A method for improving the yield, purity and activity of mitochondria of mesenchymal stem cells and application thereof

Abstract

This invention discloses a method and its application for isolating mitochondria from mesenchymal stem cells (MSCs) to improve their yield, purity, and activity. Compared to existing methods for isolating MSCs, the method of this invention effectively improves the yield and purity of MSCs, increasing the number of mitochondria by 2.96 times. The method also better maintains the activity of MSC mitochondria, significantly increasing cytochrome C content and exhibiting superior activity in treating autoimmune hepatitis. Furthermore, the method is simple, requires minimal reagents, and has broad application prospects in the field of mitochondrial transplantation therapy.

Description

Technical Field

[0001] This invention relates to the technical field of mitochondrial isolation, specifically to an isolation method and its application that improves the yield, purity, and activity of mitochondria in mesenchymal stem cells. Background Technology

[0002] Mitochondria are the energy metabolism center of eukaryotic cells, responsible for synthesizing adenosine triphosphate (ATP) through oxidative phosphorylation, providing approximately 90% of the cell's energy needs. Studies have shown that mitochondrial dysfunction is closely related to the pathogenesis of various inflammatory diseases, and significant alterations in mitochondrial structure and function can be observed in these diseases. Mitochondrial-targeted therapy has become a novel strategy for intervening in inflammatory diseases, among which mitochondrial transplantation therapy shows promising application prospects. This therapy, by delivering healthy, functional mitochondria to damaged tissues, can restore cellular energy supply, reduce oxidative stress, inhibit excessive inflammatory responses, and promote tissue repair.

[0003] Among numerous cell sources, mitochondria from stem cells (especially mesenchymal stem cells) have unique therapeutic advantages. The mitochondria from these stem cells suffer from less oxidative damage, have strong metabolic plasticity, low immunogenicity, and possess multiple functions such as tissue tropism, secretion of anti-inflammatory factors, and regulation of the immune microenvironment.

[0004] However, existing mitochondrial extraction methods are mainly designed for high-metabolic tissues such as the myocardium and liver. Research on mitochondrial extraction from stem cells is limited, and the extracted quantities are insufficient for clinical applications, severely restricting the use of stem cell-derived mitochondria in the treatment of inflammatory diseases. Chinese invention patent CN106148277A discloses a method for mitochondrial isolation from stem cells, but this method requires culturing in a high-glucose medium for 48 hours to promote mitochondrial generation. Furthermore, this method is only applicable to mesenchymal stem cells from passages P1 to P3. P1 to P3 passages represent "young" mesenchymal stem cells with strong proliferative capacity, high differentiation potential, and relatively strong mitochondrial function. As the number of passages of mesenchymal stem cells increases, the cells undergo replicative senescence, leading to a decline in differentiation potential and consequently, a decrease in mitochondrial activity.

[0005] Therefore, developing a mitochondrial extraction method specifically tailored to the characteristics of stem cells to achieve high-purity, high-yield, and high-activity mitochondrial preparation is of significant scientific importance and clinical application value. Summary of the Invention

[0006] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a method and application for isolating mitochondria to improve the yield, purity and activity of mesenchymal stem cells.

[0007] The first objective of this invention is to provide a method for isolating stem cell mitochondria that improves their yield, purity, and / or activity.

[0008] A second objective of this invention is to provide the application of stem cell mitochondria prepared by the above-described separation method in the preparation of drugs for treating autoimmune hepatitis.

[0009] This invention claims protection for the following:

[0010] A method for separating mitochondria to improve the yield, purity, and activity of stem cells, the method comprising the following steps:

[0011] S1. Collect the digested stem cells, add cell lysis buffer, vortex and incubate on ice. Repeat the vortex-on-ice incubation step 5 to 7 times, then add reaction stop solution, centrifuge and separate solid and liquid.

[0012] S2. Collect the precipitate after solid-liquid separation in step S1, add cell lysis buffer, and repeat step S1;

[0013] S3. Collect the precipitate after solid-liquid separation in step S2, add cell lysis buffer, and repeat step S1, wherein the repeated vortex-on-ice incubation step is repeated 2 to 4 times.

[0014] S4. Combine the liquids obtained from solid-liquid separation in steps S1 to S3, and then perform solid-liquid separation by centrifugation;

[0015] S5. Collect the liquid after solid-liquid separation in step S4, centrifuge and collect the precipitate. The precipitate obtained is stem cell mitochondria.

[0016] Both the cell lysis buffer and the reaction termination solution contain protease inhibitors.

[0017] Preferably, in steps S1 to S3, the centrifugation conditions are 800-1200g for 4.5-5.5 minutes.

[0018] More preferably, in steps S1 to S3, the centrifugation conditions are 1000g centrifugation for 5 minutes.

[0019] Preferably, in step S4, the centrifugation conditions are 1300-1700g for 8-12 minutes.

[0020] More preferably, in step S4, the centrifugation conditions are 1500g centrifugation for 10 minutes.

[0021] Preferably, in step S5, the centrifugation conditions are 10000-14000g for 8-12 minutes.

[0022] More preferably, in step S5, the centrifugation conditions are 12000g centrifugation for 10 minutes.

[0023] Preferably, in steps S1 to S5, the centrifugation temperature is 3 to 5°C.

[0024] More preferably, in steps S1 to S5, the centrifugation temperature is 4°C.

[0025] Preferably, in steps S1 and S2, the ratio of cell lysis buffer to stem cells is (550–650) μL: 2 × 10⁻⁶ μL. 7 The ratio of the reaction termination solution to stem cells is (550–650) μL: 2 × 10⁻⁶. 7 indivual.

[0026] More preferably, in steps S1 and S2, the ratio of the cell lysis buffer to stem cells is 600 μL: 2 × 10⁻⁶. 7 The ratio of the reaction termination solution to stem cells was 600 μL: 2 × 10⁻⁶. 7 indivual.

[0027] Preferably, in step S3, the ratio of cell lysis buffer to stem cells is (350–450) μL: 2 × 10⁻⁶ μL. 7 The ratio of the reaction termination solution to stem cells is (350–450) μL: 2 × 10⁻⁶. 7 indivual.

[0028] More preferably, in step S3, the ratio of the cell lysis buffer to stem cells is 400 μL: 2 × 10⁻⁶. 7 The ratio of the reaction termination solution to stem cells was 400 μL: 2 × 10⁻⁶. 7 indivual.

[0029] Preferably, in steps S1 to S3, the vortex conditions are 2200 to 2600 rpm for 25 to 35 seconds.

[0030] More preferably, in steps S1 to S3, the vortex condition is 2400 rpm for 30 seconds.

[0031] Preferably, in steps S1 and S2, the number of vortexes is 6.

[0032] Preferably, in step S3, the vortex is 3 times.

[0033] Preferably, in steps S1 to S3, the incubation time on ice is 1.5 to 2 minutes.

[0034] More preferably, in steps S1 to S3, the incubation time on ice is 2 minutes.

[0035] Preferably, the conditions for collecting the digested stem cells are centrifugation at 800-900g for 4.5-5.5 minutes.

[0036] More preferably, the conditions for collecting the digested stem cells are centrifugation at 850g for 5 minutes.

[0037] Preferably, in steps S1 to S3, grinding beads are added simultaneously with the addition of cell lysis buffer.

[0038] Preferably, in step S1, before adding the cell lysis buffer, the stem cells are washed with PBS 1-2 times.

[0039] Preferably, the cell lysis buffer and reaction termination solution are derived from ThermoFisher Scientific's Mitochondrial Isolation Kit for Cultured Cells, catalog number 89874.

[0040] Preferably, the protease inhibitor is PMSF.

[0041] More preferably, the stem cells are mesenchymal stem cells.

[0042] More preferably, the mesenchymal stem cells include mesenchymal stem cells derived from umbilical cord blood, mesenchymal stem cells derived from embryos, and / or mesenchymal stem cells derived from adipose tissue.

[0043] More preferably, the mesenchymal stem cells are cultured using a low-glucose DMEM medium containing fetal bovine serum and basic fibroblast growth factor.

[0044] More preferably, the stem cells are P6 to P8 generation stem cells.

[0045] Application of stem cell mitochondria prepared by any of the above separation methods in the preparation of drugs for treating autoimmune hepatitis.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] Compared to existing methods for isolating stem cell mitochondria, the method of this invention effectively improves the yield and purity of stem cell mitochondria. Furthermore, the isolation method of this invention can better maintain the activity of stem cell mitochondria, resulting in a significant increase in cytochrome C content.

[0048] Compared to stem cell mitochondria obtained using existing isolation methods, those obtained using the method of this invention exhibit superior activity in treating autoimmune hepatitis. Furthermore, the isolation method of this invention is simple and requires minimal reagents, making it a promising candidate for mitochondrial transplantation therapy. Attached Figure Description

[0049] Figure 1This is a comparison of the number of mitochondria in human umbilical cord mesenchymal stem cells (HuMSCs) extracted in Example 1 and Comparative Example 1.

[0050] Figure 2 This is a comparison of the purity of HuMSCs mitochondria extracted in Example 1 and Comparative Example 1.

[0051] Figure 3 This is a comparison chart of the cytochrome C content in the mitochondria of HuMSCs extracted in Example 1 and Comparative Example 1.

[0052] Figure 4 To improve the mitochondrial function of HuMSCs in treating concanavalin A (ConA)-induced autoimmune hepatitis; A: HE staining results and statistical diagram of liver necrosis area in each group of mice; B: Comparison of serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) in each group of mice. Detailed Implementation

[0053] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] Example 1: A method for isolating mitochondria from human umbilical cord mesenchymal stem cells (HuMSCs)

[0056] I. Experimental Methods

[0057] Mitochondria in HuMSCs were isolated using the Mitochondria Isolation Kit for Cultured Cells (ThermoFisher Scientific, catalog number 89874). Before use, 20 μL of 100 mM PMSF was added to 2 mL of Mitochondria Isolation Reagent A (cell lysis buffer), and 30 μL of 100 mM PMSF was added to 3 mL of Mitochondria Isolation Reagent C (reaction stop solution).

[0058] 1. HuMSCs were cultured in low-glucose DMEM medium containing 10% fetal bovine serum (v / v) and 2 ng / mL basic fibroblast growth factor. 2 × 10⁶ cells were collected. 7 HuMSCs that have been passaged and expanded (P6-P8 generation) were digested with 0.25% trypsin solution (v / v), centrifuged at 850g for 5 min, the supernatant was discarded, and then the cells were resuspended in PBS and washed twice, centrifuged at 850g for 5 min, and the supernatant was discarded.

[0059] 2. Resuspend the cell pellet in 600 μL of mitochondrial separation reagent A (with added PMSF), then transfer to a 2 mL EP tube and add 2 steel beads (4 mm) per tube. Vortex at 2400 rpm for 30 s, then incubate on ice for 2 min. Repeat the vortex-on-ice incubation operation 6 times. After transferring the liquid to a new 2 mL EP tube, add 600 μL of mitochondrial separation reagent C (with added PMSF), gently invert several times to mix, and centrifuge at 1000 g for 5 min at 4 °C. Transfer the supernatant to a new 1.5 mL EP tube.

[0060] 3. Add 600 μL of mitochondrial separation reagent A (with added PMSF) to the precipitate, then add 2 steel balls (4 mm) per tube. Vortex at 2400 rpm for 30 s, then incubate on ice for 2 min. Repeat the vortex-on-ice incubation operation 6 times. Transfer the liquid to a new 2 mL EP tube, add 600 μL of mitochondrial separation reagent C (with added PMSF), gently invert several times to mix, centrifuge at 1000 g for 5 min at 4 °C, and transfer the supernatant to a new 1.5 mL EP tube.

[0061] 4. Add 400 μL of mitochondrial separation reagent A (already containing PMSF) to the precipitate, and add 2 steel beads (4 mm) per tube. Vortex at 2400 rpm for 30 s, incubate on ice for 2 min, and repeat the vortex-on-ice incubation operation 3 times. Transfer the liquid to a new 2 mL EP tube, add 400 μL of separation solution C, gently invert several times to mix, centrifuge at 1000 g for 5 min at 4 °C, and transfer the supernatant to a new 1.5 mL EP tube;

[0062] 5. After mixing the supernatants obtained in steps 2 to 4, centrifuge at 1500g for 10 min at 4℃; separate the supernatant, and then centrifuge the supernatant at 12000g for 10 min at 4℃. The resulting precipitate is the HuMSCs mitochondria.

[0063] Comparative Example 1

[0064] Mitochondria were extracted from HuMSCs according to the method of Chinese invention patent CN106148277A. Mitochondria of HuMSCs were isolated using the Mitochondria Isolation Kit for Cultured Cells (brand: ThermoFisher Scientific, catalog number: 89874). Before use, 40 μL of 100 mM PMSF was added to 4 mL of mitochondrial isolation reagent A (cell lysis buffer), and 50 μL of 100 mM PMSF was added to 5 mL of mitochondrial isolation reagent C (reaction stop solution).

[0065] 1. HuMSCs were cultured in low-glucose DMEM medium containing 10% fetal bovine serum (v / v) and 2 ng / mL basic fibroblast growth factor. 2 × 10⁶ cells were collected. 7 HuMSCs that have been passaged and expanded (P6-P8 generation) were digested with 0.25% trypsin solution (v / v), centrifuged at 850g for 5 min, the supernatant was discarded, and then the cells were resuspended in PBS and washed twice, centrifuged at 850g for 5 min, and the supernatant was discarded.

[0066] 2. Resuspend the cell pellet in 2 mL of mitochondrial separation reagent A (with added PMSF), then transfer to a 7 mL EP tube. Vortex at 2400 rpm for 30 s, incubate on ice for 2 min, and repeat the vortex-on-ice incubation operation 3 times. Add 2 mL of mitochondrial separation reagent C (with added PMSF), gently mix with a pipette tip, then aliquot into four 1.5 mL EP tubes, 1 mL per tube. Immediately centrifuge at 2000 g for 10 min at 4 °C, and transfer the supernatant to a new 1.5 mL EP tube.

[0067] 3. Add 0.5 mL of mitochondrial separation reagent A (with added PMSF) to each precipitate, mix, and transfer the total 2 mL to a 7 mL EP tube. Vortex at 2400 rpm for 30 s, incubate on ice for 2 min, and repeat the vortex-on-ice incubation operation 3 times; add 2 mL of mitochondrial separation reagent C (with added PMSF), gently mix with a pipette tip, and then aliquot into four 1.5 mL EP tubes, 1 mL per tube, and immediately centrifuge at 2000 g for 10 min at 4 °C. Transfer the supernatant to a new 1.5 mL EP tube.

[0068] 4. After mixing the supernatants obtained in steps 2 and 3, centrifuge at 12000g for 10 min at 4℃, discard the supernatant, and the resulting precipitate is the HuMSCs mitochondria.

[0069] Example 2: Effect of different methods for isolating HuMSCs mitochondria on mitochondrial number

[0070] I. Experimental Methods

[0071] Use 2×10 7 HuMSCs amplified by passages (P6-P8) were used to extract mitochondria from the HuMSCs according to the methods in Example 1 and Comparative Example 1. The mitochondria were resuspended in 200 μL of mitochondrial separation reagent C (with added PMSF) to obtain a mitochondrial suspension. 300 μL of mitochondrial separation reagent C (with added PMSF) was added to a flow cytometry tube, followed by 2.5 μL of the mitochondrial suspension. After thorough mixing, the mixture was analyzed to calculate the number of HuMSCs mitochondria extracted by both methods.

[0072] II. Experimental Results

[0073] The results are as follows Figure 1 As shown, the number of HuMSCs mitochondria extracted using the method of Example 1 was significantly increased, approximately 2.96 times the number of HuMSCs mitochondria extracted using the method of Comparative Example 1.

[0074] Example 3: Effect of different methods for isolating HuMSCs mitochondria on mitochondrial purity

[0075] I. Experimental Methods

[0076] Use 2×10 7 HuMSCs amplified by passages (P6-P8) were used to extract mitochondrial precipitates of HuMSCs according to the methods in Example 1 and Comparative Example 1. Lysis buffer (RIPA lysis buffer and PMSF mixed at a volume ratio of 100:1) was added to the HuMSCs mitochondrial precipitates, and the mixture was lysed on ice for 30 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was separated to obtain the total protein extract.

[0077] Add 5×SDS loading buffer to the total protein extract, mix well, boil in water for 5 min, then separate an equal volume of the total protein extract using a 12% polyacrylamide gel (w / v) and transfer it to a PVDF membrane. Block the PVDF membrane with 5% skim milk powder solution (w / v) at room temperature for 2 h, then incubate with primary antibody (α-Tublin, COX IV) overnight at 4 °C. The next day, wash the membrane three times with 1×TBST, incubate with secondary antibody (Anti-rabbit IgG) at room temperature with shaking for 1 h, wash three times with 1×TBST, add chemiluminescence developing solution, and image using a chemiluminescence analyzer. Compare the contamination of α-Tublin cytoplasmic protein in mitochondria extracted by the two methods.

[0078] II. Experimental Results

[0079] The results are as follows Figure 2 As shown, the contamination level of cytoplasmic protein α-Tublin in the mitochondria of HuMSCs extracted using the method of Example 1 was significantly lower than that in the mitochondria of HuMSCs extracted using the method of Comparative Example 1, indicating that the mitochondria of HuMSCs extracted using the method of Example 1 had higher purity.

[0080] Example 4: Effects of different methods for isolating HuMSCs mitochondria on mitochondrial cytochrome C (Cyt C) content.

[0081] I. Experimental Methods

[0082] Use 2×10 7HuMSCs amplified by passages (P6-P8) were used to extract mitochondrial precipitates of HuMSCs according to the methods in Example 1 and Comparative Example 1. Lysis buffer (RIPA lysis buffer and PMSF mixed at a volume ratio of 100:1) was added to the HuMSCs mitochondrial precipitates, and the mixture was lysed on ice for 30 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was separated to obtain the total protein extract.

[0083] Add 5×SDS loading buffer to the total protein extract, mix well, boil in water for 5 min, then separate an equal volume of the total protein extract using a 12% polyacrylamide gel (w / v) and transfer it to a PVDF membrane. Block the PVDF membrane with 5% skim milk powder solution (w / v) at room temperature for 2 h, then incubate with primary antibodies (Cyt C, COX IV) overnight at 4°C. The next day, wash the membrane three times with 1×TBST, incubate with secondary antibody (Anti-rabbit IgG) at room temperature with shaking for 1 h, wash the membrane three times with 1×TBST, add chemiluminescence developing solution, and image using a chemiluminescence analyzer.

[0084] II. Experimental Results

[0085] The results are as follows Figure 3 As shown, the cytochrome C content of HuMSCs mitochondria extracted using the method of Example 1 was significantly higher than that of HuMSCs mitochondria extracted using the method of Comparative Example 1, indicating that the extraction method of Example 1 can better maintain the activity of HuMSCs mitochondria.

[0086] Example 5: HuMSCs mitochondria improve concanavalin A (ConA)-induced autoimmune hepatitis

[0087] I. Experimental Methods

[0088] C57BL / 6 mice (6-8 weeks old) were weighed and randomly divided into a blank control group (Control group), a hepatitis group (ConA group), and the mitochondrial therapy group (ConA-MT group) as described in Example 1. 实施例1 ), Comparative Example 1: Mitochondrial Treatment Group (ConA-MT) 对比例1 Each group consists of 3 animals, and the following treatments are performed:

[0089] Control group: Each mouse was injected with 130 μL of PBS via the tail vein;

[0090] Hepatitis group (ConA group): Each mouse was injected with 12.5 μg ConA / g BW via the tail vein to induce the hepatitis model;

[0091] Example 1: Mitochondrial Treatment Group (ConA-MT) 实施例1To establish the model, each mouse was injected via tail vein with 12.5 μg ConA / g BW. 3.5 h after modeling, the mice were injected via tail vein with 2 × 10⁻⁶ BW. 7 HuMSCs mitochondria / animal prepared in Example 1;

[0092] Comparative Example 1: Mitochondrial Treatment Group (ConA-MT) 对比例1 Each mouse was injected with 12.5 μg ConA / g BW via the tail vein. 3.5 h after model initiation, 2 × 10⁻⁶ mice were injected via the tail vein. 7 HuMSCs mitochondria prepared in Comparative Example 1 / individual;

[0093] Twelve hours after modeling, mice were sacrificed, and blood was collected from the orbital cavity. The mouse blood was placed at 4°C for 30 minutes, then centrifuged at 1600g for 15 minutes at 4°C. The supernatant (i.e., serum) was collected and centrifuged again at 1600g for 15 minutes at 4°C. The liver biochemical indicators, namely aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), were detected using an automated biochemical analyzer.

[0094] The largest lobe of the liver was placed in 4% paraformaldehyde solution (w / v) and stained with hematoxylin and eosin (HE staining) to observe the pathological damage to the liver.

[0095] II. Experimental Results

[0096] The results are as follows Figure 4 As shown, the serum ALT, AST, and LDH levels in the mitochondrial treatment group of Example 1 were significantly lower than those in the mitochondrial treatment group of Comparative Example 1. Furthermore, compared to the mitochondrial treatment group of Comparative Example 1, the mitochondrial treatment group of Example 1 exhibited less inflammatory cell infiltration around the central vein of the liver, and the area of ​​hepatocyte necrosis foci was significantly reduced. These results indicate that the HuMSCs mitochondria prepared using the method of Example 1 demonstrate superior activity in the treatment of autoimmune hepatitis.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for isolating mitochondria to improve the yield, purity, and / or activity of mesenchymal stem cells, characterized in that, The separation method includes the following steps: S1. Collect digested mesenchymal stem cells, add cell lysis buffer and 4 mm steel balls, vortex and incubate on ice. Repeat the vortex-on-ice incubation step 5 to 7 times, then add reaction stop solution, centrifuge at 800 to 1200 g for 4.5 to 5.5 min and then perform solid-liquid separation. The ratio of cell lysis buffer to mesenchymal stem cells was (550–650) μL: 2 × 10⁻⁶ μL. 7 The ratio of the reaction termination solution to mesenchymal stem cells was (550–650) μL: 2 × 10⁻⁶ μL. 7 indivual; S2. Collect the precipitate after solid-liquid separation in step S1, add cell lysis buffer and 4 mm steel beads, and repeat step S1; The ratio of cell lysis buffer to mesenchymal stem cells was (550–650) μL: 2 × 10⁻⁶ μL. 7 The ratio of the reaction termination solution to mesenchymal stem cells was (550–650) μL: 2 × 10⁻⁶ μL. 7 indivual; S3. Collect the precipitate after solid-liquid separation in step S2, add cell lysis buffer and 4 mm steel balls, and repeat step S1, wherein the repeated vortex-on-ice incubation step is repeated 2 to 4 times. The ratio of cell lysis buffer to mesenchymal stem cells was (350–450) μL: 2 × 10⁻⁶ μL. 7 The ratio of the reaction termination solution to mesenchymal stem cells is (350–450) μL: 2 × 10⁻⁶ μL. 7 indivual; S4. Combine the liquids obtained from solid-liquid separation in steps S1 to S3, centrifuge at 1300–1700 g for 8–12 min, and then perform solid-liquid separation. S5. Collect the liquid after solid-liquid separation in step S4, centrifuge at 10000-14000 g for 8-12 min and collect the precipitate. The precipitate obtained is the mitochondria of mesenchymal stem cells. Both the cell lysis buffer and the reaction termination solution contain protease inhibitors. The mesenchymal stem cells are umbilical cord mesenchymal stem cells from generation P6 to P8.

2. The application of the mesenchymal stem cell mitochondria prepared by the separation method according to claim 1 in the preparation of a drug for treating autoimmune hepatitis.

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