbers were reduced.

[0045] When the number of cells is too large and the culture time is too long, the center of the sphere appears dark when observed under a microscope. This may be due to insufficient nutrition and oxygen supply, leading to internal cell necrosis. To ensure long-term culture and good cell growth, subsequent experiments selected 1500 cells to form the kidney microsphere.

[0046] Example 3: Cell ratio optimization

[0047] On the basis of Example 1, the ratios of human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T, and human umbilical vein fusion cells EA.hy926 were changed to 1:1:1, 2:1:1, and 8:1:1; other ratios remained the same as in Example 1. The scaffold-free three-dimensional renal microsphere model prepared at 4:1:1 in Example 1 was used as a control to detect cell survival, and the steps were as follows:

[0048] After the microsphere morphology stabilized, the culture medium in the well plate was discarded, 200 μL of Hoechst solution was added to each well to stain the cell nucleus, and the cells were incubated at 37°C for 30 min. The Hoechst solution in the well plate was discarded, and 100 μL of staining solution containing 2 μmol / L Calcein-AM and 4.5 μmol / L PI fluorescent probe was added, and the cells were incubated at 37°C for 30 min. After incubation, fluorescence images were taken using a laser confocal microscope, and Image J software was used to count the total cells, live cells, and dead cells, and calculate the live cell ratio.

[0049] After 8 days of culture, the microspheres formed by different cell ratios were stained with Hoechst and Calcein / PI. The results of the live cell ratio are shown in Figure 2. Figure 3 As shown, in Example 1, when the ratio of the three types of cells is 4:1:1, the proportion of living cells is significantly higher than the other three ratios in Example 2 (1:1:1, 2:1:1 and 8:1:1).

[0050] After the cell ratio was determined, the cell microspheres were cultured for 3 days and then stained with Hoechst and Calcein / PI. The staining results were as follows: Figure 4 As shown in (A): A clear three-dimensional structure can be seen, and living cells occupy the vast majority. Figure 4(B) Live and dead cell staining at a certain cross section shows only a small number of dead cells with intact spherical shapes, demonstrating that the cells are growing well after 3 days of culture in the microspheres, and subsequent experiments can be performed on this basis.

[0051] Example 4: Application of scaffold-free three-dimensional renal microsphere model in nephrotoxicity evaluation

[0052] The scaffold-free three-dimensional renal microsphere model in Example 1 was used to evaluate the nephrotoxicity of OTA toxin, and the steps were as follows:

[0053] (1) Toxin incubation

[0054] 5 mg of OTA toxin was dissolved in 500 μL of DMSO in advance to obtain a 10 mg / mL toxin stock solution, which was then serially diluted to the working concentration during drug administration (a half-volume solution replacement method was used during drug administration in the renal microspheres, so the actual drug concentration for drug administration was twice the working concentration).

[0055] After 48 h of culture, the three-dimensional renal microsphere model was constructed and incubated with 0 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 60 μg / mL, and 100 μg / mL of OTA solution (the actual drug concentration of the microspheres was 0 μg / mL, 2 μg / mL, 4 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 120 μg / mL, and 200 μg / mL).

[0056] (2) Detection of cell viability

[0057] After incubation with the toxin for 12 h, 24 h, 48 h, and 72 h, half of the culture medium in each well was discarded, and 20 μL of CCK-8 reagent and 80 μL of fresh culture medium were added to each well. The cells were incubated at 37°C. When the culture medium turned yellow, its absorbance was measured at 450 nm using a microplate reader. Cell viability (%) = (toxin-blank) / (control-blank) × 100%.

[0058] The results are as follows Figure 5 As shown in the figure, the cell viability decreased continuously with the increase of toxin concentration and the extension of incubation time, showing a time- and dose-dependent trend.

[0059] Exposure to OTA for 12 hours significantly affected the viability of adherent cells in a dose-dependent manner. When the toxin incubation time was extended to 24 hours, 12.31 μg / mL of OTA increased the percentage of dead cells to 50%. Thereafter, the IC50 value did not change significantly with the extension of incubation time, indicating a significant dose-dependency for adherent cells, while the time-dependency was not significant after 24 hours.

[0060] In contrast, when the toxin was incubated for 12 hours, the viability of the three-dimensional microspheres was not significantly affected. However, when the toxin was incubated for 24 hours, the IC50 value of OTA increased to 69.25 μg / mL, a 462.55% increase compared to adherent cells. This phenomenon suggests that the renal microsphere model is more resistant to drug damage, and the spheroid structure, due to its unique morphology, may accumulate a certain amount of nutrients within.

[0061] Example 5: Application of scaffold-free three-dimensional renal microsphere model in nephrotoxicity evaluation

[0062] On the basis of Example 4, OTA toxin was replaced with CIT toxin, and a scaffold-free three-dimensional renal microsphere model was tested for the nephrotoxicity evaluation of CIT toxin.

[0063] The results are as follows Figure 6 As shown in the figure, the change trend of cell viability after drug addition is similar to that of OTA experiment. However, it can be seen that the cell viability after CIT incubation is significantly higher than that of OTA, indicating that CIT is less nephrotoxic than OTA.

[0064] Exposure to CIT for 12 hours significantly affected the viability of adherent cells in a dose-dependent manner. When the toxin incubation time was extended to 24 hours, 46.29 μg / mL of CIT increased the proportion of dead cells to 50%. At 48 hours, the IC50 value of CIT was 41.93 μg / mL, a decrease of only 9.42%. This indicates that the adherent cell viability was significantly dose-dependent, while the time-dependent effect was not significant after 24 hours.

[0065] When the toxin was incubated for 12 hours, the viability of the glomeruli was not significantly affected. Even at a toxin concentration of 100 μg / mL, cell viability decreased by only 18.4%. When the incubation time was extended to 24 hours, the IC50 value of CIT increased to 95.82 μg / mL, a 107% increase compared to adherent cells. The low cell viability of adherent cells may be due to the impact on nutrient metabolism, resulting in cellular nutrient deficiency and limited daughter cell production. The three-dimensional glomerular model can alleviate this problem.

[0066] Example 6: Application of scaffold-free three-dimensional renal microsphere model in the evaluation of combined nephrotoxicity of OTA and CIT

[0067] (1) Based on Examples 4 and 5, a scaffold-free three-dimensional renal microsphere model was tested for the combined nephrotoxicity evaluation of OTA and CIT toxins.

[0068] First, based on the principle of equal toxicity commonly used in toxicological studies, where the IC50 values ​​of individual effects are used as the combined exposure ratio, six dosing points were selected: 0, 1 / 16 IC50, 1 / 4 IC50, 1 / 2 IC50, 1 / 8 IC50, and IC50, to evaluate the inhibitory effect of co-exposure on cell viability. The combination index (CI) was analyzed to assess the combined toxicity of the two mycotoxins in renal cells. The results showed that the type of combined effect (synergistic, additive, or antagonistic) varied depending on the toxin concentration ratio. Figure 7 (A, C) show that the dose-response curves of co-exposure of microspheres and adherent cells compared with single toxin treatment showed that the combination group exhibited toxic synergy at low doses, and the toxic effect gradually turned into antagonism with increasing concentrations.

[0069] Secondly, toxin exposure at different exposure ratios, that is, the IC50 values ​​at different multiples of OTA concentration were fixed, and the concentration of CIT was from low to high (OTA:CIT exposure ratio = 3:1, 2:1, 1:1, 1:0, 1:2, 1:3). Figure 7 (D) shows that the combined action type of the microspheres is antagonistic at all dose points, and at 1 / 16 IC50, the CI value reaches 4.72, showing a strong antagonistic effect. Figure 7 (B) The 2D cells showed a different trend. With the increase of toxin concentration, the type of combined action changed from antagonism to additive action, and showed synergistic effect at high concentration.

[0070] (2) Based on Example 6, a scaffold-free three-dimensional renal microsphere model was tested for evaluating the combined effect of OTA and CIT toxins on the level of inflammatory factors in renal cells.

[0071] The microspheres and 2D cells were exposed to 1 / 16IC50, 1 / 8IC50, and 1 / 4IC50 of OTA and CIT for 24 hours, and the changes in the levels of seven inflammatory factors were measured. Figure 8 、 9As shown, NLRP3 expression increased with increasing toxin concentration, with significant increases in the combination group at medium and high concentrations (P < 0.001). Consequently, levels of inflammatory factors such as TNF-α, IL-6, IL-1β, and IL-18 were significantly elevated in both the combination and OTA groups. CIT also significantly increased inflammatory factors, unlike in 2D cells. The study also found that inflammatory factor levels in glomeruli were significantly lower than in adherent cells. TGF-β, a key factor in renal fibrosis, showed little change at low toxin concentrations. However, it increased significantly at medium and high toxin concentrations, reaching a maximum increase of 67.35%. CIT, on the other hand, caused almost no change in 2D cells. Furthermore, low and medium toxin concentrations caused minimal changes in IFN-γ, with the highest change in the high OTA concentration group (P < 0.001), increasing from 74.00 ng / L in the control group to 139.30 ng / L. These results indicate that the combined action of OTA and CIT induces a cellular inflammatory response, with the changes in inflammatory factor levels becoming more pronounced with increasing toxin concentration.

[0072] Although adherent cell models are widely used in toxicological evaluation, they still have many defects. Co-culturing multiple cells with different physiological functions to form a three-dimensional structure, simulating the metabolic interaction network of key functional cells in renal tissue in vitro, enhancing the synergistic effect of cell-matrix-cell three-dimensional interaction and paracrine signaling pathways, and dynamically reconstructing the specific microenvironment of renal tissue are more in line with the requirements of in vitro nephrotoxicity assessment models.

[0073] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for constructing a scaffold-free three-dimensional cell microsphere model for nephrotoxicity evaluation, characterized in that: Including steps: (1) Human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T, and human umbilical vein fusion cells EA.hy926 were mixed to obtain a mixed cell suspension; (2) culturing the mixed cell suspension to obtain a scaffold-free three-dimensional cell microsphere model; Wherein, in step (1), the ratio of the number of human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T and human umbilical vein fusion cells EA.hy926 is 1-8:1:1; The initial cell amount of the scaffold-free three-dimensional cell microsphere model in step (2) is 500 to 3000 cells.

2. The method according to claim 1, characterized in that In step (1), the quantitative ratio of human renal cortical proximal tubule epithelial cells HK-2, human embryonic kidney cells HEK293T and human umbilical vein fusion cells EA.hy926 is 4:1:

1.

3. The method according to claim 1, characterized in that The initial cell amount of the scaffold-free three-dimensional cell microsphere model in step (2) is 1500 cells.

4. The method according to claim 1, wherein The culture in step (2) is carried out for 3 to 18 days.

5. The method according to claim 1, wherein The culture in step (2) is carried out for 6 to 8 days.

6. The method according to claim 1, characterized in that The culture temperature in step (2) is 37°C.

7. The method according to claim 1, characterized in that In step (2), the culture medium was replaced by half every 48 hours.

8. A scaffold-free three-dimensional cell microsphere model constructed by the method according to any one of claims 1 to 7.

9. Use of the scaffold-free three-dimensional cell microsphere model according to claim 8 in the evaluation of nephrotoxicity.

10. The use according to claim 9, characterized in that Nephrotoxicity was evaluated as nephrotoxicity induced by OTA toxin or CIT toxin.

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