Improved mitochondrial storage liquid for protecting mitochondrial integrity

Abstract

The invention discloses an improved mitochondrial storage solution for protecting mitochondrial integrity, which is prepared by adding 1-5mM of glutathione, 1-5mM of lipoic acid and 1-5mM of NAD < + > into the existing commercial mitochondrial storage solution as improved components so as to effectively prolong the storage time of in-vitro extracted mitochondrial in the storage solution and maintain the structure and activity of the in-vitro extracted mitochondrial. According to the improved mitochondrial storage solution, the existing commercial mitochondrial storage solution is improved, so that a longer mitochondrial storage window period is provided, and the clinical application feasibility of the improved mitochondrial storage solution is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical reagents and relates to a storage solution for diluting or preserving mitochondria. The mitochondrial storage solution of the invention can effectively ensure the integrity of mitochondrial biological activity and effectively prolong the survival time of mitochondria. Background Art

[0002] As the energy factory of cells, mitochondria act as the metabolic center and signaling platform of cells, and participate in a series of important cellular processes, such as the production of adenosine triphosphate through oxidative phosphorylation, the generation and maintenance of reactive oxygen species, the biosynthesis of iron-sulfur clusters, innate immune signaling, calcium signaling, and a role in apoptosis and autophagy. Mitochondrial abnormalities can disrupt the main physiological functions of the body. Currently, more than 100 diseases caused by mitochondrial defects have been identified, including cancer, metabolic diseases, cardiovascular diseases, and nervous system diseases. Therefore, maintaining the health of mitochondrial structure and function has become the focus of attention in the prevention and treatment of human diseases.

[0003] Although some drugs (such as antioxidants) have been identified that can improve mitochondrial function and symptoms caused by mitochondrial dysfunction, most diseases caused by mitochondrial abnormalities are caused by mitochondrial DNA mutations or even deletions or mitochondrial structural damage, and there is currently a lack of effective treatments for them.

[0004] Mitochondrial transplantation has been proposed as a new strategy for treating diseases caused by mitochondrial abnormalities. It aims to deliver exogenous mitochondria into mitochondrial-deficient cells to replace damaged non-functional mitochondria with healthy functional mitochondria. This process can be likened to "replacing the old engine with a new one to restore its function."

[0005] Mitochondrial transplantation technology was first attempted by Clark and Shea in basic experiments in the field of antibiotic resistance. Subsequently, small-scale clinical research designs have been initially carried out on mitochondrial transplantation technology in the treatment of cerebral ischemia, myocardial ischemia and reperfusion injury, and postoperative cardiac rehabilitation, and it has broad application prospects in clinical practice.

[0006] During mitochondrial transplantation, maintaining the vitality of the mitochondrial donor is the key to ensuring transplantation efficiency and efficacy. The success of mitochondrial transplantation requires that the structure and function of the donor mitochondria can be effectively maintained, and in clinical applications, long-term storage and maintenance are even more necessary.

[0007] However, mitochondria are very sensitive to environmental changes. If they are not maintained in a suitable storage solution, the activity and viability of mitochondria will decline rapidly. Unfortunately, there is still no good solution to the problem of mitochondrial storage. So far, there is no method that can store and maintain mitochondrial activity for a long time.

[0008] Currently, the mitochondrial storage solution provided by commercial kits can only preserve mitochondria for 1 hour, and donor mitochondria must be used immediately after separation. This means that the short storage time of donor mitochondria cannot meet the time window required for clinical transplantation. For example, the function of donor mitochondria cannot be effectively maintained within 2-3 hours, which greatly limits the application and promotion of mitochondrial transplantation in clinical practice.

[0009] The ability of mitochondria to produce ATP and membrane potential are important indicators of the integrity of mitochondrial structure and function. Experiments have shown that the membrane potential of mitochondria stored in existing commercial storage solutions drops significantly after 3 hours. These data indicate that commercial mitochondrial storage solutions are not ideal for maintaining mitochondrial structure and function after the mitochondria have been stored for more than 1 hour. Currently in clinical research, because the storage time of donor mitochondria is limited, the main method of mitochondrial transplantation is to mix the donor mitochondria with the storage solution and immediately inject them directly into the corresponding organ. However, this short-term storage is not suitable for transplantation via the circulation route, and is even more unfavorable for promotion. Therefore, an optimal storage solution system must be established to maintain mitochondrial integrity and provide a longer survival time to ensure the feasibility of clinical application. Summary of the invention

[0010] The object of the present invention is to provide an improved mitochondrial storage solution for protecting mitochondrial integrity and providing a longer survival time to ensure the feasibility of clinical application.

[0011] The improved mitochondrial storage solution for protecting mitochondrial integrity described in the present invention is an improvement on the existing commercial mitochondrial storage solution. Specifically, the improved components glutathione, lipoic acid and NAD+ are added to the existing commercial mitochondrial storage solution, so that the glutathione concentration in the mitochondrial storage solution is 1-5mM, the lipoic acid concentration is 1-5mM, and the NAD+ concentration is 1-5mM, and the improved mitochondrial storage solution described in the present invention is prepared.

[0012] Furthermore, in the improved mitochondrial storage solution of the present invention, the concentration of glutathione is 2-5 mM, the concentration of lipoic acid is 1-3 mM, and the concentration of NAD+ is 1-3 mM.

[0013] Furthermore, in the improved mitochondrial storage solution of the present invention, the concentration of glutathione is 3 mM, the concentration of lipoic acid is 1.5 mM, and the concentration of NAD+ is 1.5 mM.

[0014] Furthermore, the improved mitochondrial storage solution after the above improvement has an osmotic pressure of 290-320 mOSmol / kg and a pH value of 6.5-7.5.

[0015] The commercial mitochondrial storage solution that can be used for the improvement of the present invention can be any existing commercially available mitochondrial storage solution, for example, FEIYUBIO mitochondrial storage solution FY40400 or Beyotime mitochondrial storage solution C3609.

[0016] The present invention improves the existing commercial mitochondrial storage solution, thereby effectively increasing the storage time of in vitro extracted mitochondria in the storage solution and maintaining the structure and activity of in vitro extracted mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the purity test result of mitochondria extracted in vitro.

[0018] Figure 2 It is the change of mitochondrial membrane potential after being stored in improved mitochondrial storage solution A and original storage solution for 1h and 3h.

[0019] Figure 3 It is a comparison of the transplantation activity of mitochondria after being stored in the improved mitochondrial storage solution A and the original storage solution for 3 hours.

[0020] Figure 4 It is the change of mitochondrial membrane potential after being stored in modified mitochondrial storage solution B and original storage solution for 1h and 3h.

[0021] Figure 5 This is the change in mitochondrial membrane potential in the improved mitochondrial storage solution of Example 7.

[0022] Figure 6 This is the change in mitochondrial membrane potential in the improved mitochondrial storage solution of Example 9. Implementation

[0023] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solution of the present invention so that those skilled in the art can well understand and utilize the present invention, rather than limiting the scope of protection of the present invention.

[0024] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0025] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art. Example

[0026] Example 1

[0027] Take the AC16 immortalized human cardiomyocytes grown in a 10 cm cell culture dish with a density of 80-90%, discard the original culture medium, add 1 ml of trypsin to each dish to digest the cells, and place them in a 37°C incubator. After 3 minutes, terminate the digestion with complete culture medium containing 10% fetal bovine serum.

[0028] Transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 min, retain the precipitate, discard the supernatant, resuspend the precipitated cells with 3 ml PBS buffer, and count.

[0029] Take 10 million cells, continue centrifugation at 1000 rpm for 3 minutes, retain the precipitate, discard the supernatant, add 2 ml of mitochondrial separation solution (Biyuntian, C3601-1) to suspend the cells, and place them in an ice bath for 10 minutes.

[0030] Transfer the cell suspension to a glass homogenizer and homogenize 25-35 times. Take 2µl of the cell homogenate, add 50µl of trypan blue staining solution, mix well, and observe the proportion of trypan blue-stained positive (blue) cells under a microscope. Stop homogenization when the proportion of positive cells is greater than 50%.

[0031] The homogenized suspension was transferred to 1.5 ml sterile enzyme-free centrifuge tubes, centrifuged at 1000 g for 10 min in a precooled centrifuge at 4°C, and the precipitate was discarded. The supernatant was transferred to a new 1.5 ml sterile enzyme-free centrifuge tube, and centrifuged at 3500 g for 10 min in a precooled centrifuge at 4°C, the supernatant was discarded, and the precipitate was retained to obtain purified mitochondria.

[0032] Figure 1 The purity of the purified mitochondria is shown. Sample 1 in the figure is the precipitate obtained after the first centrifugation, and sample 2 is the supernatant obtained after the first centrifugation; sample 3 (i.e., the framed sample) is the precipitate obtained after the second centrifugation, i.e., the complete protein sample of the cleaved mitochondria, and sample 4 is the supernatant obtained after the second centrifugation, i.e., the residue removed during the purification of mitochondria. The left and right sides are marker indicator bands for protein size, among which COX IV is the cytochrome c oxidase IV subtype, a specific protein expressed only in mitochondria, and is often used to represent mitochondria; GAPDH is glyceraldehyde-3-phosphate dehydrogenase, a protein localized in the cytoplasm, and is often used to represent the cytoplasm; Histone H3 is histone H3, a specific protein expressed only in the nucleus, and is often used to represent the nucleus.

[0033] In sample 1, the COX IV band showed that the sample contained a small amount of mitochondria, the GAPDH band showed that it contained a large amount of cytoplasmic components, and the Histone H3 band showed that it contained a small amount of nuclear components, that is, the precipitate after the first centrifugation was mainly cytoplasmic components that needed to be removed; in sample 2, the COX IV band showed that the sample contained a large amount of mitochondria, the GAPDH band showed that it contained a large amount of cytoplasmic components, and the Histone H3 band showed that it contained a large amount of nuclear components, that is, the supernatant after the first centrifugation contained mitochondria, cytoplasm and nuclear components; in sample 3, the COX IV band showed that the sample contained a large amount of mitochondria, the GAPDH band showed that it contained very small amounts of cytoplasmic components, and the Histone H3 band showed that it did not contain nuclear components, that is, the precipitate after the second centrifugation contained highly pure mitochondria; in sample 4, the COX IV band and the Histone H3 band showed that the supernatant did not contain mitochondria and nuclear components, and the GAPDH band showed that the main components were cytoplasmic contents, that is, the supernatant after the second centrifugation contained cytoplasmic components that needed to be removed.

[0034] Figure 1 The results showed that the mitochondria extracted in vitro contained almost no nuclear components and contained a small amount of cytoplasmic components. In subsequent experiments, the high-purity mitochondria obtained by the second centrifugation were used as samples.

[0035] Example 2

[0036] The mitochondrial storage solution FY40400 provided by Nantong Feiyu Biotechnology Co., Ltd. (FEIYUBIO) was used. The osmotic pressure of the mitochondrial storage solution was 298 mOSmol / kg and the pH value was 7.0.

[0037] Add 4.61 mg glutathione, 1.55 mg α-lipoic acid and 5 mg NAD+ to 5 ml mitochondrial storage solution FY40400 in sequence to prepare improved mitochondrial storage solution A.

[0038] The modified mitochondrial storage solution A contains 3mM glutathione, 1.5mM lipoic acid and 1.5mM NAD+, with an osmotic pressure of 310mOSmol / kg and a pH of 6.45.

[0039] Example 3

[0040] The mitochondria extracted in vitro in Example 1 were added to the mitochondrial storage solution FY40400 and the improved mitochondrial storage solution A prepared in Example 1 for storage, and the membrane potential of the purified mitochondria in different mitochondrial storage solutions was tested by flow cytometry at 1 h and 3 h.

[0041] Figure 2 The effects of different mitochondrial storage solutions on the membrane potential of in vitro extracted mitochondria are demonstrated.

[0042] As can be seen from the figure, there is no obvious difference in mitochondrial membrane potential when the mitochondria are placed in FY40400 and improved mitochondrial storage solution A for 1 hour; after 3 hours, the mitochondrial membrane potential in FY40400 drops rapidly, while the mitochondrial membrane potential in the corresponding improved mitochondrial storage solution A can still maintain a high level, which is significantly higher than the original storage solution. The difference is extremely significant, and the mitochondrial structure is better protected.

[0043] Example 4

[0044] The mitochondria stored in mitochondrial storage solution FY40400 and improved mitochondrial storage solution A for 3 hours in Example 3 were transplanted into normal lung epithelial Beas-2B cells in equal amounts to test the effect of mitochondria stored in different storage solutions on the level of oxidative phosphorylation in the recipient Beas-2B cells after transplantation.

[0045] One day before transplantation, Beas-2B cells were plated in Seahorse's dedicated 24-well cell plates and cultured overnight in normal complete medium (DMEM medium containing a mixture of 10% bovine serum and 1% penicillin-streptomycin). 2 hours before transplantation, the medium of Beas-2B cells was replaced with an incomplete medium (DMEM medium) and starved for 2 hours in a 37°C incubator.

[0046] After 3 h of storage in different storage solutions, the mitochondria were resuspended in complete medium. At the same time, the medium of Beas-2B cells was replaced with complete medium. The complete medium-mitochondrial suspension was transplanted at a concentration of 5 × 10 5 mitochondria were added into Beas-2B cell culture system.

[0047] Figure 3 The test results in (a) show that compared with the blank control group, the mitochondria in the original storage solution group and the improved storage solution group can increase the oxygen consumption rate (OCR) of the recipient cell Beas-2B, and compared with the original storage solution group, the mitochondria in the improved storage solution group can increase the level of OCR of the recipient cells to a greater extent, indicating that the mitochondria in the improved storage solution can better increase the overall oxygen consumption rate level of mitochondria in the recipient cells, and the mitochondria in the improved storage solution have better transplantation activity and efficiency.

[0048] The above Seahorse experiment was further statistically analyzed, and the results are as follows: Figure 3 (b)

[0049] In terms of basal respiration, both the original storage solution group and the modified storage solution group were improved compared with the blank control group, and there was no difference in the improvement level between the two. In terms of maximum respiratory reserve, the improvement of the modified storage solution group was more significant than that of the original storage solution group, indicating that the mitochondria preserved in the modified storage solution had better mitochondrial activity and function. In terms of ATP-linked respiration, both the original storage solution group and the modified storage solution group were improved compared with the blank control group, and there was no difference in the improvement level between the two.

[0050] The above results indicate that compared with the original storage solution, the mitochondria in the improved storage solution can increase the maximum respiratory reserve value of the mitochondria in the recipient cells to a greater extent and enhance their cell activity, further illustrating that the mitochondria in the improved storage solution have higher activity and transplantation efficiency than the mitochondria in the original storage solution.

[0051] Example 5

[0052] The mitochondrial storage solution C3609 provided by Shanghai Beyotime Biotechnology Co., Ltd. (Beyotime) was used. The osmotic pressure of the mitochondrial storage solution was 290 mOSmol / kg and the pH value was 7.1.

[0053] 9.22 mg of glutathione, 3.10 mg of α-lipoic acid and 9.96 mg of NAD+ were added to 10 ml of mitochondrial storage solution C3609 in sequence to prepare improved mitochondrial storage solution B.

[0054] The modified mitochondrial storage solution B contains 3mM glutathione, 1.5mM lipoic acid and 1.5mM NAD+, with an osmotic pressure of 300mOSmol / kg and a pH of 6.5.

[0055] Example 6

[0056] The mitochondria extracted in vitro in Example 1 were added to the mitochondrial storage solution C3609 and the improved mitochondrial storage solution B prepared in Example 5 for storage, and the membrane potential of the purified mitochondria in different mitochondrial storage solutions was tested by flow cytometry at 1 h and 3 h.

[0057] Figure 4 The effects of different mitochondrial storage solutions on the membrane potential of in vitro extracted mitochondria are demonstrated.

[0058] As can be seen from the figure, after the mitochondria were placed in C3609 and improved mitochondrial storage solution B for 1 hour, the membrane potential of the mitochondria in C3609 was slightly lower than that in the improved mitochondrial storage solution B; and after 3 hours, the difference was more significant, the mitochondrial membrane potential in C3609 dropped rapidly, while the corresponding mitochondrial membrane potential in the improved mitochondrial storage solution B was much higher than that in the original storage solution C3609, and the mitochondrial structure was better protected.

[0059] Example 7

[0060] The mitochondrial storage solution FY40400 provided by Nantong Feiyu Biotechnology Co., Ltd. (FEIYUBIO) was used. The osmotic pressure of the mitochondrial storage solution was 298 mOSmol / kg and the pH value was 7.0.

[0061] 7.68 mg of glutathione, 3.10 mg of α-lipoic acid and 9.95 mg of NAD+ were added to 5 ml of mitochondrial storage solution FY40400 in sequence to prepare improved mitochondrial storage solution A.

[0062] The modified mitochondrial storage solution A contains 5mM glutathione, 3mM lipoic acid and 3mM NAD+, with an osmotic pressure of 335mOSmol / kg and a pH of 6.37.

[0063] Example 8

[0064] The mitochondria extracted in vitro in Example 1 were added to the mitochondrial storage solution FY40400 and the improved mitochondrial storage solution prepared in Example 7 for storage, and the membrane potential of the purified mitochondria in different mitochondrial storage solutions was tested by flow cytometry at 1 h and 3 h.

[0065] from Figure 5 It can be seen that when the mitochondria were stored in FY40400 and the improved mitochondrial storage solution for 1 hour, although the mitochondrial membrane potential in FY40400 was slightly lower than that in the improved mitochondrial storage solution, there was no obvious difference; but after storage for 3 hours, the mitochondrial membrane potential in FY40400 declined rapidly, while the mitochondrial membrane potential in the corresponding improved mitochondrial storage solution was still able to maintain a high level, which was significantly higher than the original storage solution. The difference was significant, and the mitochondrial structure was better protected.

[0066] Example 9

[0067] The mitochondrial storage solution C3609 provided by Shanghai Beyotime Biotechnology Co., Ltd. (Beyotime) was used. The osmotic pressure of the mitochondrial storage solution was 290 mOSmol / kg and the pH value was 7.1.

[0068] Add 6.14 mg of glutathione, 2.06 mg of α-lipoic acid and 6.63 mg of NAD+ to 5 ml of mitochondrial storage solution C3609 in sequence to prepare improved mitochondrial storage solution B.

[0069] The modified mitochondrial storage solution B contains 4mM glutathione, 2mM lipoic acid and 2mM NAD+, with an osmotic pressure of 318mOSmol / kg and a pH of 6.4.

[0070] Example 10

[0071] The mitochondria extracted in vitro in Example 1 were added to the mitochondrial storage solution C3609 and the improved mitochondrial storage solution prepared in Example 9 for storage, and the membrane potential of the purified mitochondria in different mitochondrial storage solutions was tested by flow cytometry at 1 h and 3 h.

[0072] from Figure 6 It can be seen that after the mitochondria were stored in C3609 and the improved mitochondrial storage solution for 1 hour respectively, the membrane potential of the mitochondria in C3609 was slightly lower than that in the improved mitochondrial storage solution. After 3 hours of storage, the difference was more significant. The mitochondrial membrane potential in C3609 dropped rapidly, while the mitochondrial membrane potential in the corresponding improved mitochondrial storage solution was much higher than that in the original storage solution C3609. The difference was significant, and the mitochondrial structure was better protected.

[0073] The test results of the above embodiments show that the improvement of the present invention on the existing commercial mitochondrial storage solution can universally increase the window period for the preservation of mitochondria extracted in vitro and more effectively protect the structure of mitochondria.

[0074] The above embodiments of the present invention do not describe all the details in detail, nor limit the present invention to the above embodiments. Various changes, modifications, substitutions and variations made by ordinary technicians in this field without departing from the principles and purpose of the present invention should be included in the protection scope of the present invention.

Claims

1. A modified mitochondrial storage solution for protecting mitochondrial integrity, which is prepared by adding modified ingredients glutathione, lipoic acid and NAD+ to a commercial mitochondrial storage solution, so that the glutathione concentration in the mitochondrial storage solution is 1-5 mM, the lipoic acid concentration is 1-5 mM, and the NAD+ concentration is 1-5 mM.

2. The improved mitochondrial storage solution according to claim 1, wherein the concentration of glutathione is 2-5 mM, the concentration of lipoic acid is 1-3 mM, and the concentration of NAD+ is 1-3 mM.

3. The improved mitochondrial storage solution according to claim 1, wherein the concentration of glutathione is 3 mM, the concentration of lipoic acid is 1.5 mM, and the concentration of NAD+ is 1.5 mM.

4. The improved mitochondrial storage solution according to claim 1, 2 or 3, wherein the osmotic pressure of the improved mitochondrial storage solution is 290-320 mOSmol / kg and the pH value is 6.5-7.

5.

5. The improved mitochondrial storage solution according to claim 1, 2 or 3, characterized in that The commercial mitochondrial storage solution is FEIYUBIO mitochondrial storage solution FY40400 or Beyotime mitochondrial storage solution C3609.

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