JP7497554B2 - Uses of cell culture supernatant of human decidual mesenchymal stem cells - Google Patents

Description

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

Diabetes Mellitus (DM) is one of the top ten causes of death in the world. According to statistics from the Taiwan Ministry of Health and Welfare's National Health Administration, there are approximately 2 million DM patients in Taiwan. In 2016, 9,960 patients died from DM, a 4.5% increase from the previous year.

According to statistics, 15-25% of diabetic patients will develop diabetic foot ulcers (DFU) in their lifetime. DFU is the most common problem in the disease process and the main reason for hospitalization of DM patients.

Diabetes impairs normal angiogenesis, resulting in serious complications and forcing many DM patients to undergo limb amputation.

Mesenchymal stromal cells (MSCs) have been widely studied as an ideal cell source for regenerative medicine, and they can promote angiogenesis through paracrine effects. Evidence also suggests that transplantation of MSCs can accelerate wound closure, improve clinical symptoms, and avoid amputation.

The inner walls of blood vessels in healthy humans are smooth, but when they are damaged nonspecifically (for example, by damage caused by many factors such as high blood pressure, tobacco hydrocarbons, cholesterol, high blood sugar, inflammation, and injury), plaque accumulates at the damaged site, damaging the endothelial cells, and lipids penetrate into the endothelium and media. When lipids are peroxidized, an immune response is triggered in macrophages, which promotes the secretion of a series of cytokines, resulting in inappropriate proliferation of smooth muscle in the media of the vascular wall, and continued thickening of the plaque. If this continues for a long period of time, ulcers, bleeding, or calcification will appear in the media of the blood vessel, and ultimately fibrous plaque will be formed, making the surface of the blood vessel uneven. Furthermore, platelets will act on this to form a thrombus, leading to vascular occlusion.

Because the tissues below the site of obstruction cannot obtain sufficient nutrients and oxygen, pathological changes such as ischemia and gangrene appear. This is collectively known as peripheral arterial occlusive disease.

In diabetic patients, poor blood sugar control over a long period of time accelerates arteriosclerosis and thickening of the vascular wall basement membrane, leading to a deterioration in tissue oxygen permeability to the blood and increased blood clotting function. This makes it easier for blood clots to form, causing lumen stenosis and blockage, resulting in lower limb ischemia.

The terms "a" or "one" are used in this specification to describe elements and components of the present invention, but this term is merely for convenience of description and to present the basic idea of the present invention. Furthermore, such descriptions should be interpreted as including one or at least one. In addition, unless expressly indicated otherwise, the singular form is meant to include the plural form. In addition, when used in conjunction with the term "comprises" in the claims, the term "a" may mean one or more than one.

A pharmaceutical composition for preventing or treating an ischemic disease, comprising an effective amount of human decidual mesenchymal stem cell supernatant and a pharma- ceutical acceptable vector or excipient.

The supernatant of human decidual mesenchymal stem cells is determined by the following steps. That is, a serum-free stem cell culture medium is provided to subculture stem cells. The culture medium contains a stem cell medium, 0.9-1.1% insulin-transferrin-selenium, and 9-11 ng/ml epidermal growth factor. Culture is performed for 4-12 days in the serum-free stem cell culture medium. Then, 40-80 mg/ml trehalose and 10-30 mg/ml dextran are collected and added to the cultured serum-free stem cell culture medium, and the serum-free stem cell culture medium is filtered.

The effective amount of human decidual mesenchymal stem cell supernatant can be determined by the weight of freeze-dried powder of the supernatant. In the examples of the present invention, the amounts of freeze-dried powder of the supernatant added are 0.1879 g, 0.22 g, and 0.247 g.

An 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 number of extracellular vesicles in the supernatant of human decidual mesenchymal stem cells is 1×10 ⁶ to 1×10 ¹¹ particles per ml.

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 ml.

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

"Pharmaceutically acceptable vector" or "excipient" or "pharmaceutically acceptable vector or excipient" or "bioavailable vector" or "biologically available vector or excipient" includes, but is not limited to, solvents for preservation, dispersants, coatings, antibacterial agents, antifungal agents, or absorption retardants for formulation preparation, and any other known compounds. Generally, these vectors or excipients themselves do not have activity to treat diseases. Pharmaceutical compositions or formulations prepared by combining the novel compounds or derivatives thereof disclosed in the present invention with pharmaceutically acceptable vectors or excipients do not cause side effects, allergies, or other inappropriate reactions in animals or humans. Therefore, the combination of the novel compounds or derivatives thereof disclosed in the present invention and pharmaceutically acceptable vectors or excipients is applicable to human clinical practice. Pharmaceutical compositions or formulations containing the novel compounds or derivatives of the present invention can achieve therapeutic effects by intravenous injection, oral administration, inhalation, or partial administration via the nose, rectum, vagina, or sublingual.

The present invention also provides a use of a drug for preventing or inhibiting ischemic disease.

FIG. 1 shows an analysis image of a mouse hindlimb ischemia model using a Doppler perfusion imager. FIG. 2 is an analysis diagram of the number of extracellular vesicles in cell culture supernatant of human decidual mesenchymal stem cells. FIG. 3 shows a diabetic mouse hindlimb ischemia model treated with the supernatant of human decidual mesenchymal stem cells.

Cell Culture of Human Decidual Mesenchymal Stem Cells The steps of the present invention were carried out in a dust-free environment. Subculture was carried out using a stem cell culture medium that was not mixed with serum, using a cell proliferation culture method with aseptic manipulation. During the culture period, the stem cell culture medium was replaced with fresh one every three days. The solution contained MCDB201 formulation medium, insulin transferrin selenium (ITS) at a concentration of 0.9-1.1%, and epidermal growth factor (EGF) at a concentration of 9-11 ng/ml, and was cultured for 4-12 days.

The cell culture medium was formulated to be a chemically defined medium that was serum-free, non-animal-derived, and phenol red-free, with each additive selected from non-animal-derived substances. Therefore, there was no need to consider the risk of infection or allergic reactions in humans caused by animal or serum substances. In addition, it was possible to directly collect the supernatant when the cells reached an optimal state and apply it to the subsequent steps of the present invention.

In the stem cell culture, the cells were transplanted into a T175 flask at 1×10 ⁴ /cm ^2. When the cell density reached 90% or more, the supernatant with a total protein concentration of 200-300 μg/ml was made available for collection, and a freeze-drying process was carried out.

To maintain the stability of the active factor in the supernatant, 60 mg/ml of trehalose and 20 mg/ml of dextran were added and mixed uniformly, and then filtered through a membrane filter with a pore size of 0.22 μM or less. 2 ml of the filtered supernatant was then filled into a cryo-resistant container.

The freeze-dryer was pre-cooled to -30°C (taking 1-2 hours) and the prepared samples were placed into the vacuum chamber. Then, 2-3 minutes after the chamber temperature had dropped to -50°C (rapidly freezing into a solid), negative pressure was activated to reduce the pressure inside the chamber to 200 Torr and this negative pressure was maintained for 60 hours. This caused the ice to sublimate into water vapor, removing moisture from the material to be dried. Once complete, the chamber temperature was raised to 10°C and the freeze-dried powder samples were removed and packed into cans for storage at -20 to -80°C.

Number of extracellular vesicles in cell culture supernatant of human decidual mesenchymal stem cells In the examples of the present invention, the total number of extracellular vesicles in the supernatant of human decidual mesenchymal stem cells was ^9.7x107 , ^8.6x108 , ^1.1x109 , ^1.5x109 , ^2.3x109 , and ^1.45x109 .

Streptozotocin-induced diabetes mouse model C57BL/6J mice were anesthetized with 2.5% isoflurane gas and femoral artery ligation was performed. After surgery, 100 μL of 0.75% bupivacaine was intraperitoneally injected to relieve pain for 3 days, and blood flow data of the mouse lower limbs was analyzed using a Doppler perfusion imager. After completion of femoral artery ligation of the mouse, and before suturing the skin, the freeze-dried powder of the supernatant was redissolved in 400 μL of PBS, and the redissolved cell culture supernatant of human decidual mesenchymal stem cells was injected intramuscularly or intraperitoneally.

For the streptozotocin-induced diabetic mouse model, 40 mg/kg of streptozotocin was intraperitoneally injected for five consecutive days starting from the first day of the experiment. Then, on the 14th day of the experiment, the mice were fasted and their urinary glucose levels were measured using Roche (Diastix). After the urinary glucose level was higher than 3 for two consecutive days, blood glucose was measured after six hours of fasting, and if the measurement result was 300 mg/dl, the induction was considered successful.

Mice were anesthetized with 2.5% isoflurane gas and subjected to femoral artery ligation. After surgery, 100 μL of 0.75% bupivacaine was intraperitoneally injected to relieve pain for 3 days, and blood flow data of the mouse lower limbs was analyzed by Doppler perfusion imager.

When femoral artery ligation of the mice was completed, and before suturing the skin, the freeze-dried powder of the supernatant was reconstituted in 400 μL of PBS, and 60 μL of the reconstituted supernatant was intramuscularly injected into the inner and outer muscles of the lower leg and thigh, for a total of 180 μL. On the other hand, in the abdominal injection group, 180 μL of the reconstituted supernatant was injected into the abdominal cavity of the experimental animals all at once after surgery.

As shown in the image results in Figure 1, blood flow was reduced in the right paws of the mice after surgery, but by the 7th and 14th days after treatment, blood flow in the right paws of the treatment group had recovered to a higher level than that of the control group.

As shown in the analysis result of the number of extracellular vesicles in the cell culture supernatant of human decidual mesenchymal stem cells in Figure 2, when the supernatant was analyzed by nanoparticle tracking analysis (NTA), the supernatant contained 7.25 x ¹⁰⁸ extracellular vesicles/ml. Since the lyophilized powder was prepared by drying 2 ml of the supernatant, each supernatant contained 1.45 x ¹⁰⁹ extracellular vesicles.

As shown in Figure 3, when a diabetic mouse hindlimb ischemia model was treated with the supernatant of human decidual mesenchymal stem cells, diabetic symptoms were induced in C57BL/6J mice with streptozotocin, and then hindlimb ischemia surgery was performed, followed by intramuscular and intraperitoneal injections.

After surgery, image data was collected using a Doppler perfusion imager for 14 consecutive days, and the results were quantified. When the supernatant was injected intramuscularly or intraperitoneally, higher blood flow data was observed than in the control group.

In summary, diabetic mice are inferior to healthy mice in their ability to naturally recover blood flow. In this condition, administration of the supernatant of human decidual mesenchymal stem cells effectively improved the blood flow supply effect. Data are shown as mean ± standard deviation. DM: diabetic mice, IM: intramuscular injection, IP: peritoneal injection, EX: supernatant.

Claims (8)

A supernatant of human decidual mesenchymal stem cells, comprising:
(a) providing a serum-free stem cell culture medium to subculture human decidual mesenchymal stem cells, the serum-free stem cell culture medium comprising a stem cell medium, 0.9-1.1% insulin-transferrin-selenium, and 9-11 ng/ml epidermal growth factor;
(b) culturing human decidual mesenchymal stem cells in the serum-free stem cell culture medium for 4 to 12 days;
(c) collecting the serum-free stem cell culture medium obtained in step (b) after culturing the human decidual mesenchymal stem cells, adding 40 to 80 mg/ml of trehalose and 10 to 30 mg/ml of dextran;
(d) filtering the culture medium obtained in step (c) to obtain a supernatant of human decidual mesenchymal stem cells;
The supernatant of human decidual mesenchymal stem cells prepared in the steps of (a) and (b). 2. The supernatant of human decidual mesenchymal stem cells according to claim 1, wherein the number of extracellular vesicles produced is 1×10 ⁶ to 1×10 ¹¹ particles per ml. The supernatant of human decidual mesenchymal stem cells according to claim 2, wherein the number of extracellular vesicles produced is 1×10 ⁷ to 1×10 ¹⁰ particles per ml. The supernatant of human decidual mesenchymal stem cells according to claim 1, wherein the serum-free stem cell culture medium is a MCDB201 formulated medium. The human decidual mesenchymal stem cell supernatant according to claim 1, wherein the human decidual mesenchymal stem cell supernatant is stored by freeze-drying. Use of a pharmaceutical composition in the manufacture of a medicament for treating or preventing an ischemic disease, comprising:
The pharmaceutical composition comprises the supernatant of human decidual mesenchymal stem cells according to claim 1, and further comprises a pharma- ceutically acceptable vector, diluent or excipient. The use according to claim 6, wherein the ischemic disease is a diabetic foot ulcer. The use according to claim 6, wherein the administration route is intramuscular injection or intraperitoneal injection.