Construction, identification and application of MRL / lpr lupus mouse kidney organoids

Abstract

A method for identifying MRL / lpr lupus renal organoids, specifically collecting organoid culture medium to be tested, detecting the levels of ANA and anti-ds-DNA antibodies in the culture medium, and performing histomorphological identification of the organoids. The present invention identifies the histomorphology of MRL / lpr lupus mouse renal organoids by detecting the levels of ANA and anti-ds-DNA antibodies, as well as HE staining and immunofluorescence, thereby detecting whether the MRL / lpr lupus renal organoids have been successfully constructed.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a construction and identification method of MRL / lpr lupus mouse kidney organoid and its application. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that is more common in women than men across all age groups and populations, with the highest prevalence among women and men during the reproductive years. SLE can affect any organ, but it commonly damages the kidneys. Lupus nephritis (LN) is a common complication of SLE, with 10% to 20% of LN patients developing end-stage renal disease within five years of diagnosis, a life-threatening condition.

[0003] The pathogenesis of lupus nephritis (LN) is complex and is currently believed to be related to genetics, neuroendocrine factors, environmental factors (ultraviolet light, drugs), and viral infections. These factors lead to the deposition of in situ immune complexes in renal tissue, causing excessive proliferation of lymphoid tissue, the formation of autoantibodies, and the activation of complement and multiple cytokines within the kidney. This leads to abnormal proliferation and differentiation of parenchymal and interstitial cells, such as glomerular mesangial cells, endothelial cells, and podocytes, and consequently altered renal function. The pathogenesis of LN involves multiple pathogenic mechanisms. The extrarenal etiology of systemic lupus nephritis is based on a diverse combination of genetic variants that impair the mechanisms that normally ensure immune tolerance to nuclear autoantigens.

[0004] The diagnosis and treatment of LN are challenging and primarily rely on renal biopsy, with no reliable non-invasive LN biomarkers. To date, the complete remission rate for LN after 6 months of standard treatment is only 20% to 30%, which is far from satisfactory. Furthermore, the adverse effects of immunosuppressants, particularly glucocorticoids, further impair the prognosis of LN.

[0005] Biologic agents targeting autoimmune and inflammatory pathways offer hope for the treatment of refractory lupus. However, animal models are essential for drug screening and efficacy evaluation. The MRL / Lpr lupus mouse is an animal model of an autoimmune disease similar to human systemic lupus erythematosus (SLE). However, the high cost and long development cycles of MRL / lpr lupus mice make them unsuitable for large-scale drug screening and evaluation.

[0006] Organoids are a newly developed in vitro three-dimensional culture technology. Compared with conventional cell line culture, the tissue structure and function formed by the organoid culture system are more complex. They have the advantages of high fit, stable passage, short culture cycle, and high success rate. They are currently the ideal in vitro culture model. Because they maintain the key characteristics of the parent tissue, organoids can be used for drug screening, disease modeling, etc., contributing to personalized treatment. Therefore, the construction of MRL / lpr lupus mouse kidney organoids is of great significance for the study of systemic lupus erythematosus nephritis. However, there is currently no better technical solution for the construction of lupus nephritis organoids. There is also no method to identify lupus nephritis organoids. Summary of the Invention

[0007] The present invention aims to provide a method for identifying MRL / lpr lupus kidney organoids.

[0008] The present invention provides a culture medium for constructing MRL / lpr lupus mouse kidney organoids. The culture medium of the present invention optimizes the culture medium components and has a good supporting and promoting effect on the cultivation of MRL / lpr lupus mouse kidney organoids.

[0009] The present invention aims to provide a method for constructing kidney organoids of MRL / lpr lupus mice.

[0010] The present invention aims to provide the application of the above-mentioned MRL / lpr lupus mouse kidney organoids.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] A method for identifying MRL / lpr lupus renal organoids, characterized by collecting the culture medium of the organoid to be tested, detecting the levels of ANA and anti-ds-DNA antibodies in the culture medium, and performing histomorphological identification of the organoids.

[0013] Furthermore, the levels of ANA and anti-ds-DNA antibodies in the renal organoid culture medium were detected by ELISA experimental scheme. When the expression of ANA and ds-DNA genes was detected in the culture medium, the organoids cultured in the culture medium were MRL / lpr lupus renal organoids.

[0014] Furthermore, the organoids to be tested were collected, fixed, embedded in paraffin, and sliced. Then, HE staining and immunofluorescence staining were used to detect the expression level of podocyte functional protein nephrin, and the tissue morphology of the organoids to be tested was identified.

[0015] A MRL / lpr lupus kidney organoid culture medium, characterized in that the MRL / lpr lupus kidney organoid culture medium is composed of a basal culture medium, a pH buffer, an antioxidant, a serum substitute, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator and a primary cell antibiotic.

[0016] Furthermore, the growth factors consist of Noggin, EGF, FGF 10 and Wnt-3a.

[0017] Furthermore, the basal culture medium is Advanced DMEM / F12 basal culture medium, the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the TGF-β receptor inhibitor is A83-01, the ROCK inhibitor is Y-27632; the cAMP pathway activator is Forskolin; and the primary cell antibiotic is primocin.

[0018] Furthermore, the MRL / lpr lupus kidney organoid culture medium also includes triple antibodies: penicillin / streptomycin / gentamicin.

[0019] Furthermore, in the MRL / lpr lupus kidney organoid culture medium, the concentration of B27 supplement is 0.8~1.2 x, the concentration of L-Glutamax is 0.8~1.2 x, the concentration of HEPES is 0.8~1.2 x, the concentration of A83-01 is 4~6 μM, the concentration of N-acetylcysteine ​​is 1.0~1.5 mM, the concentration of Noggin is 90~110 ng / mL, the concentration of EGF is 40~60 ng / mL, the concentration of Wnt-3a is 90~110 ng / mL, the concentration of Forskolin is 8~12 μM, the concentration of FGF 10 is 18~22 ng / mL, the concentration of Y-27632 is 8~12 μM, the concentration of primocin is 90~110 μg / mL, and the concentration of the triple antibody is 90~110 U / mL.

[0020] A method for constructing MRL / lpr lupus kidney organoids is characterized by: obtaining a tissue sample, sequentially shearing the tissue, digesting, filtering, lysing red blood cells, washing, and counting to obtain a cell solution; mixing the cell solution with Matrigel matrix gel, and then inoculating it into a well plate. After the inoculation is completed, the plate is allowed to stand until the Matrigel matrix gel solidifies; after the Matrigel matrix gel solidifies, MRL / lpr lupus kidney organoid culture medium is added for culturing to obtain MRL / lpr lupus mouse kidney organoids.

[0021] Furthermore, the MRL / lpr lupus kidney organoid culture medium is composed of a basal culture medium, a pH buffer, an antioxidant, a serum replacement, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator, and primary cell antibiotics.

[0022] Furthermore, the growth factors consist of Noggin, EGF, FGF 10 and Wnt-3a.

[0023] Furthermore, the basal culture medium is Advanced DMEM / F12 basal culture medium, the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the TGF-β receptor inhibitor is A83-01, the ROCK inhibitor is Y-27632; the cAMP pathway activator is Forskolin; and the primary cell antibiotic is primocin.

[0024] Furthermore, the MRL / lpr lupus kidney organoid culture medium also includes triple antibodies: penicillin / streptomycin / gentamicin.

[0025] Furthermore, in the MRL / lpr lupus kidney organoid culture medium, the concentration of B27 supplement is 0.8~1.2 x, the concentration of L-Glutamax is 0.8~1.2 x, the concentration of HEPES is 0.8~1.2 x, the concentration of A83-01 is 4~6 μM, the concentration of N-acetylcysteine ​​is 1.0~1.5 mM, the concentration of Noggin is 90~110 ng / mL, the concentration of EGF is 40~60 ng / mL, the concentration of Wnt-3a is 90~110 ng / mL, the concentration of Forskolin is 8~12 μM, the concentration of FGF 10 is 18~22 ng / mL, the concentration of Y-27632 is 8~12 μM, the concentration of primocin is 90~110 μg / mL, and the concentration of the triple antibody is 90~110 U / mL.

[0026] Most specifically, a method for constructing MRL / lpr lupus kidney organoids is characterized by comprising the following steps:

[0027] (1) Obtain MRL / lpr lupus renal tissue and cut it into 1~2 mm pieces 3After the digestion is complete, add the digestion solution and digest in a shaking incubator at 37°C until the cells are digested into 2-10 cell clusters. After digestion, add an appropriate amount of PBS to stop the digestion, pass through a 100 μm sieve, and then wash the filter twice with 1 mL of PBS. Centrifuge the filtrate. After centrifugation, discard the supernatant of the filtrate, lyse and resuspend with 2-3 mL of red blood cell lysis buffer, let it stand at 4°C for 5 minutes, then add 2-3 times the volume of red blood cell lysis buffer to stop the lysis, and centrifuge at 4°C, 1000 rpm for 5 minutes. Discard the upper layer of red blood cell lysis buffer, resuspend the cell pellet with PBS, and wash 1 to 2 times. Count the cells during the last wash. When used in a 24-well plate, evenly divide 25 w cells into 1.5 ml centrifuge tubes per well and centrifuge. When used in a 96-well plate, evenly divide 5 w cells into 1.5 ml centrifuge tubes per well and centrifuge.

[0028] (2) After centrifugation, discard the supernatant and resuspend the cell pellet with 75%~80% Matrigel. After uniform resuspension, add 50 μL of Matrigel to each well of a 24-well plate (use the middle 8 wells of each well); add 20 μL of Matrigel to each well of a 96-well plate (use the middle 60 wells of each well). Invert the 24-well plate and 96-well plate with Matrigel drops and place them in a 37°C incubator for 30 min to wait for the Matrigel to solidify.

[0029] (3) After the matrix gel solidifies, remove the 24-well plate and the 96-well plate. Add 500 μL of culture medium to each well of the 24-well plate and 200 μL of MRL / lpr lupus renal organoid culture medium to each well of the 96-well plate. Seal the surrounding wells with PBS and replace the MRL / lpr lupus mouse renal organoid culture medium every 3 to 5 days.

[0030] The MRL / lpr lupus kidney organoids constructed using the above method are used in screening LN-related drugs or disease modeling.

[0031] The present invention has the following technical effects:

[0032] The method for identifying MRL / lpr lupus mouse kidney organoids of the present invention comprises collecting culture medium and detecting the levels of ANA and anti-ds-DNA antibodies in the culture medium of the MRL / lpr lupus mouse kidney organoids by an ELISA experimental method; and identifying the histomorphology of the MRL / lpr lupus mouse kidney organoids by HE staining and immunofluorescence, thereby detecting whether the MRL / lpr lupus mouse kidney organoids are successfully constructed.

[0033] The culture medium of the present invention optimizes its composition and effectively supports and promotes the culture of renal organoids from MRL / lpr lupus mice. Observation of the renal organoids under a bright-field microscope revealed distinct organoid-typical vacuolar morphology on day 14, with good preservation of tissue consistency. This culture medium optimizes the growth density, culture medium composition, and culture conditions of lupus nephritis organoids, successfully establishing lupus nephritis organoids. This lays the foundation for further research and application in LN. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Routine detection of body weight, ANA and anti-ds-DNA antibody levels in serum of MRL / lpr lupus female mice in Example 2.

[0035] Figure 2 : Morphological images of MRL / lpr lupus kidney organoids stably cultured in the MRL / lpr lupus kidney organoid culture medium under bright field microscope on the 14th day in Example 2 ((a), magnification 10 times; (b), magnification 20 times) and morphological images under bright field microscope on the 5th day after passaging ((c), magnification 40 times).

[0036] Figure 3 : HE staining and immunohistochemistry results of MRL / lpr lupus kidney organoids in Example 2.

[0037] Figure 4 : The levels of ANA and anti-ds-DNA antibodies in the culture medium of renal organoids from MRL / lpr lupus mice at different time periods in Example 2.

[0038] Figure 5 :Different growth factor combinations affect the growth status of lupus renal organoids, (a): no Wnt-3a in the growth factor; (b): Wnt-3a is replaced by HGF in the growth factor; (c): HGF is added to the growth factor.

[0039] Figure 6 : Growth status of lupus kidney organoids when the concentration of ROCK inhibitor Y-27632 in MRL / lpr lupus kidney organoid culture medium is 10 mmol.

[0040] Figure 7 : Growth status of lupus kidney organoids after the cAMP pathway activator Forskolin was replaced with Sp-5,6-DCl-cBIMPS.

[0041] Figure 8 : Growth status of lupus kidney organoids after additional addition of R-spondins to the culture medium. DETAILED DESCRIPTION

[0042] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0043] Example 1

[0044] A MRL / lpr lupus kidney organoid culture medium, specifically composed of a basal culture medium, a pH buffer, L-glutamine, an antioxidant, a serum replacement, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator, and a primary cell antibiotic;

[0045] The basal medium was Advanced DMEM / F12, the pH buffer was HEPES buffer, the antioxidant was N-acetylcysteine, the serum replacement was B-27 supplement, the TGF-β receptor inhibitor was A83-01, the growth factors consisted of Noggin, EGF, FGF 10, and Wnt-3a, the ROCK inhibitor was Y-27632, the cAMP pathway activator was Forskolin, and the primary cell antibiotics were primocin and penicillin / streptomycin / gentamicin. The components, brands, and concentrations of the MRL / lpr lupus kidney organoid culture medium are shown in Table 1.

[0046] Table 1:

[0047]

[0048] Example 2

[0049] A method for constructing MRL / lpr lupus kidney organoids comprises the following steps:

[0050] (1) Obtain kidney tissue from a spontaneous MRL / lpr lupus female mouse model, approximately 30 weeks old. Wash the tissue 1-2 times with PBS (containing 100 U / mL penicillin / streptomycin / gentamicin) and mince into 1-2 mm pieces. 3After the cells are digested into small pieces, type IV collagenase digestion solution is added and digested in a shaking incubator at 37°C until they are digested into 2-10 cell clusters; after digestion, an appropriate amount of PBS is added to stop the digestion, the cells are passed through a 100 μm sieve, and the filter is washed twice with 1 mL of PBS. The filtrate is centrifuged; after centrifugation, the supernatant is discarded and the cells are lysed and resuspended with 2-3 mL of red blood cell lysis buffer. After standing at 4°C for 5 minutes, 2-3 times the volume of red blood cell lysis buffer is added to stop the lysis, and the cells are centrifuged at 1000 rpm at 4°C for 5 minutes; the upper layer of red blood cell lysis buffer is discarded, the cell pellet is resuspended with PBS, and washed 1 to 2 times; the cells are counted during the last wash. When used in a 24-well plate, 25 w cells are evenly distributed into a 1.5 ml centrifuge tube per well and centrifuged; when used in a 96-well plate, 5 w cells are evenly distributed into a 1.5 ml centrifuge tube per well and centrifuged;

[0051] (2) After centrifugation, discard the supernatant and resuspend the cell pellet with 75%~80% Matrigel. After uniform resuspension, add 50 μL of Matrigel to each well of a 24-well plate (use the middle 8 wells of each well); add 20 μL of Matrigel to each well of a 96-well plate (use the middle 60 wells of each well). Invert the 24-well plate and 96-well plate with Matrigel drops and place them in a 37°C incubator for 30 min to wait for the Matrigel to solidify.

[0052] (3) After the matrix gel solidifies, remove the 24-well plate and the 96-well plate. Add 500 μL of culture medium to each well of the 24-well plate and 200 μL of the MRL / lpr lupus renal organoid culture medium in Example 1 to each well of the 96-well plate. Seal the surrounding wells with PBS and replace the MRL / lpr lupus mouse renal organoid culture medium every 3 to 5 days.

[0053] The daily index detection of the spontaneous MRL / lpr lupus female mouse model in Example 2 of the present invention is about 30 weeks old. The daily detection of the changes in the weight, anti-dsDNA, urine protein antibodies and ANA levels of the MRL / lpr lupus female mice is as follows: Figure 1 shown.

[0054] According to the construction method in Example 2, MRL / lpr lupus kidney organoids were obtained after 14 days of culture. The morphology of MRL / lpr lupus kidney organoids under a bright field microscope is as follows: Figure 2 As shown, on day 14, the typical vacuolar morphology of organoids was evident.

[0055] Subculture after 14-21 days of culture:

[0056] Aspirate and discard the old culture medium, add preheated Dispase II working solution, 200 μL / well, and incubate in the incubator for 1 hour. After incubation, add 0.5 mL Advanced DMEM / F12 culture medium to the well plate, use a 1 mL pipette tip to blow the matrix gel off the well plate, collect the matrix gel in a 15 mL centrifuge tube, and centrifuge at 4°C, 1000 rpm for 5 minutes. Discard the supernatant, add 1 mL TrypLE enzyme, and use a 1 mL pipette tip to blow for 3-5 minutes. Add 1 mL Advanced DMEM / F12 to terminate the digestion, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, add 1 mL Advanced DMEM / F12 to resuspend and wash, transfer to a 1.5 mL EP tube, and centrifuge at 4°C, 1000 rpm for 5 minutes.

[0057] Discard some Advanced DMEM / F12, leaving the required volume, gently disperse the organoid tissue, add Matrigel and mix thoroughly, to a final concentration of 75% (Matrigel should be kept at 4°C throughout the entire process);

[0058] Add Matrigel containing organoids into 24-well and 96-well plates; place them in an incubator upside down for 30 minutes and then add complete culture medium.

[0059] After multiple passages, the cultured MRL / lpr lupus kidney organoids can still grow stably. The morphology of the MRL / lpr lupus kidney organoids under a bright field microscope after passage in the MRL / lpr lupus mouse kidney organoid culture medium in Example 1 is as follows: Figure 2 (c) shows that on day 5, there is obvious typical vacuolar morphology of organoids.

[0060] Example 3

[0061] The specific steps for identifying the organoids constructed above are as follows:

[0062] (1) The MRL / lpr lupus mouse kidney organoids obtained in Example 2 were embedded and sectioned, and conventional HE staining and immunohistochemical staining were performed to detect the expression of podocyte function protein nephrin.

[0063] like Figure 3 As shown, HE staining results showed that the organoids and original MRL / lpr lupus kidney tissue had similar morphology. Immunofluorescence results showed that MRL / lpr mouse kidney organoids expressed high levels of nephrin, a podocyte functional protein.

[0064] (2) The culture medium of the MRL / lpr lupus mouse kidney organoid culture obtained in Example 2 was collected at different time stages, and the levels of ANA and anti-ds-DNA antibodies in the culture medium of the MRL / lpr lupus kidney organoid were detected by ELISA.

[0065] Antinuclear antibodies (ANA), also known as anti-nucleic acid antigen antibodies, are a group of autoantibodies produced against DNA, RNA, proteins, or molecular complexes of these substances within the cell nucleus. Anti-ds-DNA antibodies are characteristic markers of systemic lupus erythematosus (SLE), present in the serum of over 95% of patients with active SLE. The titer of these antibodies is closely correlated with the activity of SLE; higher titers indicate a greater likelihood of active SLE. As lupus kidney organoids grow, the concentrations of these two antibodies secreted in the culture medium increase.

[0066] The results are as follows Figure 4 As shown in Figure 2, our constructed kidney organoids can secrete ANA and anti-ds-DNA antibodies. Notably, the levels of ANA and anti-ds-DNA antibodies secreted by kidney organoids gradually increased with culture time, suggesting that the efficacy of SLE drugs can be evaluated by measuring autoantibody secretion in the culture medium.

[0067] Comparative Example 1:

[0068] Regarding the impact of different growth factor combinations in the culture medium on the culture results of MRL / lpr lupus kidney organoids:

[0069] When preparing the MRL / lpr lupus kidney organoid culture medium, the growth factors used are as follows:

[0070] (1) The growth factors are a combination of Noggin, EGF, and FGF 10;

[0071] (2) HGF was used instead of Wnt-3a, and the growth factors were a combination of Noggin, EGF, FGF 10, and HGF;

[0072] (3) The growth factors are a combination of Noggin, EGF, FGF 10, Wnt-3a and HGF.

[0073] After 14 days of stable culture in the culture medium, the results were as follows Figure 5As shown in the figure, when Wnt-3a is omitted from the growth factor mix, the growth of lupus kidney organoids is affected, with both growth and growth rate significantly inhibited. When Wnt-3a is replaced with HGF, the growth of lupus kidney organoids is also affected to some extent. However, the addition of HGF does not significantly affect the growth of lupus kidney organoids. These results demonstrate that the growth factor Wnt-3a is crucial for the culture of lupus kidney organoids, indicating that the growth factor combination of Noggin, EGF, FGF-10, and Wnt-3a acts synergistically, and each component of this essential combination is indispensable.

[0074] Comparative Example 2:

[0075] MRL / lpr lupus kidney organoid culture medium includes basal culture medium, L-glutamine, pH buffer, antioxidants, serum replacement, TGF-β receptor inhibitor, growth factors, ROCK inhibitor, cAMP pathway activator, etc. Among them, when the cAMP pathway activator Forskolin is replaced with Sp-5,6-DCl-cBIMPS.

[0076] The results are as follows Figure 6 As shown in the figure, when Forskolin was replaced with Sp-5,6-DCl-cBIMPS, the density and growth state of lupus kidney organoids obtained after 14 days of stable culture in the culture medium were affected, and the growth state was relatively slow, that is, the cAMP pathway activator Forskolin was replaced with Sp-5,6-DCl-cBIMPS, which inhibited the formation of MRL / lpr lupus kidney organoids.

[0077] Comparative Example 3:

[0078] The concentrations of the various components in the MRL / lpr lupus kidney organoid culture medium also had a significant effect on the culture results of the MRL / lpr lupus kidney organoids. When the concentration of the ROCK inhibitor Y-27632 was changed from 10 μM in Example 1 to 10 mM, we found that the growth state of the lupus kidney organoids was negatively affected by the increase in the concentration of the ROCK inhibitor Y-27632. When the concentration of the ROCK inhibitor Y-27632 was increased to 10 mM, the organoid results after 14 days of stable culture in the culture medium were as follows: Figure 7 As shown, it can be seen that increasing the concentration of ROCK inhibitor Y-27632 inhibited the formation of MRL / lpr lupus kidney organoids.

[0079] Furthermore, R-spondins (RSPOs), enhancers of Wnt / β-catenin signaling, are routinely added. Therefore, during this study, we also attempted to add R-spondins. Results after 14 days of stable culture in the culture medium are shown in Figure 8. We found that the growth of lupus kidney organoids was relatively slow. This indicates that the addition of R-spondins inhibited the formation of MRL / lpr lupus kidney organoids.

Claims

1. A MRL / lpr lupus kidney organoid culture medium, characterized by: The MRL / lpr lupus kidney organoid culture medium is composed of a basal culture medium, L-glutamine, a pH buffer, an antioxidant, a serum replacement, a TGF-β receptor inhibitor A83-01, growth factors, a ROCK inhibitor, a cAMP pathway activator, and primary cell antibiotics. The growth factors are composed of Noggin, EGF, FGF 10, and Wnt-3a. The concentration of Noggin is 90-110 ng / mL, the concentration of EGF is 40-60 ng / mL, the concentration of Wnt-3a is 90-110 ng / mL, the concentration of FGF 10 is 18-22 ng / mL, and the concentration of A83-01 is 4-6 μM.

2. The MRL / lpr lupus kidney organoid culture medium according to claim 1, wherein: The basal culture medium is Advanced DMEM / F12 basal culture medium, the pH buffer is HEPES buffer, the antioxidant is N-acetylcysteine, the serum substitute is B-27supplement, the ROCK inhibitor is Y-27632, the cAMP pathway activator is Forskolin, and the primary cell antibiotic is primocin.

3. The MRL / lpr lupus kidney organoid culture medium according to claim 2, wherein: In the MRL / lpr lupus kidney organoid culture medium, the concentration of B27 supplement is 0.8~1.2 x, the concentration of L-Glutamax is 0.8~1.2 x, the concentration of HEPES is 0.8~1.2 x, the concentration of N-acetyl cysteine ​​is 1.0~1.5 mM, the concentration of Forskolin is 8~12 μM, the concentration of Y-27632 is 8~12 μM, and the concentration of primocin is 90~110 μg / mL.

4. A method for constructing MRL / lpr lupus kidney organoids based on the culture medium of claim 3, characterized in that: After obtaining the tissue sample, the cell solution is obtained by shearing the tissue, digesting, filtering, lysing the red blood cells, washing, and counting. The cell solution is mixed with Matrigel matrix gel and then inoculated into the well plate. After the inoculation is completed, it is allowed to stand until the Matrigel matrix gel solidifies. After the Matrigel matrix gel solidifies, MRL / lpr lupus kidney organoid culture medium is added for culture to obtain MRL / lpr lupus mouse kidney organoids.

5. The method for constructing an MRL / lpr lupus kidney organoid according to claim 4, wherein: The steps include: (1) Obtain MRL / lpr lupus renal tissue and cut it into 1~2 mm pieces 3 After the digestion is complete, add the digestion solution and digest in a shaking incubator at 37°C until the cells are digested into 2-10 cell clusters. After digestion, add an appropriate amount of PBS to stop the digestion, pass through a 100 μm sieve, and then wash the filter twice with 1 mL of PBS. Centrifuge the filtrate. After centrifugation, discard the supernatant of the filtrate, lyse and resuspend with 2-3 mL of red blood cell lysis buffer, let it stand at 4°C for 5 minutes, then add 2-3 times the volume of red blood cell lysis buffer to stop the lysis, and centrifuge at 4°C, 1000 rpm for 5 minutes. Discard the upper layer of red blood cell lysis buffer, resuspend the cell pellet with PBS, and wash 1 to 2 times. Count the cells during the last wash. When used in a 24-well plate, evenly divide 25 w cells into 1.5 ml centrifuge tubes per well and centrifuge. When used in a 96-well plate, evenly divide 5 w cells into 1.5 ml centrifuge tubes per well and centrifuge. (2) After centrifugation, discard the supernatant and resuspend the cell pellet with 75%~80% Matrigel. After uniform resuspension, add 50 μL of Matrigel to each well of a 24-well plate; add 20 μL of Matrigel to each well of a 96-well plate. Invert the 24-well plate and 96-well plate with Matrigel drops and place them in a 37°C incubator for 30 min to wait for the Matrigel to solidify. (3) After the matrix gel solidifies, remove the 24-well plate and the 96-well plate. Add 500 μL of culture medium to each well of the 24-well plate and 200 μL of MRL / lpr lupus renal organoid culture medium to each well of the 96-well plate. Seal the surrounding wells with PBS and replace the MRL / lpr lupus mouse renal organoid culture medium every 3 to 5 days.

6. Use of the MRL / lpr lupus kidney organoid constructed by the method according to claim 5 in screening LN-related drugs or disease modeling.

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