Preparation method and application of myocardial cell mitochondria directionally differentiated by iPSC

Abstract

The invention discloses a preparation method and application of IPSC directionally differentiated myocardial cell mitochondria, and relates to the technical field of mitochondria, human induced pluripotent stem cells (iPSC) are directionally differentiated into myocardial cells (iPSC-CMs), and the mitochondria is separated and extracted from the iPSC-CMs. The preparation method comprises the following steps: inducing and differentiating iPSC into iPSC-CMs by utilizing a Wnt signal channel regulation and control differentiation system, purifying by a metabolism selection method, and then separating mitochondria by a homogenate and differential centrifugation method. The mitochondria prepared by the method can be effectively ingested by renal tubular epithelial cells, and the oxidative injury of the renal tubular epithelial cells and / or the renal ischemia-reperfusion injury can be remarkably relieved by inhibiting accumulation of reactive oxygen species (ROS), improving ATP synthesis capability, recovering mitochondrial membrane potential, inhibiting apoptosis and other mechanisms.

Description

Technical Field

[0001] This invention relates to the field of mitochondrial technology, and more specifically to a method for preparing and applying mitochondria of IPSC-directed differentiated cardiomyocytes. Background Technology

[0002] Kidney transplantation is the most effective treatment for patients with end-stage renal disease (ESRD), significantly prolonging survival and improving quality of life. However, ischemia-reperfusion injury (IRI), an unavoidable consequence of transplantation, severely impacts early functional recovery and long-term survival of the transplanted kidney, and is a major cause of delayed graft function (DGF) and chronic transplanted kidney dysfunction. Currently, there are no FDA-approved drugs specifically for the treatment of renal IRI.

[0003] The kidney is a highly metabolic organ, with the proximal tubular epithelial cells (TECs) having the highest mitochondrial content. Its energy primarily comes from fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). During ischemia, the interruption of oxygen supply leads to impaired electron transport chain function; ATP production decreases; and Na+... + / K + Pump inactivation; intracellular Ca 2+ Overload; abnormal opening of the mitochondrial permeability transition pore (mPTP). During reperfusion, electron leakage intensifies; reactive oxygen species are generated in large quantities; mitochondrial membrane potential is lost; cytochrome c is released; and apoptosis and the inflammatory cascade are activated. Numerous studies have shown that mitochondrial dysfunction is a key node in the occurrence and development of renal renal injury (IRI). Renal IRI refers to the damage caused by the restoration of perfusion after tissue blood flow interruption; it is a pathological process. In in vitro experiments, oxidative damage to the TEC (transmitter organelle) and hypoxia-reoxygenation damage can be used to simulate the cellular pathological process during IRI.

[0004] In recent years, mitochondrial transplantation has attracted widespread attention as a novel organelle therapy strategy. This technique involves isolating functional mitochondria from healthy tissue and delivering them to damaged tissue to replenish mitochondrial numbers and restore metabolic function. Previous studies have shown that mitochondria can transfer between cells; mitochondrial transplantation can improve functional recovery in a myocardial renal tubular injury (IRI) model; mitochondrial supplementation can alleviate various metabolic diseases; and mitochondrial transplantation has shown a certain protective effect in an in vitro renal tubular injury model. However, current research mainly focuses on mitochondria derived from skeletal muscle or autologous tissue, which suffers from insufficient donor source stability, difficulties in standardizing preparation, and low purity. Furthermore, its application in renal IRI remains in the exploratory stage.

[0005] Cardiomyocytes possess the following characteristics: high mitochondrial content; strong oxidative phosphorylation capacity; and active energy metabolism. Induced pluripotent stem cells (iPSCs) are pluripotent stem cells reprogrammed from terminally differentiated somatic cells through the introduction of specific transcription factors. Unlike classic embryonic stem cell technology and somatic cell nuclear transfer technology, iPSC technology does not use embryonic cells or oocytes, thus avoiding ethical issues. Furthermore, iPSC technology allows for the preparation of proprietary stem cells using the patient's own somatic cells, significantly reducing the likelihood of immune rejection. Under certain conditions, iPSCs can be directed to differentiate into cardiomyocytes. If iPSC-induced differentiated cardiomyocytes can be used as a donor source, combined with standardized isolation and quality control systems, it is hoped that highly active, scalable mitochondrial preparations can be obtained, thus providing a new technological pathway for IRI treatment.

[0006] There are currently no reports on using iPSC-directed differentiated cardiomyocytes as a standardized mitochondrial donor source; systematically verifying the role of this source of mitochondria in a TEC oxidative damage and ischemia-reperfusion model; and constructing a mitochondrial preparation system with clear quality control indicators.

[0007] Therefore, it is necessary to propose a method for preparing and applying IPSC-directed differentiation of cardiomyocyte mitochondria to solve the above problems. Summary of the Invention

[0008] The purpose of this invention is to provide a method for isolating highly active mitochondria from human induced pluripotent stem cells (iPSCs) directed differentiation into cardiomyocytes (iPSC-CMs); and to provide the application of said mitochondria in alleviating oxidative damage and / or ischemia-reperfusion injury of renal tubular epithelial cells.

[0009] To achieve the above objectives, the present invention specifically adopts the following technical solution:

[0010] The method for preparing mitochondria of iPSC-directed differentiated cardiomyocytes includes the following steps:

[0011] a. Directing the differentiation of human induced pluripotent stem cells (iPSCs) into cardiomyocytes (iPSC-CMs);

[0012] b. Isolate and extract mitochondria from the iPSC-CMs.

[0013] Further, step a includes:

[0014] a1. Human iPSCs were inoculated into matrix gel-coated culture dishes;

[0015] a2. When the cell density reaches 85%–95%, the differentiation system regulated by the Wnt signaling pathway is used for induction.

[0016] a3. Cardiac cells that differentiate into spontaneously beating cardiomyocytes after 8–12 days;

[0017] a4. iPSC-CMs with a purity ≥85% were obtained by metabolic selection.

[0018] Further, step b includes:

[0019] b1. Digest and collect iPSC-CMs, and wash with pre-cooled phosphate buffer;

[0020] b2. Add mitochondrial separation buffer and 2 mm diameter steel beads, and homogenize in a tissue homogenizer by shaking at 30 Hz for 30 s.

[0021] b3. Centrifuge at 1000g for 5 min to remove cell nuclei and debris, and collect the supernatant;

[0022] b4. Centrifuge the supernatant at 3500g to 12000g for 10 min and collect the resulting precipitate as the mitochondrial component;

[0023] b5. Resuspend the mitochondrial precipitate in mitochondrial storage solution.

[0024] Furthermore, the preparation method further includes quality control of the obtained mitochondria, the quality control including one or more of the following:

[0025] a. Observation of the integrity of the mitochondrial double membrane using transmission electron microscopy;

[0026] b. Mito Tracker staining confirmed the integrity of the mitochondrial membrane potential;

[0027] c. Cytotoxicity test.

[0028] Furthermore, the concentration of the mitochondrial preparation is 5 × 10⁻⁶. 5per mL.

[0029] The application of mitochondria prepared according to the above method in the preparation of drugs for alleviating oxidative damage to renal tubular epithelial cells and / or renal ischemia-reperfusion injury.

[0030] Furthermore, the mitochondria can be taken up by renal tubular epithelial cells and their mitochondrial function can be restored.

[0031] Furthermore, the mitochondria exert their protective function through one or more of the following mechanisms: inhibiting the accumulation of intracellular reactive oxygen species; enhancing ATP synthesis capacity; restoring mitochondrial membrane potential; and inhibiting apoptosis.

[0032] Furthermore, the mitochondria are delivered to the recipient's renal cortex via subcapsular injection.

[0033] A pharmaceutical composition for treating oxidative damage and / or ischemia-reperfusion injury of renal tubular epithelial cells, characterized in that it comprises the above-described mitochondrial formulation and a pharmaceutically acceptable carrier.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention can significantly reduce ROS levels; restore mitochondrial membrane potential; improve ATP synthesis capacity; reduce the proportion of apoptosis; improve kidney tissue damage scores; does not involve cell transplantation, significantly reducing the risk of immune rejection; has no risk of tumor formation; and is easy to prepare on a large scale. Attached Figure Description

[0036] Figure 1 Immunofluorescence imaging of IPSC-CMs (α-actinin, cTNT).

[0037] Figure 2 Electron micrographs of mitochondria after co-culturing with HK-2.

[0038] Figure 3 The results of mitotracker live cell staining after co-culturing mitochondria with HK-2 cells.

[0039] Figure 4 For mitochondrial cck8 toxicity testing.

[0040] Figure 5 The results of CCK8 assay for cell proliferation levels after co-culturing mitochondria with HK-2 cells under oxidative damage conditions.

[0041] Figure 6 The results of ROS flow cytometry for co-culturing mitochondria with HK-2 cells to prevent oxidative damage.

[0042] Figure 7The results of GSH testing on the prevention of oxidative damage by co-culturing mitochondria with HK-2.

[0043] Figure 8 Results of Mitosox co-culture with HK-2 to prevent oxidative damage.

[0044] Figure 9 The results of CCK8 assay were obtained after co-culturing mitochondria with HK-2 cells under hypoxic-recombinant conditions.

[0045] Figure 10 The results of ROS flow cytometry for mitochondrial co-culture with HK-2 cells to prevent hypoxia-reoxygenation injury.

[0046] Figure 11 GSH results for mitochondria co-cultured with HK-2 to prevent hypoxia-reoxygenation injury.

[0047] Figure 12 Results of Mitosox co-culture with HK-2 to prevent hypoxia-reoxygenation injury. Detailed Implementation

[0048] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0049] Please see Figures 1 to 12 Example 1

[0050] Mitochondria isolated and counted from IPSC-CMs and observed under electron microscopy.

[0051] 1. IPSCs differentiate into iPSC-CMs;

[0052] IPSCs were differentiated into iPSC-CMs for subsequent experiments. The specific method is as follows:

[0053] (1) Human induced pluripotent stem cells (iPSCs) were seeded into 6-well culture plates coated with matrix gel and cultured using complete stem cell culture medium.

[0054] (2) When the cell density reaches 85% to 95%, start the directed differentiation program according to the instructions of the myocardial differentiation kit and induce myocardial differentiation by regulating the Wnt signaling pathway.

[0055] (3) The cells can be observed to beat spontaneously 8 to 12 days after differentiation. Change the myocardial purification medium and culture for 3 to 5 days to improve the purity of the myocardial cells.

[0056] (4) Immunofluorescence staining of cardiomyocytes. Cardiomyocytes were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Triton-X100 for 10 min at room temperature, blocked with 4% bovine serum albumin for 1 h at room temperature, and incubated overnight at 4°C with Oct4 (1:200), cTnT (1:200), α-actinin (1:250), and DAP1 (1:200). Goat anti-rabbit secondary antibody was added and incubated at room temperature in the dark for 2 h. 4′,6-diamidino-2-phenylindole (DAPI) was added to stain the nuclei for 10 min at room temperature. The cells were washed three times with PBS for 5 min between each step. Fluorescence images were captured under a LEICA:PDMI4000B inverted fluorescence microscope. The results are shown below. Figure 1 As shown in the figure, the results indicate that the cells are spindle-shaped and express myocardial-specific marker proteins, indicating successful differentiation.

[0057] 2. Mitochondrial isolation

[0058] (1) Cardiac cardiomyocytes were digested with trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in pre-cooled PBS, and placed on ice for 3 min. The cells were homogenized, 1 mL of pre-cooled mitochondrial extraction buffer was added, along with 3 steel beads with a diameter of 2 mm. The mixture was then shaken at 30 Hz for 30 s in a tissue homogenizer. The cells were centrifuged at 1000 g for 5 min to remove cell nuclei and debris. The supernatant was collected and centrifuged at 3500 g for 10 min. The resulting precipitate was the mitochondrial component. The cells were resuspended in 20 μL of mitochondrial storage solution and placed on ice for later use.

[0059] (2) Protein concentration was detected using the BCA method, and mitochondrial particles were counted using a Countstar automated cell counter. The mitochondrial concentration was adjusted to 5 × 10⁻⁶. 5 per mL.

[0060] Structural integrity assessment (transmission electron microscopy): A small amount of isolated and purified mitochondria was prepared according to standard transmission electron microscopy sample preparation procedures: the mitochondrial precipitate was fixed with 2.5% glutaraldehyde, dehydrated with graded ethanol, embedded, and ultrathinly sectioned. The ultrastructure of the mitochondria was observed using a transmission electron microscope. Figure 2 As shown in the electron microscopy images, the isolated mitochondria have a clear and complete double membrane structure and cristae, and are plump with no obvious swelling or damage, indicating that the mitochondrial structure is in good integrity.

[0061] Membrane potential detection (MitoTracker staining): After co-culturing the isolated mitochondria with HK-2 cells for 4 hours, the co-culture medium was removed, and medium containing MitoTracker Red CMXRos was added. The cells were incubated at 37°C for 30 minutes. After washing, the MitoTracker fluorescence signal in the mitochondria of HK-2 cells was observed using a fluorescence microscope. Figure 3 As shown, MitoTracker staining results indicate that HK-2 cells have taken up mitochondria with intact membrane potential, which exhibit punctate or short rod-shaped fluorescence in the cytoplasm, indicating that the transplanted mitochondria maintained good membrane potential activity.

[0062] 3. Mitochondrial toxicity test

[0063] HK-2 cells were seeded into 6-well plates, and different concentrations of mitochondria were co-cultured with the cells. Cell proliferation activity was assessed using CCK8 assays to verify the non-toxicity of the exogenous mitochondria. Figure 4 The results showed that exogenous mitochondria did not produce significant toxicity to HK-2 cells within the above concentration range (P>0.05).

[0064] 4. Results of co-culturing mitochondria with HK-2 cells to prevent oxidative damage using CCK-8, ROS flow cytometry, GSH, and Mitosox.

[0065] (1) Method

[0066] HK-2 cells were added to 6-well plates and cultured until 90% confluence was reached before the experiment. Cells were divided into four groups: ① Blank group (cultured in normal culture medium); ② Control group (cultured in normal culture medium); ③ Hydrogen peroxide-damaged group (co-cultured with 125 μM hydrogen peroxide for 2 h); ④ Group 1 (cultured with 5 × 10⁻⁶ cells / well). 5 After adding 0.2 ml of mitochondrial solution at a concentration of / ml and co-culturing with cells for 2 hours as a preventative treatment, the cells were then co-cultured with 125 μM hydrogen peroxide for 2 hours.

[0067] Take a 96-well plate, digest HK-2 cells with trypsin, centrifuge to collect the cells, and discard the liquid. Resuspend the cells in complete culture medium and count them using a CountStar instrument. Aim for a cell count of 5 × 10⁶ cells / well. 4 Add approximately / ml to a 96-well plate. Add the corresponding volume of cell suspension and 5×10⁶ ml of [unclear text - possibly a typo, should be 500 ml]. 5 Mitochondria were co-cultured at a concentration of / ml in 3 wells. 200µl of PBS was added around the perimeter. A blank control group (containing only complete culture medium) was also included in 3 wells. After 24 hours, the culture medium was discarded and the culture was kept dark. 100µl of complete culture medium containing 10% CCK8 was added to each well. After 2 hours, the culture was analyzed at 450nm.

[0068] Dilute the DCFH-DA stock solution to a final concentration of 10 μM using serum-free medium. Remove the cell culture medium, wash once with PBS, and add 100 μL of the diluted DCFH-DA probe to each well. Incubate at 37°C in the dark for 20–30 min. Remove the probe solution, wash twice with PBS to remove any unexploded probes. Digest the cells with trypsin, resuspend in PBS to obtain a single-cell suspension, and detect DCF fluorescence intensity. Analyze the average fluorescence intensity.

[0069] Dilute 1 mM GSH standard with buffer to prepare gradient solutions of 0, 1, 5, 10, and 20 μM. Collect four groups of cells, wash with PBS, add lysis buffer, lyse on ice for 10 min, centrifuge at 12000g, 4℃ for 10 min, and collect the supernatant. Add 50 μL of standard / sample and 50 μL of Thiolite Green working solution to a 96-well plate, and incubate at room temperature in the dark for 10–60 min. Detect the fluorescence intensity at 490 nm excitation / 525 nm emission using a microplate reader, and plot a standard curve with standard concentration on the x-axis and fluorescence intensity on the y-axis.

[0070] MitoSOX Red stock solution was diluted to a final concentration of 5 μM using serum-free medium. The cells were then covered with the probe working solution and incubated at 37°C in the dark for 10–15 minutes. The probe working solution was then removed, and the cells were gently washed three times with pre-warmed PBS or HBSS buffer to thoroughly remove any free probes that had not entered the mitochondria. Cells were digested with trypsin to obtain a single-cell suspension, and the mean fluorescence intensity was analyzed by flow cytometry.

[0071] (2) Results

[0072] The CCK-8 assay results indicated that, under oxidative damage conditions, the cell proliferation level detected by CCK-8 assay after co-culturing mitochondria with HK-2 cells was higher in the co-cultured group than in the oxidative damage group. Figure 5 As shown.

[0073] ROS experiment results showed that cells treated with hydrogen peroxide and oxygenation showed significantly more ROS damage than the control group. The experimental group treated with mitochondrial prophylaxis showed a significant leftward shift in the ROS peak, indicating reduced ROS damage. Figure 6 As shown.

[0074] GSH experiment results showed that cells treated with hydrogen peroxide and oxygen oxidative damage had lower GSH concentrations than the control group, while the GSH concentration in the mitochondrial pretreatment group was higher than that in the oxidative damage group, indicating that oxidative damage was alleviated. Figure 7 As shown.

[0075] The Mitosox experiment results showed that the mitochondrial superoxide anion concentration in cells treated with hydrogen peroxide damage was lower than that in the control group, while the mitochondrial superoxide anion concentration was higher in the experimental group that underwent mitochondrial pretreatment, indicating that intracellular mitochondrial function was improved. Figure 8 As shown.

[0076] 5. Results of co-culturing mitochondria with HK-2 cells to prevent hypoxia-reoxygenation injury using CCK-8, ROS flow cytometry, GSH, and Mitosox.

[0077] (1) Method

[0078] After digesting Hk-2 cells with trypsin and centrifuging (1000 rpm, 5 min), resuspend them in complete culture medium and count them using a count starter. Adjust the cell density and seed them into 6-well plates, with 3 × 10⁶ cells per well. 5 Cells were seeded and incubated for 24 hours. The complete culture medium was discarded, and the cells were washed twice with PBS. The medium was replaced with glucose-free and serum-free medium, and the 6-well plate was transferred to a tri-gas incubator. Hypoxia was observed for 12 hours. After hypoxia, the medium was discarded, the cells were washed twice with PBS, and the medium was replaced with complete medium. The 6-well plate was then transferred back to a normal incubator, and reoxygenation was carried out for 6 hours.

[0079] Take a 96-well plate, digest HK-2 cells with trypsin, centrifuge to collect the cells, and discard the liquid. Resuspend the cells in complete culture medium and count them using a CountStar instrument. Aim for a cell count of 5 × 10⁶ cells / well. 4 Add approximately / ml to a 96-well plate. Add the corresponding volume of cell suspension and 5×10⁶ ml of [unclear text - possibly a typo, should be 500 ml]. 5 Mitochondria were co-cultured at a concentration of / ml in 3 wells. 200µl of PBS was added around the perimeter. A blank control group (containing only complete culture medium) was also included in 3 wells. After 24 hours, the culture medium was discarded and the culture was kept dark. 100µl of complete culture medium containing 10% CCK8 was added to each well. After 2 hours, the culture was analyzed at 450nm.

[0080] Dilute the DCFH-DA stock solution to a final concentration of 10 μM using serum-free medium. Remove the cell culture medium, wash once with PBS, and add 100 μL of the diluted DCFH-DA probe to each well. Incubate at 37°C in the dark for 20–30 min. Remove the probe solution, wash twice with PBS to remove any unexploded probes. Digest the cells with trypsin, resuspend in PBS to obtain a single-cell suspension, and detect DCF fluorescence intensity. Analyze the average fluorescence intensity.

[0081] Dilute 1 mM GSH standard with buffer to prepare gradient solutions of 0, 1, 5, 10, and 20 μM. Collect four groups of cells, wash with PBS, add lysis buffer, lyse on ice for 10 min, centrifuge at 12000g, 4℃ for 10 min, and collect the supernatant. Add 50 μL of standard / sample and 50 μL of Thiolite Green working solution to a 96-well plate, and incubate at room temperature in the dark for 10–60 min. Detect the fluorescence intensity at 490 nm excitation / 525 nm emission using a microplate reader, and plot a standard curve with standard concentration on the x-axis and fluorescence intensity on the y-axis.

[0082] MitoSOX Red stock solution was diluted to a final concentration of 5 μM using serum-free medium. The cells were then covered with the probe working solution and incubated at 37°C in the dark for 10–15 minutes. The probe working solution was then removed, and the cells were gently washed three times with pre-warmed PBS or HBSS buffer to thoroughly remove any free probes that had not entered the mitochondria. Cells were digested with trypsin to obtain a single-cell suspension, and the mean fluorescence intensity was analyzed by flow cytometry.

[0083] (2) Results

[0084] The results of CCK8 assay on cell proliferation levels after co-culturing mitochondria and HK-2 cells under hypoxic-reoxygenation conditions indicated that the proliferative activity of cells co-cultured with mitochondria and HK-2 cells was higher than that in the hypoxic-reoxygenation injury group. Figure 9 As shown.

[0085] ROS experiment results showed that cells treated with hypoxia-reoxygenation injury suffered more ROS damage than the control group. In the experimental group treated with mitochondrial prophylaxis, the peak value shifted significantly to the left, indicating reduced ROS damage. Figure 10 As shown.

[0086] GSH experiment results showed that cells treated with hypoxia-reoxygenation injury had lower GSH concentrations than the control group, while the GSH concentration in the mitochondrial pretreatment group was higher than that in the oxidative damage group, indicating that the damage was alleviated. Figure 11 As shown.

[0087] The Mitosox experiment results showed that cells treated with hypoxia-reoxygenation injury had lower mitochondrial superoxide anion concentrations than the control group, while the experimental group that underwent mitochondrial pretreatment had higher mitochondrial superoxide anion concentrations, indicating improved mitochondrial function. Figure 12 As shown.

[0088] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A method for preparing mitochondria of iPSC-directed differentiated cardiomyocytes, characterized in that, Includes the following steps: a. Directing the differentiation of human induced pluripotent stem cells (iPSCs) into cardiomyocyte iPSC-CMs; b. Isolate and extract mitochondria from the iPSC-CMs.

2. The method for preparing mitochondria of iPSC-directed differentiated cardiomyocytes according to claim 1, characterized in that, Step a includes: a1. Human iPSCs were inoculated into matrix gel-coated culture dishes; a2. When the cell density reaches 85%–95%, the differentiation system regulated by the Wnt signaling pathway is used for induction. a3. Cardiac cells that differentiate into spontaneously beating cardiomyocytes after 8–12 days; a4. iPSC-CMs with a purity ≥85% were obtained by metabolic selection.

3. The method for preparing mitochondria of iPSC-directed differentiated cardiomyocytes according to claim 1, characterized in that, Step b includes: b1. Digest and collect iPSC-CMs, and wash with pre-cooled phosphate buffer; b2. Add mitochondrial separation buffer and 2 mm diameter steel beads, and homogenize in a tissue homogenizer by shaking at 30 Hz for 30 s. b3. Centrifuge at 1000g for 5 min to remove cell nuclei and debris, and collect the supernatant; b4. Centrifuge the supernatant at 3500g to 12000g for 10 min and collect the resulting precipitate as the mitochondrial component; b5. Resuspend the mitochondrial precipitate in mitochondrial storage solution.

4. The method for preparing mitochondria of iPSC-directed differentiated cardiomyocytes according to claim 1, characterized in that, The preparation method further includes quality control of the obtained mitochondria, wherein the quality control includes one or more of the following: a. Observation of the integrity of the mitochondrial double membrane using transmission electron microscopy; b. Mito Tracker staining confirmed the integrity of the mitochondrial membrane potential; c. Cytotoxicity test.

5. The method for preparing mitochondria of iPSC-directed differentiated cardiomyocytes according to claim 4, characterized in that, The concentration of the mitochondrial preparation is 5 × 10⁻⁶. 5 per mL.

6. The use of mitochondria prepared by the method according to claim 1 in the preparation of medicaments for alleviating oxidative damage to renal tubular epithelial cells and / or renal ischemia-reperfusion injury.

7. The application according to claim 6, characterized in that, The mitochondria are delivered to the recipient's renal cortex via subcapsular injection.

8. A pharmaceutical composition for treating oxidative damage and / or ischemia-reperfusion injury of renal tubular epithelial cells, characterized in that, Includes the mitochondrial formulation according to claim 5; and a pharmaceutically acceptable carrier.

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