TW202323520A - Use of human decidual mesenchymal stem cell culturing supernatants - Google Patents

Abstract

Disclosed herein is the cell culturing supernatant from human decidual mesenchymal stem cell culturing that are therapeutic to a subject having an ischemic disease, for example, diabetic foot ulcer. Through the establishment of a mouse lower limb ischemia model, treatment with cell culturing supernatant from human decidual mesenchymal stem cell culturing can improve the blood supply of the lower limbs. In the streptozotocin induced diabetic model, treatment with cell culturing supernatant from human decidual mesenchymal stem cell culturing can also improve the blood supply of the lower limbs. The invention can be applied to diabetic patients to reduce the incidence of amputation by promoting angiogenesis.

Description

Use of the cell culture supernatant of human decidual mesenchymal stem cells

The invention discloses the use of a cell culture supernatant of human decidual mesenchymal stem cells.

Diabetes Mellitus (DM) is one of the top ten causes of death worldwide. According to statistics from the National Health Service of Taiwan’s Ministry of Health and Welfare, there are about 2 million DM patients in Taiwan, and 9,960 patients died of DM in 2016, an increase of 4.5% compared to last year.

According to statistics, 15-25% of diabetic patients suffer from diabetic foot ulcers (Diabetic Foot Ulcer, DFU) in their lifetime. DFU is considered to be the most important problem in the pathogenesis process and the main reason for hospitalization of DM patients.

To date, due to impairment of normal angiogenesis due to diabetes, a large number of DM patients require amputation, resulting in serious complications.

Mesenchymal stromal cells (MSCS) have been widely studied as an ideal cell source for regenerative medicine, which can promote angiogenesis through paracrine effect. There is also evidence that MSCs transplantation can accelerate wound closure and improve clinical symptoms, Avoid amputation.

The inner walls of blood vessels in healthy people are smooth. If they are damaged by non-specific damage, such as high blood pressure, hydrocarbons from cigarettes, cholesterol, high blood sugar, inflammation, trauma, etc., fatty plaques will accumulate in the blood vessels. Injury, which in turn causes damage to vascular endothelial cells, causing lipids to penetrate into the middle layer of vascular endothelium. After lipid peroxidation, macrophages will be induced to produce an immune response, prompting a series of cytokine secretion, resulting in improper proliferation of smooth muscle in the middle layer of the blood vessel wall, Fatty plaque continues to thicken, and after a long time, ulcers, bleeding or calcification will appear in the middle layer of blood vessels, and finally fibrous plaques will form, making the surface of blood vessels uneven, and then interact with platelets to form thrombus, resulting in blood vessel blockage.

The tissue below the blocked site will suffer from pathological changes such as ischemia or gangrene due to the inability to receive sufficient nutrients and oxygen. This is collectively referred to as peripheral arterial occlusive disease.

Among them, if diabetic patients have poor blood sugar control for a long time, it will accelerate arteriosclerosis and thicken the basal layer of the blood vessel wall, resulting in poor tissue blood oxygen permeability, enhanced blood coagulation function, and easy thrombosis, which in turn will cause stenosis and blockage of the lumen. Cause lower extremity ischemia.

The term "a" or "an" in this specification is used to describe the elements and components of the present invention. This term is only for the convenience of description and to give the basic concept of the present invention. Further, this description should be understood as including one or at least one , and references to the singular include the plural unless clearly stated otherwise. When used together with the word "comprising" in the claims, the word "a" can mean one or more than one.

A pharmaceutical composition for preventing or treating ischemic diseases, comprising: effective amount of human decidual mesenchymal stem cell supernatant; and a pharmaceutically acceptable carrier or excipient.

The supernatant of human decidual mesenchymal stem cells is determined by the following steps: providing a serum-free stem cell culture medium for subculture of stem cells, wherein the culture medium contains: a stem cell culture medium; a 0.9% to 1.1% insulin-transferrin - selenium; and a 9ng/ml to 11ng/ml epidermal growth factor; culture in the serum-free stem cell culture medium for 4 to 12 days; collect and add a 40mg/ml to 80mg/ml trehalose and a 10mg/ml to 30 mg/ml dextran in the cultured serum-free stem cell culture fluid; and filtering the serum-free stem cell culture fluid.

The supernatant of an effective amount of human decidual mesenchymal stem cells can be defined by the weight of the supernatant liquid freeze-dried powder. In an embodiment of the present invention, the added amount of the supernatant liquid freeze-dried powder is 0.1879 grams, 0.22 grams, and 0.247 grams. Heavy.

The effective amount of the supernatant of human decidual mesenchymal stem cells can be defined by the number of extracellular vesicles. In an embodiment of the present invention, the total amount of extracellular vesicles in the supernatant of human decidual mesenchymal stem cells is 1 per milliliter. ×10 ⁶ to 1×10 ¹¹ particles.

In another embodiment of the present invention, the total number of extracellular vesicles in the supernatant of human decidual mesenchymal stem cells is 1×10 ⁷ to 1×10 ¹⁰ particles per milliliter.

In one embodiment of the invention, the ischemic disease is diabetic foot ulcer.

"pharmaceutically acceptable carrier" or "excipient" or "pharmaceutically acceptable carrier or excipient" or "bioavailable (bioavailable) carrier" or "bioavailable carrier or excipient" Including, but not limited to, solvents for preservation, dispersants, coatings, antimicrobials, antifungals or absorption delaying agents for formulation and any other known compounds. Generally, these carriers or excipients have no disease-treating activity by themselves. By using the present invention The pharmaceutical composition or formulation prepared by combining the disclosed novel compound or its derivative with a pharmaceutically acceptable carrier or excipient will not cause side effects, allergies or other inappropriate reactions in animals or humans. Therefore, the combination of the novel compound disclosed in the present invention or its derivative and a pharmaceutically acceptable carrier or excipient can be applied to human clinical practice. Pharmaceutical compositions or formulations comprising the novel compounds of the present invention or derivatives thereof can be administered intravenously, orally, inhaled or administered nasally, rectally, vaginally or sublingually for therapeutic effect.

In another aspect, the present invention provides a use of a medicament for preventing or treating ischemic diseases.

Figure 1 is an analysis of a mouse lower limb ischemia model with a Doppler blood flow imager.

Figure 2 is an analysis chart of the number of extracellular vesicles in the culture supernatant of human decidual mesenchymal stem cells.

Figure 3 is a graph showing the treatment of the diabetic mouse lower limb ischemia model with the supernatant of human decidual mesenchymal stem cells.

Cell culture of human decidual mesenchymal stem cells

Implement the steps of the present invention in a dust-free environment space, use the aseptic operation cell proliferation culture method, use the stem cell culture solution without mixing any serum to carry out the subculture of the cells, and replace the fresh stem cell culture solution every three days during the culture period, wherein the solution Contains MCDB201 formula medium, insulin-transferrin-selenium (insulin transferrin selenium, ITS) with a concentration of 0.9% to 1.1%, and epidermal growth factor (epidermal growth factor) with a concentration of 9ng/ml to 11ng/ml growth factor, EGF), cultured for 4 to 12 days.

Among them, the added substances of this cell culture liquid formula must be selected from non-animal sources, which are chemically defined medium composed of no serum, no animal sources and no phenol red, so there is no need to consider whether animals or serum substances may cause infection or infection of the human body. Allergic reaction is triggered, and the supernatant can be collected directly when the cells are in the best state for subsequent application of the present invention.

When the stem cell culture is planted in T175 Flask at 1×10 ⁴ /cm ² and the cell density reaches over 90%, the supernatant with a total protein concentration of 200-300 μg/ml can be collected and freeze-dried.

Freeze-dried preparation

In order to maintain the stability of the active factor in the supernatant, 60 mg/ml trehalose and 20 mg/ml dextran were added to the supernatant, mixed evenly, and then filtered with a filter membrane with a pore size below 0.22 μM. Fill 2ml of the filtered supernatant in a cryogenic container.

The freeze dryer needs to be pre-cooled to -30°C (it takes one to two hours), put the prepared sample into the vacuum box, cool the box to -50°C for two to three minutes (rapidly freeze into a solid state), and start The negative pressure pumps the box to 200 torr, and maintains the negative pressure for 60 hours, so that the ice is sublimated into water vapor, and the moisture in the dried object is removed. After completion, raise the temperature of the box to 10°C, collect the freeze-dried powder samples, seal the cans, and send them to -20°C to -80°C for storage.

The number of extracellular vesicles in the culture supernatant of human decidual mesenchymal stem cells

In an embodiment of the present invention, the total number of extracellular vesicles in the supernatant of human decidual mesenchymal stem cells is 9.7x10 ⁷ , 8.6x10 ⁸ , 1.1x10 ⁹ , 1.5x10 ⁹ , 2.3x10 ⁹ , 1.45×10 ⁹ .

Streptozotocin diabetic mouse model

C57BL/6J mice were anesthetized with 2.5% isoflurane gas for femoral artery ligation, and 0.75%, 100 μL bupivacaine was injected intraperitoneally for three days to relieve pain after operation, and Doppler A blood flow imager was used to analyze and record the blood flow data of the mouse lower limbs. Before the mouse skin was sutured after femoral artery ligation, the supernatant freeze-dried powder was redissolved in 400 μL PBS, and the redissolved human decidual mesenchymal stem cell culture supernatant was injected intramuscularly or intraperitoneally.

The streptozotocin diabetic mouse model is to inject 40mg/kg streptozotocin into the abdominal cavity continuously for 5 days starting from the first day of the experiment, and the mice are fasted on the 14th day of the experiment and detected by Roche (Diastix). Urine sugar value, when the urine sugar value is higher than 3 for two consecutive days, measure the blood sugar after 6 hours of fasting, and the induction is successful when the test result is greater than 300mg/dl.

Doppler flow imager analysis

Mice were anesthetized with 2.5% isoflurane gas for femoral artery ligation, and 0.75%, 100 μL bupivacaine was injected intraperitoneally for three days after the operation to relieve pain, and Doppler blood flow imaging was performed Analyze and record blood flow data of mouse lower limbs.

Before the skin was sutured after femoral artery ligation, the supernatant freeze-dried powder was redissolved in 400 μL PBS, and intramuscularly injected 60 μL on the inner and outer muscles of the calf and thigh respectively, with a total of 180 μL supernatant Reconstituted solution; In the intraperitoneal injection group, 180 μL of the supernatant reconstituted solution was injected into the abdominal cavity of the experimental animal once after the operation for the test.

According to the image results in Figure 1, it was found that the blood flow in the right foot of the mouse decreased after the operation, but it was observed that the blood flow in the right foot of the treatment group was more restored than that of the control group on the 7th day and the 14th day after treatment.

According to the results of analysis of the number of extracellular vesicles in the cell culture supernatant of human decidual mesenchymal stem cells in Figure 2, the supernatant was analyzed by Nanoparticle Tracking Analysis (NTA) and showed that the supernatant contained 7.25 ×10 ⁸ /ml extracellular vesicles (extracellular vesicles), since the freeze-dried powder is obtained by drying 2ml supernatant, each supernatant contains 1.45×10 ⁹ extracellular vesicles.

According to the diagram of the results of treatment of the lower limb ischemia model of diabetic mice with the supernatant of human decidual mesenchymal stem cells in Figure 3, the diabetic symptoms of C57BL/6J strain mice were induced with streptozotocin, and then the lower limb ischemia operation was performed and given intramuscular and intraperitoneal injections.

The image data were collected and quantified by Doppler blood flow imager for 14 consecutive days after the operation. The results showed that blood flow data higher than those in the control group could be observed in the supernatant injected intramuscularly or intraperitoneally.

Overall, compared with healthy mice, diabetic mice have poorer blood flow recovery ability. In this state, the administration of human decidual mesenchymal stem cell supernatant can effectively improve blood flow supply. Data represent mean ± standard deviation. DM: diabetic mice; IM: intramuscular injection; IP: intraperitoneal injection; EX: supernatant.

Claims (8)

A human decidual mesenchymal stem cell supernatant, wherein the human decidual mesenchymal stem cell supernatant is determined by the following steps: (a) providing a serum-free stem cell culture medium for subculture of stem cells, wherein the serum-free stem cell culture medium comprises: a stem cell culture medium; - 0.9% to 1.1% insulin-transferrin-selenium; and - 9ng/ml to 11ng/ml epidermal growth factor; (b) culturing with the serum-free stem cell culture medium for 4 to 12 days; (c) collecting and adding a 40mg/ml to 80mg/ml trehalose and a 10mg/ml to 30mg/ml dextran in the cultured serum-free stem cell culture medium; and (d) filtering the cultured serum-free stem cell culture fluid. The supernatant of human decidual mesenchymal stem cells as described in item 1 of the patent application, wherein the number of extracellular vesicles produced is 1×10 ⁶ to 1×10 ¹¹ particles per milliliter. The supernatant of human decidual mesenchymal stem cells as described in item 2 of the patent application, wherein the number of extracellular vesicles produced is 1×10 ⁷ to 1×10 ¹⁰ particles per milliliter. The human decidual mesenchymal stem cell supernatant as described in item 1 of the patent application, wherein the serum-free stem cell culture medium is MCDB201 formula medium. The human decidual mesenchymal stem cell supernatant as described in item 1 of the patent application, wherein the human decidual mesenchymal stem cell supernatant is preserved by freeze-drying. A pharmaceutical composition for the preparation of medicines for the treatment or prevention of ischemic diseases, which The pharmaceutical composition is determined according to the steps described in item 1 of the patent application, which may further include a pharmaceutically acceptable carrier, diluent or excipient. The use as described in item 6 of the scope of the patent application, wherein the ischemic disease is diabetic foot ulcer. For the purposes described in item 6 of the scope of the patent application, the route of administration is intramuscular injection or intraperitoneal injection.

Images