Mitochondria obtained from extracellular fluid, method for preparing same, and use thereof

Abstract

The present invention relates to mitochondria obtained from an extracellular fluid. More specifically, the present invention relates to a method for isolating mitochondria from an extracellular fluid obtained by centrifuging a liposuction-derived fraction (LDF), and to a use thereof. It was observed that the mitochondria obtained from the extracellular fluid maintained normal functions, and MT-SVF introduced into a stromal vascular fraction (SVF) exhibited excellent functions in angiogenesis and anti-inflammatory effects. Accordingly, it is suggested that, according to the present invention, mitochondria derived from an extracellular fluid and mitochondria isolated from an extracellular fluid and introduced into SVF obtained from adipose tissue can be used as a composition for preventing or treating ischemic diseases and skin diseases, and for improving skin conditions.

Description

Mitochondria obtained from extracellular fluid, method of preparation thereof, and uses

[0001] The present invention relates to mitochondria obtained from extracellular fluid, a method for producing the same, and uses.

[0002] Mitochondria (MT) are organelles essential for the survival of eukaryotic cells, involved in the synthesis and regulation of adenosine triphosphate (ATP) as an energy source. Mitochondria are associated with the control of various metabolic pathways in vivo, such as cell signaling, cell differentiation, and apoptosis, as well as the cell cycle and cell growth.

[0003] Therefore, damage to mitochondria can lead to various diseases, and most known mitochondrial disorders are attributed to hereditary or acquired mutations in mitochondrial DNA. For example, mitochondrial function can be altered by swelling due to abnormal mitochondrial membrane potential, oxidative stress caused by reactive oxygen species or free radicals, and defects in oxidative phosphorylation for mitochondrial energy production (Schapira AH et al., Lancet, 1;368(9529):70-82, 2006).

[0004] Meanwhile, research and development utilizing mitochondria as active ingredients is increasing, and there are growing attempts to use them industrially in medical settings. In particular, there is a need for further verification regarding the technical limitations of the purity of isolated mitochondria and the immunological safety of allogeneic transplantation.

[0005] Therefore, there is a need for research and development of technology that can easily and rapidly isolate autologous mitochondria and apply them to various diseases.

[0006] Accordingly, the inventors of the present invention researched a method for isolating mitochondria that can avoid immune rejection and isolate them easily and quickly. As a result, they completed the present invention by isolating autologous mitochondria from liposuction samples obtained through liposuction surgery and analyzing their characteristics.

[0007] To achieve the above objective, one aspect of the present invention provides a method for obtaining mitochondria from extracellular fluid.

[0008] Another aspect of the present invention provides mitochondria isolated from extracellular fluid.

[0009] Another aspect of the present invention provides a stem cell into which mitochondria isolated from extracellular fluid have been introduced.

[0010] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of ischemic diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0011] Another aspect of the present invention provides a composition for preventing or treating skin diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0012] Another aspect of the present invention provides a composition for improving skin condition comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0013] Another aspect of the present invention provides a method for preventing or treating ischemic diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0014] Another aspect of the present invention provides a method for preventing or treating skin diseases comprising mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced as active ingredients.

[0015] Another aspect of the present invention provides an MT-SVF in which mitochondria isolated from extracellular fluid are introduced into an SVF obtained from adipose tissue.

[0016] The autologous mitochondria according to the present invention were obtained by utilizing liposuction material obtained through liposuction surgery. The mitochondria are obtained from the extracellular fluid obtained by centrifuging the obtained liposuction material, and as autologous mitochondria that perform normal functions, they can be used as a therapeutic agent for various diseases.

[0017] Figure 1 is a schematic diagram showing the process of separating the stromal vascular fraction (SVF) and mitochondria after liposuction.

[0018] Figure 2 is a diagram showing the materials and process for preparing extracellular fluid-derived mitochondria and stromal vascular fraction (SVF).

[0019] Figure 3 is a graph showing the amount of extracellular fluid-derived mitochondrial protein.

[0020] Figure 4a is a schematic diagram of a method for isolating plasma-derived mitochondria.

[0021] Figure 4b is a schematic diagram of a method for isolating extracellular fluid-derived mitochondria.

[0022] Figure 5a is a graph showing the size of plasma-derived mitochondria.

[0023] Figure 5b is a graph showing the activity of the electron transport chain, ATP production capacity, and citric acid synthase activity of plasma-derived mitochondria.

[0024] Figure 6a is a graph showing the size of extracellular fluid-derived mitochondria.

[0025] Figure 6b is a graph showing the activity of the electron transport chain, ATP production capacity, and citric acid synthase activity of extracellular fluid-derived mitochondria.

[0026] Figure 6c is a diagram showing the results of confirming extracellular fluid-derived mitochondria using the anti-TOM20 antibody.

[0027] Figure 6d is a diagram showing the results of membrane potential analysis of extracellular fluid-derived mitochondria.

[0028] Figure 7a is a schematic diagram showing the differentiation process of monocytes.

[0029] Figure 7b is a diagram showing the results of confirming the differentiation process of monocytes to confirm the anti-inflammatory effect of plasma-derived or extracellular fluid-derived mitochondria.

[0030] Figure 8 is a graph comparing the electron transport chain activity, ATP production capacity, and citric acid synthase activity of extracellular fluid-derived mitochondria and plasma-derived mitochondria.

[0031] Figure 9 is a diagram showing the results of confirming extracellular fluid-derived mitochondria introduced into the stromal vascular fraction (SVF) using an anti-TOM20 antibody.

[0032] Figure 10a is a schematic diagram showing the process of confirming the angiogenic ability of extracellular fluid-derived mitochondria introduced into the stromal vascular fraction of HUVEC (Human umbilical vein endothelial cell).

[0033] Figures 10b and 10c are photographs and graphs showing a comparison of the angiogenic ability of extracellular fluid-derived mitochondria introduced into the stromal vascular fraction of HUVECs.

[0034] Figure 11a is a schematic diagram showing the process of confirming the skin regenerative ability of extracellular fluid-derived mitochondria introduced into the stromal vascular fraction of HDF (Human dermal fibroblast).

[0035] Figures 11b and 11c are photographs and graphs showing a comparison of the skin regenerative capacity of extracellular fluid-derived mitochondria introduced into the stromal vascular fraction of HDF.

[0036] Figure 12a is a schematic diagram showing the process of confirming the inflammatory regulatory ability of extracellular fluid-derived mitochondria introduced into the stromal vascular fraction of THP-1 monocytes.

[0037] Figures 12b and 12c are results and graphs comparing the inflammatory regulatory ability of extracellular fluid-derived mitochondria introduced into the stromal vascular fraction of THP-1 monocytes.

[0038] One aspect of the present invention provides a method for obtaining mitochondria from extracellular fluid.

[0039] The term "extracellular fluid (ECF)" used in the present invention may refer to all body fluids located outside of cells, and its main component is the interstitial fluid surrounding the cells. Extracellular fluid accounts for about one-third of body fluids and is known to be composed of interstitial fluid, plasma, lymph fluid, etc. In the present invention, extracellular fluid refers to the portion of extracellular fluid obtained from liposuction, and may refer to a state in which all the body fluids located outside of cells described above are mixed with saline solution, anesthetic solution, etc. used during the liposuction procedure. Mitochondria can be obtained from the extracellular fluid through various known methods, such as centrifugation, enzymatic treatment, or immunocapture.

[0040] The method for obtaining the above mitochondria may be performed by centrifugation to maintain mitochondrial activity. Specifically, the extracellular fluid may be centrifuged at a rate of 100xg to 30,000xg, 500xg to 25,000xg, 1,000xg to 20,000xg, 2,000xg to 15,000xg, 3,000xg to 10,000xg, or 4,000xg to 8,000xg. The time for centrifugation may be 30 seconds to 1 hour, 1 minute to 50 minutes, 2 minutes to 40 minutes, 3 minutes to 30 minutes, 4 minutes to 20 minutes, or 5 minutes to 10 minutes, and may be appropriately adjusted depending on the number of centrifugations and the content of the sample. In one embodiment, extracellular fluid can be placed in a 50 mL tube and centrifuged at 20,000 xg for 5 minutes to obtain mitochondria.

[0041] The term "lipoaspirate-derived fraction (LDF)" as used in this invention may refer to the liquid component of liposuction obtained during the liposuction surgical procedure. The lipoaspirate-derived fraction contains various cells, including stem cells, perivascular cells (Pericytes), and endothelial cells, and is known to have potential uses such as skin regeneration and cell therapy.

[0042] In the present invention, the extracellular fluid may be the liposuction fraction.

[0043] In the present invention, the liposuction fraction may be obtained by centrifuging lipoaspirate obtained through liposuction surgery. Specifically, the lipoaspirate may be centrifuged at a speed of 100xg to 5,000xg, 200xg to 4,000xg, 300xg to 3,000xg, 400xg to 2,000xg, or 500xg to 1,000xg. The time for centrifugation may be 30 seconds to 1 hour, 1 minute to 50 minutes, 2 minutes to 40 minutes, 3 minutes to 30 minutes, 4 minutes to 20 minutes, or 5 minutes to 10 minutes, and may be appropriately adjusted according to the number of centrifugations and the content of the sample. In one embodiment, the liposuction sample can be placed in a 50 mL tube and centrifuged at 2,000 xg for 5 minutes to obtain the separated lower layer, which is the liposuction fraction.

[0044] Another aspect of the present invention provides mitochondria isolated from extracellular fluid.

[0045] The above extracellular fluid is the same as described above.

[0046] The mitochondria separated from the above extracellular fluid can be separated from the extracellular fluid through various known methods, such as centrifugation, enzymatic treatment, or immunocapture.

[0047] The method of separating the above mitochondria from the extracellular fluid can be performed by centrifugation to maintain mitochondrial activity. Specifically, the extracellular fluid can be centrifuged at a rate of 100xg to 30,000xg, 500xg to 25,000xg, 1,000xg to 20,000xg, 2,000xg to 15,000xg, 3,000xg to 10,000xg, or 4,000xg to 8,000xg. The time for centrifugation can be 30 seconds to 1 hour, 1 minute to 50 minutes, 2 minutes to 40 minutes, 3 minutes to 30 minutes, 4 minutes to 20 minutes, or 5 minutes to 10 minutes, and can be appropriately adjusted depending on the number of centrifugations and the content of the sample. In one embodiment, mitochondria can be separated from the extracellular fluid by placing the extracellular fluid into a 50 mL tube and centrifuging at 20,000 xg for 5 minutes.

[0048] The mitochondria isolated from the above extracellular fluid may be used in combination with mitochondria derived from extracellular fluid or mitochondria obtained from extracellular fluid.

[0049] In the present invention, the extracellular fluid may be a liposuction fraction.

[0050] The above liposuction fraction is the same as described above.

[0051] In the present invention, the mitochondria may have a size of about 100 nm to about 3,500 nm. Specifically, it may be about 100 nm to about 3,500 nm, about 200 nm to about 3,000 nm, about 300 nm to about 2,500 nm, about 400 nm to about 2,000 nm, about 500 nm to about 1,500 nm, or about 600 nm to about 1,000 nm. In one embodiment, the average may be 610.3 nm, and reasonably, 610.3 ± 403.3 nm.

[0052] Another aspect of the present invention provides a stem cell into which mitochondria isolated from extracellular fluid have been introduced.

[0053] The extracellular fluid and mitochondria isolated from the extracellular fluid are the same as those described above.

[0054] The above stem cell may be any one selected from the group consisting of mesenchymal stem cells, adipose-derived stem cells, endothelial progenitor cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, tissue-derived stem cells, and combinations thereof.

[0055] In the present invention, the isolated mitochondria may be centrifuged together with stem cells to deliver the mitochondria into the stem cells. Specifically, the isolated mitochondria and stem cell mixture may be centrifuged at a rate of 100xg to 20,000xg, 100xg to 5,000xg, 200xg to 4,000xg, 300xg to 3,000xg, 400xg to 2,000xg, or 500xg to 1,000xg. The centrifugation time may be 30 seconds to 1 hour, 1 minute to 50 minutes, 2 minutes to 40 minutes, 3 minutes to 30 minutes, 4 minutes to 20 minutes, or 5 minutes to 10 minutes, and may be appropriately adjusted according to the number of centrifugations and the content of the sample.

[0056] In the present invention, the stem cells may be included in the stromal vascular fraction (SVF) obtained by centrifuging adipose tissue after treating it with collagenase.

[0057] The term "stromal vascular fraction (SVF)" used in the present invention may refer to a complex of various cells and growth factors derived from adipose tissue. It is composed of various stem cells, endothelial cells, perivascular cells, macrophages, etc., and the stromal vascular fraction can be obtained through known methods, such as centrifugation or enzymatic treatment of adipose tissue. In one embodiment, it may be obtained by centrifugation, and specifically, centrifugation may be performed at a speed of 100xg to 20,000xg, 100xg to 5,000xg, 200xg to 4,000xg, 300xg to 3,000xg, or 400xg to 4,000xg. The time for which centrifugation is performed can be 30 seconds to 1 hour, 1 minute to 50 minutes, 2 minutes to 40 minutes, 3 minutes to 30 minutes, 4 minutes to 20 minutes, or 5 minutes to 10 minutes, and can be appropriately adjusted depending on the number of centrifugations and the content of the sample, etc.

[0058] The above adipose tissue may consist of adipose tissue obtained from an intermediate layer of adipose tissue separated by centrifuging the liposuction material obtained during the liposuction surgery process.

[0059] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of ischemic diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0060] The above extracellular fluid, mitochondria isolated from the extracellular fluid, stem cells, and stem cells into which mitochondria isolated from the extracellular fluid have been introduced are identical to those described above.

[0061] The above-mentioned ischemic disease is a condition in which irreversible damage occurs to cells constituting tissues due to a decrease in blood supply, triggering processes known as the ischemic cascade, which results in the permanent loss of function of the brain, heart, or peripheral tissues. For example, the above-mentioned ischemic disease may include severe lower limb ischemia, ischemic stroke, ischemic heart disease, or ischemic colitis. In this case, the above-mentioned ischemic disease may be a disease caused by mitochondrial abnormalities and includes all ischemic cell disorders resulting from a decline in mitochondrial function.

[0062] As used herein, the term "prevention" may comprehensively mean preventing a disease in advance or reducing the likelihood or frequency of occurrence by administering the above pharmaceutical composition in a pharmaceutically effective amount. For example, it may mean reducing the probability of occurrence or the probability of recurrence in patients who are at risk of developing ischemic disease or who have previously developed it. The above "pharmaceutical effective amount" has the same meaning as "therapeutic effective amount" and can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, gender, the patient's sensitivity to drugs, the route of administration, the method of administration, the frequency of administration, the duration of treatment, the combination, or drugs used concurrently.

[0063] As used in this specification, the term "treatment" may comprehensively refer to improving a disease by administering the above-mentioned pharmaceutical composition in a pharmaceutically effective amount, providing alleviation or healing of disease symptoms in a time shortened compared to natural healing, and improving a single symptom or most symptoms caused by the disease. The above-mentioned pharmaceutically effective amount is the same as described above. The pharmaceutical composition of the present invention may serve as a composition for treating ischemic diseases in itself, or it may be applied as an adjuvant for treatment of said diseases by being administered together with other pharmacological components. Accordingly, the above-mentioned "treatment" includes the meaning of "adjuvant treatment."

[0064] As used herein, the term "administration" means introducing a specific substance into an individual by an appropriate method, and the route of administration of the composition may be any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.

[0065] In addition, the above-mentioned pharmaceutical composition for prevention or treatment may improve mitochondrial disease by enhancing the mitochondrial activity of the cells to which the composition is applied, by including as an active ingredient mitochondria isolated from extracellular fluid in which cell activity is maintained and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0066] In addition, regarding the above composition, mitochondria isolated from the extracellular fluid may be included at a concentration of 0.1 μg / mL to 500 μg / mL, 0.1 μg / mL to 200 μg / mL, or 0.2 μg / mL to 10 μg / mL. By including mitochondria within the above ranges, it is easy to adjust the mitochondrial dose during administration, and the degree of improvement in ischemic symptoms of the affected area may be further enhanced.

[0067] In addition, with respect to the above composition, the stem cells into which mitochondria isolated from the extracellular fluid have been introduced are 1 x 10 5 0.1 μg to 500 μg, 0.2 μg to 450 μg, or 1 μg to 300 μg of mitochondria may be introduced per cell, and the composition may improve cell function in the affected area of ​​ischemic disease upon administration by including cells to which foreign mitochondria have been delivered in such amounts.

[0068] Another aspect of the present invention provides a composition for preventing or treating skin diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0069] The above extracellular fluid, mitochondria isolated from extracellular fluid, stem cells, stem cells into which mitochondria isolated from extracellular fluid have been introduced, and prevention and treatment are the same as described above.

[0070] The above skin diseases may be selected from the group consisting of dermatitis, acne, wounds, skin wrinkles, skin aging, weakened skin elasticity, dry skin, sensitive skin, acne, hair loss, and skin pigmentation, and combinations thereof, but are not limited thereto.

[0071] Another aspect of the present invention provides a composition for improving skin condition comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0072] The above extracellular fluid, mitochondria isolated from the extracellular fluid, stem cells, and stem cells into which mitochondria isolated from the extracellular fluid have been introduced are identical to those described above.

[0073] The above-mentioned improvement of skin condition may be selected from the group consisting of improvement of dermatitis, improvement of acne, inhibition of wrinkle formation, inhibition of skin aging, improvement of skin elasticity, whitening, moisturization, inhibition of hair loss, and combinations thereof, but is not limited thereto. Additionally, it may be characterized by protecting the skin from the deterioration or loss of skin cell function, improving skin condition, or preventing or improving skin diseases.

[0074] In the present invention, the composition for improving skin condition may be an injectable agent.

[0075] The above-mentioned injectable may be an aqueous injectable, a non-aqueous injectable, an aqueous suspension injectable, a non-aqueous suspension injectable, or a solid injectable used by dissolving or suspending, but is not limited thereto. Depending on the type, the injectable may include at least one of distilled water for injection, vegetable oil (e.g., peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, estradiol benzoate, bismuth subsalicylate, sodium arsenobenzol, or streptomycin sulfate, and may optionally include a stabilizer or a preservative.

[0076] Another aspect of the present invention provides a use for the prevention or treatment of ischemic diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0077] The above extracellular fluid, mitochondria isolated from extracellular fluid, stem cells, stem cells into which mitochondria isolated from extracellular fluid have been introduced, ischemic disease, prevention and treatment are the same as described above.

[0078] Another aspect of the present invention provides a method for preventing or treating ischemic diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

[0079] The above extracellular fluid, mitochondria isolated from extracellular fluid, stem cells, stem cells into which mitochondria isolated from extracellular fluid have been introduced, ischemic disease, prevention and treatment are the same as described above.

[0080] Another aspect of the present invention provides a use for preventing or treating skin diseases comprising mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced as an active ingredient.

[0081] The above extracellular fluid, mitochondria isolated from extracellular fluid, stem cells, stem cells into which mitochondria isolated from extracellular fluid have been introduced, skin diseases, prevention and treatment are the same as described above.

[0082] Another aspect of the present invention provides a method for preventing or treating skin diseases comprising mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced as active ingredients.

[0083] The above extracellular fluid, mitochondria isolated from extracellular fluid, stem cells, stem cells into which mitochondria isolated from extracellular fluid have been introduced, skin diseases, prevention and treatment are the same as described above.

[0084] Another aspect of the present invention provides an MT-SVF in which mitochondria isolated from extracellular fluid are introduced into a stromal vascular fraction (SVF) obtained from adipose tissue.

[0085] The mitochondria isolated from the above SVF and extracellular fluid are the same as those described above.

[0086] MT-SVF, in which mitochondria isolated from extracellular fluid are introduced into SVF obtained from the above-mentioned adipose tissue, can introduce mitochondria isolated from extracellular fluid into SVF through various known methods such as centrifugation, co-culture, electroporation, liposome-mediated delivery, or introduction methods using cell-permeable peptides.

[0087] The method of introducing mitochondria isolated from extracellular fluid into the SVF obtained from the above adipose tissue can be performed by centrifugation to maintain mitochondrial activity.

[0088] Specifically, the SVF and mitochondrial mixture can be centrifuged at a rate of 100xg to 30,000xg, 500xg to 25,000xg, 1,000xg to 20,000xg, 2,000xg to 15,000xg, 3,000xg to 10,000xg, or 4,000xg to 8,000xg. The centrifugation time can be 30 seconds to 1 hour, 1 minute to 50 minutes, 2 minutes to 40 minutes, 3 minutes to 30 minutes, 4 minutes to 20 minutes, or 5 minutes to 10 minutes, and can be appropriately adjusted according to the number of centrifugations and the content of the sample.

[0089] The above adipose tissue may consist of adipose tissue obtained from an intermediate layer of adipose tissue separated by centrifuging the liposuction material obtained during the liposuction surgery process.

[0090] The present invention will be explained in more detail below through the following examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0091] Preparation Example 1. Obtaining SVF and extracellular fluid from adipose tissue

[0092] As shown in Figures 1 and 2, 530 mL of lipoaspirate obtained by liposuction was centrifuged to separate it into a stromal vascular fraction (SVF) and extracellular fluid (LDF). After centrifugation, 120 mL of extracellular fluid in the lower layer was separated, and 410 mL of adipose tissue in the middle layer was separated.

[0093] In addition, the obtained extracellular fluid was centrifuged to obtain mitochondria, and the amount of obtained mitochondria was measured using the BCA protein quantification method. As a result, as shown in Figure 3, it was confirmed that approximately 80 μg was present per 1 mL of extracellular fluid.

[0094] In addition, after centrifuging the lipoaspirate, the adipose tissue was treated with collagenase and centrifuged. Subsequently, RBCs and impurities were removed and the tissue was washed to finally obtain SVF. When the number of cells in the obtained SVF was counted, it was found to be 5 x 10⁶ 8 The number of cells was measured.

[0095] Example 1. Obtaining plasma-derived or extracellular fluid-derived mitochondria

[0096] Example 1.1. Obtaining plasma-derived mitochondria

[0097] To obtain plasma-derived mitochondria, the donor's blood was first placed in a 50 mL tube and centrifuged at 2,000 xg for 5 minutes. After the first centrifugation, the plasma was separated and obtained in a new 50 mL tube, and centrifuged at 20,000 xg for 5 minutes.

[0098] As shown in Fig. 4a, plasma-derived mitochondria were obtained from the pellet obtained after secondary centrifugation.

[0099] Example 1.2. Obtaining mitochondria derived from extracellular fluid

[0100] Liposuction was performed to obtain adipose tissue for obtaining extracellular fluid-derived mitochondria. The liposuction site of the donor was disinfected with Betadine solution, and a 0.5 mm incision was made using a No. 11 blade. An appropriate amount of liposuction composition (Tumescent) was injected through the incision, and the patient waited for at least 20 minutes for anesthesia. Subsequently, adipose tissue was collected using a 50 mL syringe connected to an aspiration cannula and stored in a 50 mL tube. The adipose tissue (lipoaspirate) was subjected to primary centrifugation at 2,000 xg for 5 minutes, after which the extracellular fluid was separated and obtained in a new 50 mL tube. Impurities were removed from the separated extracellular fluid. Subsequently, secondary centrifugation was performed at 20,000 xg for 5 minutes.

[0101] As shown in Fig. 4b, extracellular fluid-derived mitochondria were obtained from the pellet obtained after secondary centrifugation.

[0102] Example 2. Analysis of plasma-derived or extracellular fluid-derived mitochondria

[0103] To confirm the size of the plasma-derived or extracellular fluid-derived mitochondria isolated and obtained in Example 1 above, the particle size and distribution were measured using dynamic light scattering equipment. As a result, it was confirmed that the size of the plasma-derived mitochondria was 383 nm (Fig. 5a), and the size of the extracellular fluid-derived mitochondria was 610 nm (Fig. 6a).

[0104] In addition, to analyze the activity of plasma-derived or extracellular fluid-derived mitochondria, the activity of the electron transport chain, ATP production capacity, and citric acid synthase activity were verified. At this time, heat-treated and damaged mitochondria were used as a control to verify and compare the activities in the same manner. As a result, it was confirmed that the activity of plasma-derived mitochondria was maintained (Fig. 5b), and the activity of extracellular fluid-derived mitochondria was maintained (Fig. 6b).

[0105] To confirm the extracellular fluid-derived mitochondria obtained in Example 1 above, the presence of mitochondria was confirmed using an anti-TOM20 antibody, which is an antibody against the mitochondrial outer membrane protein TOM20. As a result, as shown in Figure 6c, it was confirmed that they were present normally.

[0106] In addition, the membrane potential of the extracellular fluid-derived mitochondria was measured using TMRE staining to confirm that they perform normal functions. As a result, it was confirmed that they perform normal functions as shown in Figure 6d.

[0107] Example 3. Confirmation of anti-inflammatory effects of plasma-derived or extracellular fluid-derived mitochondria

[0108] To confirm the anti-inflammatory effect of plasma-derived or extracellular fluid-derived mitochondria, human monocyte cell line THP-1 cells were treated with PMA (Phorbol 12-myristate 13-acetate) and reacted for 48 hours. Subsequently, plasma-derived or extracellular fluid-derived mitochondria isolated and obtained in Example 1 were treated and reacted for 48 hours.

[0109] Subsequently, cells were recovered and mRNA analysis was performed via PCR, and as shown in Figure 7b, it was confirmed that the expression of CD206, a marker of M2 macrophages with anti-inflammatory functions, increased in THP-1 cells treated with plasma-derived or extracellular fluid-derived mitochondria.

[0110] Example 4. Comparative analysis of plasma-derived mitochondria and extracellular fluid-derived mitochondria

[0111] The activity of plasma-derived mitochondria and extracellular fluid-derived mitochondria isolated and obtained in Example 1 above was compared. To analyze the activity of mitochondria, the activity of the electron transport chain, ATP production capacity, and citric acid synthase activity were confirmed. At this time, mitochondria derived from lipoaspirate-derived fraction (LDF) and mitochondria derived from extracellular fluid are the same expression.

[0112] As a result, as shown in Figure 8, a comparative analysis of LDF-derived mitochondria and plasma-derived mitochondria confirmed that the activity of LDF-derived mitochondria was superior.

[0113] Example 5. Preparation of MT-SVF in which extracellular fluid-derived mitochondria were introduced into SVF obtained from adipose tissue

[0114] SVF and extracellular fluid were obtained from adipose tissue in the same manner as in Preparation Example 1 above. Subsequently, extracellular fluid-derived mitochondria were obtained in the same manner as in Example 1.2. The mixture of obtained SVF and extracellular fluid-derived mitochondria was centrifuged to introduce extracellular fluid-derived mitochondria into the SVF.

[0115] To confirm whether extracellular fluid-derived mitochondria were introduced into SVF obtained from adipose tissue, 1 x 10⁶ of the SVF cell population 5 50 μg of extracellular fluid-derived mitochondria attached to anti-TOM20 antibodies were delivered per cell. As a result, as shown in Figure 9, it was confirmed that mitochondria were successfully introduced into the SVF.

[0116] Example 6. Efficacy of MT-SVF in which extracellular fluid-derived mitochondria were introduced into SVF obtained from adipose tissue

[0117] Example 6.1. Confirmation of MT-SVF angiogenesis in HUVEC

[0118] To confirm the angiogenic ability of MT-SVF, 1 x 10⁶ HUVECs (Human umbilical vein endothelial cells) 4 1x10 of dog cells and SVF cell groups 4 Cells were mixed and dispensed into 96-well plates coated with 50 μL of Matrigel. The experimental groups consisted of HUVEC, HUVEC + SVF, HUVEC + 5 μg-SVF of mitochondria, HUVEC + 50 μg-SVF of mitochondria, and HUVEC + growth factor bFGF 50 ng / mL. After dispensing each experimental group into a 96-well plate and waiting 4 hours, the tube length was measured by microscopic observation and quantitatively analyzed using the Image J program.

[0119] As a result, as shown in Figures 10b and 10c, it can be confirmed that the angiogenic efficacy of the experimental group in which MT-SVF, in which mitochondria were introduced into SVF, was superior to that of the experimental group in which SVF was inoculated alone.

[0120] Example 6.2. Confirmation of MT-SVF skin regenerative ability in HDF

[0121] To confirm the skin regenerative capacity of MT-SVF, Human dermal fibroblast (HDF) cells were cultured in a 12-well plate. At this time, artificial scratches were created in the HDF monolayer, and 1 x 10⁶ SVF cell populations were placed in each well. 5 Cells were inoculated. The experimental groups consisted of HDF, HDF + SVF, HDF + 5 μg-SVF of mitochondria, HDF + 50 μg-SVF of mitochondria, HDF + 50 ng / mL of growth factor bFGF, and HDF w / o FBS (non-FBS). Each experimental group was inoculated into a 12-well plate, and after 24 hours, the progress of wound healing was observed under a microscope and quantitatively analyzed using the Image J program.

[0122] As a result, as shown in Figures 11b and 11c, it was confirmed that the skin regeneration efficacy of both the experimental group treated with SVF alone and the experimental group treated with MT-SVF, in which mitochondria were introduced into SVF, was excellent.

[0123] Example 6.3. Confirmation of inflammation regulatory ability through macrophage differentiation process

[0124] To confirm the anti-inflammatory activity of MT-SVF, the human monocyte cell line THP-1 was seeded into 24-well plates and cultured. Each well was treated with PMA and cultured for 48 hours to induce differentiation into M0-type macrophages. The differentiated M0-type macrophages were treated with SVF, LPS, LPS-SVF, LPS-MT-SVF, and IL-4 / 13, respectively, and cultured for 48 hours. The SVF was 1 x 10⁶ in the SVF cell population. 5 The cells were 50 μg of SVF-introduced mitochondria (MT). mRNA analysis was performed by PCR of cells cultured in each well, and the expression of CD11b (M0-type macrophage marker), CD86 (M1-type macrophage marker), and CD206 (M2-type macrophage marker) was confirmed.

[0125] As a result, as shown in Figures 12b and 12c, it was confirmed that SVF treatment induces differentiation of M2 macrophages. It was confirmed that treatment with LPS alone induces differentiation into M1 macrophages, and that simultaneous treatment with LPS and SVF inhibits M1 differentiation. In particular, it was confirmed that LPS-MT-SVF, which is SVF with introduced mitochondria and simultaneous treatment with LPS, is more effective than LPS-SVF.

Claims

1. Method for obtaining mitochondria from extracellular fluid.

2. In Paragraph 1, A method for obtaining mitochondria, wherein the extracellular fluid is a lipoaspirate-derived fraction (LDF).

3. In Paragraph 2, A method for obtaining mitochondria, wherein the above-mentioned liposuction fraction is obtained by centrifuging a liposuction sample obtained through liposuction surgery.

4. In Paragraph 1, A method for obtaining mitochondria, wherein the mitochondria are separated by centrifuging the extracellular fluid.

5. Mitochondria isolated from extracellular fluid.

6. In Paragraph 5, The above extracellular fluid is a liposuction fraction, and the mitochondria separated from the extracellular fluid.

7. In Paragraph 6, The above mitochondria are mitochondria isolated from extracellular fluid, characterized by having a size of about 100 nm to about 3,500 nm.

8. Stem cells into which mitochondria isolated from extracellular fluid have been introduced.

9. In Paragraph 8, A stem cell into which mitochondria are introduced, wherein the above-mentioned separated mitochondria are centrifuged together with the stem cell to deliver the mitochondria into the stem cell.

10. In Paragraph 8, The above stem cells are mitochondrial-introduced stem cells, which are stromal vascular fraction (SVF) obtained by centrifuging adipose tissue after treating it with collagenase.

11. A pharmaceutical composition for the prevention or treatment of ischemic diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

12. A composition for preventing or treating skin diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

13. A composition for improving skin condition comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

14. In Paragraph 13, The above-mentioned composition for improving skin condition is an injectable composition for improving skin condition.

15. A method for the prevention or treatment of ischemic disease comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

16. A method for preventing or treating skin diseases comprising, as an active ingredient, mitochondria isolated from extracellular fluid and / or stem cells into which mitochondria isolated from extracellular fluid have been introduced.

17. MT-SVF in which mitochondria isolated from extracellular fluid were introduced into SVF obtained from adipose tissue.

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