Macrophage-kidney organoid co-culture method for simulating kidney immune microenvironment in vitro

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a macrophage-kidney organoid co-culture method for simulating a kidney immune microenvironment in vitro. The invention provides a method for co-culturing kidney organoid and macrophages in vitro, which can effectively simulate the immune microenvironment of the kidney, and is similar to that in the in-vivo proinflammatory reaction, the macrophages infiltrate into specific parts of kidney tissues and are self-polarized into proinflammatory M1 type macrophages to be accumulated in the kidney. The co-culture system can provide a model closer to an in-vivo model for in-vitro research of a kidney immune microenvironment, and lays a foundation for research of a regulation mechanism of a kidney disease inflammation signal.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a macrophage-kidney organoid co-culture method for simulating a kidney immune microenvironment in vitro. BACKGROUND

[0002] Macrophages play an important role in the kidney and are a common type of immune cell in damaged kidney tissue. Inflammation induced by acute kidney injury is a protective response, and persistent inflammatory response promotes the progression of fibrosis and eventually leads to end-stage renal disease. Kidney inflammation involves the activation of immune system cells and intrinsic kidney cells, and acute kidney injury can lead to the accumulation of activated M1 pro-inflammatory macrophages in the kidney (related literature: Meng X M, Nikolic-Paterson D J, Lan H Y. Inflammatory Processes in Renal Fibrosis. Nature Reviews Nephrology. 2014, 10 (9): 493-503.).

[0003] To obtain a kidney immune microenvironment involving macrophages in vitro, the following challenges have always existed: 1. Due to different developmental lineages, it is difficult to obtain immune cells such as macrophages in the kidney organoid obtained by differentiation of hPSCs, which are similar to the microenvironment in vivo; 2. The differentiation of organoids usually requires a culture medium rich in growth factors, some of which can affect the activity of immune cells. For example, fibroblast growth factors (FGF) added during the differentiation of organoids can affect the response of immune cells. Therefore, to successfully simulate the kidney immune microenvironment in vitro, establishing a culture system in which both kidney organoids and immune cells can normally differentiate and mature is an effective method to achieve this goal.

[0004] In recent years, stable induced mature macrophages (iMacs) have been successfully obtained by differentiating hPSCs, which exhibit phagocytic and polarized properties in vitro (relevant literature: Baoqiang Kang, Qi Xing, Yuhua Huang, Huaisong Lin, Jiaojiao Peng, Zhishuai Zhang, Mingquan Wang, Xinrui Guo, Xing Hu, Shuoting Wang, Junwei Wang, Minghui Gao, Yanling Zhu, and Guangjin Pan, Large-scale generation of IL-12 secreting macrophages from human pluripotent stem cells for cancer therapy, Molecular Therapy: Methods & Clinical Development Vol. 32 March 2024).

[0005] Kidney organoids obtained by differentiating hPSCs have developed to the extent that the required differentiation inducers are increasingly simple, especially Przepiorski et al. developed a simpler method of obtaining kidney organoids in 3D culture compared to previously reported methods, and the second stage of differentiation culture medium can obtain kidney organoids without additional addition of exogenous factors (relevant literature: Aneta Przepiorski, Veronika Sander, Tracy Tran, Jennifer A. Hollywood, Brie Sorrenson, Jen-Hsing Shih, Ernst J. Wolvetang, Andrew P. McMahon, Teresa M. Holm, and Alan J. Davidson. A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells. Stem Cell Reports (2018) 11:470-484.), which will provide a realizable basis for co-culture of kidney organoids and macrophages, and is the best tool for in vitro research of immune-mediated kidney diseases, which can better reveal the role of macrophages in regulating kidney cell differentiation and kidney diseases, and is conducive to further understanding the mechanism of occurrence and development of kidney diseases.

[0006] It is of great significance to establish a stable co-culture system of kidney organoids and macrophages for in-depth study of the inflammatory regulation mechanism of kidney diseases. However, there is no co-culture model of kidney organoids and macrophages in vitro in the field at present, because it is difficult to differentiate immune cells of different lineages during the differentiation of kidney organoids, and the addition of macrophages in kidney organoids is limited by the in vitro differentiation culture conditions, which cannot guarantee the normal differentiation of kidney organoids and the survival, recruitment and function of macrophages. SUMMARY

[0007] The first aspect of the present application aims to provide a macrophage-kidney organoid co-culture method simulating the immune microenvironment of the kidney.

[0008] The second aspect of the present application aims to provide a kidney organoid infiltrated with macrophages.

[0009] The third aspect of the present application aims to provide the use of the co-culture method of the first aspect of the present application and the kidney organoid of the second aspect of the present application.

[0010] In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides a macrophage-kidney organoid co-culture method simulating the immune microenvironment of the kidney, comprising the following steps: 1) adding mature macrophages induced by hPSC to kidney organoids at the early stage of differentiation; 2) using co-culture medium for culture.

[0011] In some embodiments of the present application, the co-culture medium comprises: basal medium DMEM, KOSR 10-20% (v / v), NEAA 0.7-1.3% (v / v), Glutamax 0.7-1.3% (v / v), HEPES 0.7-1.3% (v / v), PVA: 1.7-3.3 mg / mL, 1-2% (v / v) FBS and 25-75 ng / mL M-CSF.

[0012] In some embodiments of the present application, the culture method of the kidney organoid is: hPSCs were digested into low-adherent 6-well cell culture plates and cultured with the first stage differentiation medium BPEL (base medium IMDM: 41% (v / v), F12: 45% (v / v), PFHMII: 5% (v / v), additives: BSA: 2.2-2.8 mg / mL, ITS-X: 0.07-0.13% (v / v), aMTG: 0.002-0.005% (v / v), AA2P: 0.03-0.07 mg / mL, Glutamax: 0.7-1.3% (v / v), CD Lipid concentrate: 0.7-1.3% (v / v), PVA: 1.7-3.3 mg / mL, CHIR99021: 1-10 mM, Y-27632: 2-10 mM and b-Mercaptoethanol: 0.07-0.13 mM) for 3 days, then continued with the second stage differentiation medium Stage II (base medium DMEM, additives: KOSR: 10-20% (v / v), NEAA: 0.7-1.3% (v / v), Glutamax: 0.7-1.3% (v / v), HEPES: 0.7-1.3% (v / v) and PVA: 1.7-3.3 mg / mL) for 2 days to obtain day 5 early kidney organoids.

[0013] In some embodiments of the present application, the culture method of the mature macrophages is: hPSCs were cultured with F12 + 40 ng / mL BMP4 + 30 ng / mL ACTIVIN A + 20 ng / mL bFGF + 6 mM CHIR99021 + 10 mM LY294002 medium for 2 days, then changed to F12 + 40 ng / mL VEGF + 50 ng / mL bFGF medium for 3 days, and the cells were digested and suspended in F12 + 10 ng / mL SCF + 50 ng / mL thrombopoietin + 10 ng / mL IL-3 + 50 ng / mL IL-6 + 50 ng / mL FLT3 medium for 5 days, then changed to myeloid differentiation medium StemPro + 50 ng / mL FLT3L + 50 ng / mL M-CSF + 25 ng / mL GM-CSF for 12 days, and finally cultured with macrophage maturation medium RPMI-1640 + 10% FBS + 50 ng / mL M-CSF for 7-10 days to obtain mature macrophages.

[0014] In some embodiments of the present application, the kidney organoids are day 5 differentiated kidney organoids.

[0015] In some embodiments of the present application, the ratio of the number of the kidney organoids and the mature macrophages is 1: (4000-6000); preferably 1:5000.

[0016] In some embodiments of the present application, the culture time is 5-7 days.

[0017] In some embodiments of the present application, the culture method is static culture.

[0018] In a second aspect of the present application, a kidney organoid infiltrated with macrophages is provided, which is obtained by the co-culture method of the first aspect of the present application.

[0019] In some embodiments of the present application, the macrophages infiltrate the interstitium and the basement membrane of the kidney organoid.

[0020] In a third aspect of the present application, the co-culture method of the first aspect of the present application and the kidney organoid infiltrated with macrophages of the second aspect of the present application are used in at least one of the following applications: 1) in the preparation of a model simulating the interaction between immune cells and kidney.

[0021] 2) in the preparation of a kidney disease model.

[0022] In some embodiments of the present application, the kidney disease includes kidney inflammation caused by kidney injury and kidney fibrosis caused by persistent inflammation. According to the Bulk RNA sequencing results, the model can stably simulate kidney inflammation caused by kidney injury in vitro, the macrophages infiltrating the kidney organoid are polarized into pro-inflammatory M1 type, and the expression of pro-inflammatory cytokine IL-1 receptor (IL-1R) in the kidney organoid is significantly up-regulated. This co-culture model provides a stable in vitro research model for the regulation of inflammatory signaling and drug screening of kidney injury.

[0023] The present application has the following beneficial effects: The present application provides a method for co-culturing kidney organoids and macrophages in vitro, which can effectively simulate the immune microenvironment of the kidney, similar to the specific site of macrophage infiltration into the kidney tissue in the pro-inflammatory response in vivo, and the accumulation of pro-inflammatory M1 type macrophages in the kidney. The co-culture system can provide a more in vivo model for the in vitro study of the immune microenvironment of the kidney, and lay a foundation for the study of the regulation mechanism of inflammatory signaling of kidney disease. BRIEF DESCRIPTION OF DRAWINGS

[0024] The present application will be further described below in conjunction with the drawings and examples, in which: Figure 1Results of 3D suspension culture kidney organoids differentiated from hPSCs and mature macrophages differentiated from hPSC-DsRed (UH10-DsRed), wherein: A, kidney organoids differentiated from hPSCs at early stage day 5; B, flow cytometry identification of iMac characteristic protein markers CD11b and CD14 expression of iMac differentiated from hPSC-DsRed; C, immunofluorescence staining identification of iMac characteristic protein markers CD11b and CD14 expression results of hPSC-DsRed.

[0025] Figure 2 Results of co-culture medium formula screening, wherein: A, bright field and immunofluorescence identification of CD14 expression of adherent cells after culturing iMac for 3 days with iMac medium; B, bright field and immunofluorescence identification of CD14 expression of adherent cells after culturing iMac for 3 days with kidney organoid medium Stage II; C, bright field and immunofluorescence identification of CD14 expression of adherent cells after culturing iMac for 3 days with kidney organoid medium Stage II added with M-CSF.

[0026] Figure 3 Results of co-culture cell ratio selection. 100 kidney organoids were selected and co-cultured with 100000, 300000, 500000 and 800000 iMac cells for 6 days, and the imaging results are shown.

[0027] Figure 4 Imaging results of different co-culture methods and co-culture time, wherein A is the bright field and fluorescence imaging results of 4 days, 6 days, 8 days and 10 days of culture in two ways of dynamic culture in a shaking flask and static culture in a low-adhesion 6-well cell culture plate; B is the quantification of the proportion of iMac infiltrating into the kidney organoid through the imaging results of A.

[0028] Figure 5 Results of effective infiltration and co-localization of macrophages in kidney organoids, wherein A proves that iMac does not infiltrate in these sites by labeling kidney distal tubule ECAD, connecting tube GATA3 and kidney glomerular podocyte PODXL; B proves that iMac infiltrating into kidney organoids is located in the interstitium and basement membrane sites by labeling kidney organ interstitium MEIS1 / 2 / 3 and basement membrane COL1A1.

[0029] Figure 6 Bulk RNA sequencing results of macrophage-kidney organoid. Comparison of macrophages infiltrating into kidney organoids and iMac cells before co-culture proves that the infiltrated macrophages undergo pro-inflammatory M1 polarization. DETAILED DESCRIPTION

[0030] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0031] Related terms of the present application: Macrophage; fibroblast growth factors (FGF); human pluripotent stem cell (hPSC); induced mature macrophages (iMacs); kidney organoids.

[0032] Example 1: hPSC differentiation to obtain kidney organoids The differentiation method of Przepiorski et al. was used. First, hPSCs were digested into low-adhesion 6-well cell culture plates, and cultured with the first-stage differentiation medium BPEL (IMDM: 41% (v / v), F12: 45% (v / v), PFHMII: 5% (v / v), additives: BSA: 2.2-2.8 mg / mL, ITS-X: 0.07-0.13% (v / v), aMTG: 0.002-0.005% (v / v), AA2P: 0.03-0.07 mg / mL, Glutamax: 0.7-1.3% (v / v), CD Lipid concentrate: 0.7-1.3% (v / v), PVA: 1.7-3.3 mg / mL, CHIR99021: 1-10 mM, Y-27632: 2-10 mM and β-Mercaptoethanol: 0.07-0.13 mM.) for 3 days, and then the second-stage differentiation medium Stage II (base medium DMEM, additives: KOSR: 10-20% (v / v), NEAA: 0.7-1.3% (v / v), Glutamax: 0.7-1.3% (v / v), HEPES: 0.7-1.3% (v / v) and PVA: 1.7-3.3 mg / mL) was used to continue inducing differentiation for 2 days to obtain early kidney organoids at day 5.

[0033] The results are shown in Figure 1 As shown in FIG. A, kidney organoids in 3D suspension culture were obtained after hPSCs were differentiated for 5 days.

[0034] Example 2 hPSC differentiation to obtain kidney organoids To facilitate real-time observation, the iMac used in this example was obtained from the differentiation induction method of published articles (relevant literature: Baoqiang Kang, Qi Xing, Yuhua Huang, Huaisong Lin, Jiaojiao Peng, Zhishuai Zhang, Mingquan Wang, Xinrui Guo, Xing Hu, Shuoting Wang, Junwei Wang, Minghui Gao, Yanling Zhu, and Guangjin Pan, Large-scale generation of IL-12 secreting macrophages from human pluripotent stem cells for cancer therapy, Molecular Therapy: Methods & Clinical Development Vol. 32 March 2024. https: / / doi.org / 10.1016 / j.omtm.2024.101204.). Mature macrophages with red fluorescence were obtained. hPSC-DsRed cells were cultured in F12+40 ng / mL BMP4+30 ng / mL ACTIVIN A+20 ng / mL bFGF+6 mM CHIR99021+10 mM LY294002 medium for 2 days, then replaced with F12+40 ng / mL VEGF+50 ng / mL bFGF medium for 3 days, and the cells were digested and suspended in F12+10 ng / mL SCF+50 ng / mL thrombopoietin+10 ng / mL IL-3+50 ng / mL IL-6+50 ng / mL FLT3 medium for 5 days, then replaced with myeloid differentiation medium StemPro+50 ng / mL FLT3L+50 ng / mL M-CSF+25 ng / mL GM-CSF for 12 days, and finally cultured in macrophage maturation medium RPMI-1640+10% FBS+50 ng / mL M-CSF for 7-10 days to obtain mature macrophages.

[0035] The differentiation of macrophages was determined by flow cytometry and immunofluorescence staining of macrophage-specific expression proteins CD11b and CD14.

[0036] The results are as follows Figure 1As shown in B and C, in the flow identification, UH10, iMac differentiated from the hPSC-DsRed cell strain with red fluorescence, and iMac obtained from unedited hPSC, compared in macrophage maturation medium, both CD11b and CD14 were expressed. Immunofluorescence staining results also showed that UH10-iMac macrophages expressed CD11b and CD14.

[0037] Example 3 Medium selection for co-culture of kidney organoids and macrophages Medium candidates are as follows: 1) Mature macrophage iMac medium: RPMI-1640 + 10% (v / v) FBS + 50 ng / mL M-CSF.

[0038] 2) Kidney organoid differentiation medium Stage II: base medium DMEM, additives: KOSR: 15% (v / v), NEAA: 1% (v / v), Glutamax: 1% (v / v), HEPES: 1% (v / v), and PVA: 2.5 mg / mL.

[0039] 3) Combined medium: base medium DMEM, additives: KOSR: 15% (v / v), NEAA: 1% (v / v), Glutamax: 1% (v / v), HEPES: 1% (v / v), PVA: 2.5 mg / mL, 1.5% (v / v) FBS, and 50 ng / mL M-CSF.

[0040] Microscopic imaging of iMac after three days of culture in mature macrophage iMac medium, kidney organoid differentiation medium Stage II, and combined medium, respectively, and immunofluorescence staining of macrophage-specific expression protein CD14.

[0041] The results, as shown in Figure 2 , mature macrophage medium proliferated rapidly and adhered well after 3 days of culture of iMac. Kidney organoid second-stage differentiation medium Stage II cultured iMac for 3 days, most of the cells were apoptotic, and a small amount adhered. Combined medium cultured iMac for 3 days, with obvious proliferation and adhesion. Immunofluorescence staining of adherent cells were CD14 + macrophages. Considering that the kidney organoids in the co-culture system need to continue to differentiate and mature, the best co-culture medium is finally selected as the combined medium.

[0042] Example 4 Screening of co-culture ratio of kidney organoids and macrophages Screening ratio: One hundred kidney organoids differentiated on day 5 were grouped together, and 100,000, 300,000, 500,000, and 800,000 macrophages were added to each group and co-cultured in 6-well cell culture plates. After 6 days, fluorescence microscopy imaging was performed.

[0043] The results are as follows Figure 3 As shown, after co-culturing 100 kidney organoids and 500,000 iMac cells for 6 days, macrophages were able to successfully enter the kidney organoids and there were relatively few macrophages free in the culture medium. Therefore, a ratio of 100 kidney organoids on day 5 and 500,000 iMac cells was selected for co-culturing.

[0044] Example 5: Co-culture method and culture duration test This embodiment considers that the blood shear force in the in vivo microenvironment helps macrophages migrate into the kidney tissue. Therefore, shake-flask culture was used in vitro to provide shear force to the co-culture system, while static co-culture in cell culture plates was also employed to compare the advantages and disadvantages of different culture methods. Furthermore, because kidney organoids lack a rich vascular network during in vitro development, the culture time is limited. The optimal co-culture time needs to be determined to ensure the best differentiation state of both kidney organoids and macrophages.

[0045] Specifically, in this embodiment, 500,000 macrophages were added to 100 kidney organoids on day 5 of differentiation, and then transferred to shake flasks for culture. The magnetic rotor speed was set to 90 rpm to simulate the in vivo shear stress microenvironment. Half of the medium was changed every 2 days. The culture medium was a conjugated medium: basal medium DMEM, with the following additives: KOSR: 15% (v / v), NEAA: 1% (v / v), Glutamax: 1% (v / v), HEPES: 1% (v / v), PVA: 2.5 mg / mL, 1.5% (v / v) FBS and 50 ng / mL M-CSF.

[0046] The experiment on culture duration was divided into four groups: 4 days, 6 days, 8 days, and 10 days.

[0047] The cells were cultured statically in 6-well low-adhesion cell culture plates under the same co-culture conditions and divided into four groups for 4, 6, 8 and 10 days of culture.

[0048] The results are as follows Figure 4 As shown, static culture is more conducive to macrophage infiltration than dynamic culture in shake flasks, and the kidney organoid differentiation state is best on day 6 of static co-culture, during which macrophages can also infiltrate the kidney organoids efficiently.

[0049] Example 6: Identification of effective infiltration and co-localization of macrophages within renal organoids After adding 500,000 macrophages to 100 kidney organoids on day 5 of differentiation, they were cultured in conjugated medium with half the medium changed every 2 days. After 6 days of culture, the co-cultured kidney organoids were harvested, frozen sections were prepared and immunofluorescence stained for observation. The distal duct (ECAD), connecting duct (GATA3), glomerular podocytes (PODXL), interstitial cells (MEIS1 / 2 / 3), and basement membrane (COL1A1) of the kidney organoids were stained to observe the specific location of macrophages infiltrating the kidney organoids.

[0050] The results are as follows Figure 5 As shown, similar to the infiltration and polarization process of macrophages in kidney tissue in vivo, macrophages in the co-culture system can effectively infiltrate kidney organoids and locate in the interstitial cells (MEIS1 / 2 / 3) and basement membrane (COL1A1), but do not appear in the renal tubular (ECAD and GATA3) region or the glomerular podocyte (PODXL) region.

[0051] Bulk RNA sequencing was performed on organoids collected after co-culture to detect changes in the major expressed genes of macrophages before and after co-culture.

[0052] The results are as follows Figure 6 As shown, compared with iMacs cultured alone, iMacs entering kidney organoids exhibited decreased expression of M0-type genes such as CD11b, CD14, and CD68, increased expression of M1-type gene CD40, and decreased expression of M2-type genes CD206, CD163, and IRF4. Simultaneously, the expression of the pro-inflammatory cytokine IL-1 receptor IL-1R was significantly increased, consistent with the process of inducing further leukocyte aggregation through the secretion of pro-inflammatory factors in renal inflammation. This indicates that macrophages entering kidney organoids show a certain tendency to polarize towards the pro-inflammatory M1 type, but have not yet polarized towards the pro-fibrotic M2 type, similar to the fact that macrophages infiltrating in the early stages of in vivo injury are predominantly M1 type.

[0053] The results above demonstrate that this invention establishes a stable in vitro co-culture method for simulating the renal immune microenvironment. This method effectively simulates the early stages of renal injury, where macrophages infiltrate the kidney, leading to the accumulation of activated M1 pro-inflammatory macrophages. This model stably simulates renal inflammation caused by kidney injury in vitro, with macrophages infiltrating renal organoids polarizing into the pro-inflammatory M1 type and significantly upregulating the expression of the pro-inflammatory cytokine IL-1 receptor (IL-1R) in renal organoids. This co-culture model provides a stable in vitro research model for the regulation of inflammatory signals in renal injury and drug screening.

[0054] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for co-culturing macrophages and kidney organoids to mimic the renal immune microenvironment, comprising the following steps: 1) Add mature macrophages obtained by hPSC in the early differentiation stage of kidney organoids; 2) Use co-culture medium for culturing.

2. The co-culture method according to claim 1, characterized in that: The co-culture medium includes: basal medium DMEM, KOSR 10-20% (v / v), NEAA 0.7-1.3% (v / v), Glutamax 0.7-1.3% (v / v), HEPES 0.7-1.3% (v / v), PVA: 1.7-3.3 mg / mL, 1-2% (v / v) FBS and 25-75 ng / mL M-CSF.

3. The co-culture method according to claim 1, characterized in that: The kidney organoids mentioned are kidney organoids that have differentiated on day 5.

4. The co-culture method according to claim 1, characterized in that: The ratio of the number of kidney organoids to mature macrophages is 1:(4000~6000).

5. The co-culture method according to claim 1, characterized in that: The culture time is 5 to 7 days.

6. The co-culture method according to claim 1, characterized in that: The cultivation method is static cultivation.

7. A kidney organoid infiltrated with macrophages, characterized in that: The kidney organoids infiltrated with macrophages were prepared by the co-culture method according to any one of claims 1 to 6.

8. The kidney organoid according to claim 7, characterized in that: The macrophages infiltrate the interstitium and basement membrane of the renal organoids.

9. The application of at least one of the co-culture methods according to any one of claims 1 to 6, and the renal organoids infiltrated with macrophages according to any one of claims 7 to 8, in 1) to 2): 1) Application in the preparation of a model simulating immune cell-kidney interaction. 2) Application in the preparation of kidney disease models.

10. The application according to claim 9, characterized in that: The kidney disease includes kidney inflammation.

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