Stem cell preparation, method of making and use in preventing and treating lung injury

Abstract

The present application provides a method for preparing a stem cell preparation, comprising: 1) isolating and primary culturing bone marrow mesenchymal stem cells; 2) subculturing the bone marrow mesenchymal stem cells, and adding lacto-N-neohexaose and adrenaline to the bone marrow mesenchymal stem cell culture medium during the P5 and / or P6 generation culture; and 3) collecting the bone marrow mesenchymal stem cells and suspending them in physiological saline containing vitamin C, glucose and trehalose. The stem cell preparation provided by the present application can be used for the prevention and / or treatment of lung injury.

Description

Technical Field

[0001] This invention relates to stem cell preparations including bone marrow mesenchymal stem cells, and also to methods for their preparation and their use in the prevention and / or treatment of lung injury. Background Technology

[0002] Bone marrow mesenchymal stem cells are important members of the stem cell family, originating from the mesoderm in early development. Stem cells possess multi-differentiation potential and can proliferate over a long period, making them of significant medical value.

[0003] Radiation-induced lung injury is a common complication of radiotherapy for thoracic tumors. The ionizing effect of high-energy rays activates water molecules in the body, thereby generating a large number of free radicals and reactive oxygen species (ROS), which damage lung tissues such as alveolar epithelial cells and vascular endothelial cells, ultimately leading to radiation-induced lung injury.

[0004] Bacterial lipopolysaccharide (LPS)-induced acute lung injury is a common complication of trauma or bacterial infection, causing a large number of deaths worldwide each year, and there are currently no effective means to reduce the mortality rate.

[0005] A few reports have been published on the use of mesenchymal stem cell exosomes for the prevention and treatment of lung injury. For example, Chinese Patent Publication CN118436686A describes the application of mesenchymal stem cell-derived exosomes in the preparation of drugs for treating acute lung injury, and CN117243977A describes the use of isoxazole-9 combined with mesenchymal stem cells in the preparation of drugs for the prevention or treatment of acute lung injury. However, there have been no attempts to directly use autologous or homologous mesenchymal stem cells for the prevention and treatment of lung injury in humans or animals. Summary of the Invention

[0006] In one aspect, the present invention provides a method for preparing stem cell preparations, comprising: 1) Isolate and primary culture bone marrow mesenchymal stem cells; 2) Passage the bone marrow mesenchymal stem cells, and add lactose-N-neohexose and epinephrine to the bone marrow mesenchymal stem cell culture medium during P5 and / or P6 passage culture; and 3) Collect the bone marrow mesenchymal stem cells and suspend them in physiological saline containing vitamin C, glucose and trehalose.

[0007] In some embodiments, the concentration of lactose-N-neohexose in the bone marrow mesenchymal stem cell culture medium is 2-20 μM.

[0008] In some embodiments, the concentration of lactose-N-neohexose in the bone marrow mesenchymal stem cell culture medium is 2-10 μM.

[0009] In some embodiments, the concentration of the adrenaline in the bone marrow mesenchymal stem cell culture medium is 1-5 μM.

[0010] In some embodiments, the concentration of bone marrow mesenchymal stem cells in the stem cell preparation is 5 x 10⁻⁶. 6 per mL.

[0011] On the other hand, the present invention provides a stem cell preparation prepared by the above method.

[0012] On the other hand, the present invention provides the use of the above-mentioned stem cell preparation in the preparation of a medicament for the prevention and / or treatment of lung injury in subjects.

[0013] In some implementations, the lung injury is LPS-induced or radiation-induced lung injury.

[0014] In some embodiments, the bone marrow mesenchymal stem cells included in the stem cell preparation are or are derived from the subject's own stem cells.

[0015] In some implementations, the subject is a mammal, such as a mouse or a human. Attached Figure Description

[0016] Figure 1 The results of TNF-α concentration detection in bronchoalveolar lavage fluid of mice treated with LPS are shown. In this figure and the following figures, Control refers to the control group, Model refers to the model group, 1# refers to the stem cell preparation 1# treatment group, 2# refers to the stem cell preparation 2# treatment group, and 3# refers to the stem cell preparation 3# treatment group. p < 0.05; p < 0.01; p < 0.001, all relative to the model group.

[0017] Figure 2 The results show the concentration of IL-6 in the bronchoalveolar lavage fluid of mice treated with LPS.

[0018] Figure 3 The results of IL-1β concentration detection in bronchoalveolar lavage fluid of LPS-treated mice are shown.

[0019] Figure 4 The results of serum TNF-α concentration detection in LPS-treated mice are shown.

[0020] Figure 5 The results of serum IL-6 concentration detection in LPS-treated mice are shown. Detailed Implementation

[0021] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0022] In this document, the term “and / or” should be understood to mean either one of the options or both of the options.

[0023] In this document, the terms “include” and “include” generally mean to include the explicitly specified elements, but do not exclude other elements, and also include situations where such elements constitute the whole.

[0024] Unless otherwise stated, all percentages and proportions in this document are weight percentages.

[0025] In this document, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0026] Lacto-N-neohexaose (LNnH) has the following structure, CAS Registry No. 64003-52-7:

[0027] Our study found that cultured autologous (or homologous) mouse mesenchymal stem cells (MSCs) have a preventive and / or therapeutic effect on lung injury and liver injury in mice (see separate application). This effect depends on the addition of appropriate amounts of lactose-N-neohexose and epinephrine to the MSC culture medium. Lactose-N-neohexose and epinephrine are typically added at the P5 and / or P6 passages of stem cell culture; adding them too early can affect the subsequent cell proliferation rate, while excessively passaged stem cells have relatively poor therapeutic effects. The concentration of LNnH added to the culture medium ranges from 2-20 μM, preferably 2-10 μM, for example, about 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. The concentration of epinephrine added to the culture medium ranges from 1-5 μM, for example, about 1, 2, 3, 4, or 5 μM.

[0028] The present invention will be described in detail below through specific embodiments.

[0029] Example 1: Preparation of mouse bone marrow mesenchymal stem cells Under aseptic conditions, femurs and tibias were harvested from C57BL / 6 mice. After removing other tissues, the bone marrow cavity was repeatedly flushed with PBS buffer using a syringe to collect bone marrow fluid. The bone marrow fluid was centrifuged at 4°C (500g, 10 min), and the supernatant was discarded. The precipitate was resuspended in mouse mesenchymal stem cell culture medium (MesenCult™ Expansion Kit, STEMCELL) and cultured in a culture flask at 37°C and 5% CO2 for approximately 48 hours. The cells were carefully washed twice with PBS to remove non-adherent cells, and the culture medium was changed every 3 days. Cells were passaged when the confluence reached approximately 85%. At passage P4, a portion of the cells was analyzed by flow cytometry. Mouse bone marrow mesenchymal stem cells were considered qualified if the positivity rates of CD29 and CD44 were greater than 95% and the positivity rates of CD31, CD11b, and CD45 were less than 2%, and the culture continued. During P5-P6 generation culture, lactose-N-neohexose (LNnH) and adrenaline were added to the culture medium to a final concentration of 5 μM. When the P6 generation cells reached approximately 80% confluence, they were digested with trypsin (2.5 g / L) for 2 minutes. The resulting cell suspension was centrifuged at 500 g for 10 min at 4°C, resuspended in PBS, and centrifuged again at 500 g for 10 min. The cell concentration was then reduced to approximately 2 x 10⁻⁶ cells / mL. 6 per mL.

[0030] Example 2 (Comparative Example): Preparation of Mouse Bone Marrow Mesenchymal Stem Cells Mouse bone marrow mesenchymal stem cell PBS suspension was prepared according to the method in Example 1, except that lactose-N-neohexose and adrenaline were not added during the P5-P6 generation culture.

[0031] Example 3 (Comparative Example): Preparation of Mouse Bone Marrow Mesenchymal Stem Cells Mouse bone marrow mesenchymal stem cell PBS suspension was prepared according to the method in Example 1, except that lactose-N-neohexose was not added during P5-P6 generation culture.

[0032] Example 4: Preparation of stem cell preparations The mesenchymal stem cell PBS suspensions prepared in Examples 1-3 were added to 100 mL of physiological saline containing 0.2 g vitamin C, 5 g glucose, and 0.5 g trehalose, so that the concentration of mesenchymal stem cells was 5 x 10⁻⁶. 6 Mix each stem cell at a concentration of 100 cells / mL until homogeneous. The resulting stem cell preparations are numbered 1-3# and stored at 4°C for later use.

[0033] Example 5: Mouse Lung Injury Experiment 1) LPS-induced lung injury Forty-five C57BL / 6 mice were randomly divided into five groups of nine mice each after achieving a balanced weight: a control group, a model group, and stem cell preparation group 1, 2, and 3. Except for the control group, mice were administered LPS (dispersed in physiological saline) via tracheal instillation at a dose of 0.6 mg / kg, while the control group received an equal volume of physiological saline. Starting from the second day after LPS administration (day 1), mice in stem cell preparation groups 1, 2, and 3 were injected intravenously with stem cell preparations 1-3 prepared in Example 4 at a dose of 0.5 mL for four consecutive days. The control and model groups received no administration. The number of dead mice in each group was counted daily. On day 6, all surviving mice were anesthetized, and bronchoalveolar lavage fluid was collected. After centrifugation, the supernatant was collected, and the concentrations of cytokines TNF-α, IL-6, and IL-1β in the supernatant were determined using an ELISA kit.

[0034] The total number of deaths and survival rates of mice in each group on day 6 are shown in Table 1 below.

[0035] Table 1. Number of mice that died and survival rate after LPS treatment

[0036] Based on the survival rate of mice, the stem cell preparation 1# treatment group was close to the control group, while the stem cell preparations 2# and 3# treatment groups were close to the model group.

[0037] In the process of lung injury, certain cytokines, especially pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, play a crucial role, and the therapeutic effect can be reflected by detecting the expression or secretion levels of these cytokines. The results of these cytokine concentration measurements show... Figure 1-3 From Figure 1-3 The results showed that the concentrations of TNF-α, IL-6, and IL-1β in mice in the stem cell preparation 1# group were significantly lower than those in other groups except the control group. In mice in the stem cell preparation 2# and 3# groups, only the IL-6 concentration was lower than that in the model group. These results were largely consistent with the mouse survival rate results, demonstrating that stem cell preparation 1# has excellent therapeutic effects on lung injury.

[0038] 2) Radiation-induced lung injury Forty C57BL / 6 mice were randomly divided into four groups of 10 mice each after achieving a balanced weight: a model group, a stem cell preparation 1# group, a stem cell preparation 2# group, and a stem cell preparation 3# group. Mice in the stem cell preparation 1#, 2#, and 3# groups were injected via tail vein with stem cell preparations 1-3# prepared in Example 4 at a dose of 0.5 mL, once every two days for eight injections. Mice in the model group were injected with an equal volume of physiological saline. The day after the last injection, all mice underwent radiation treatment. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital saline solution at a dose of 45 mg / kg. The limbs of the mice were fixed with tape, and the chest area (excluding the chest) was shielded with a lead plate. A single 25 Gy cobalt-60 irradiation was applied to the chest. On day 7 post-radiation, blood was collected from the submandibular vein, centrifuged, and serum was obtained. The concentrations of cytokines TNF-α and IL-6 in the serum were measured using an ELISA kit. The survival of mice in each group was observed until day 14 post-radiation, at which point the experiment was terminated.

[0039] The total number of deaths and survival rates of mice in each group on day 14 after radiation treatment are shown in Table 2 below.

[0040] Table 2. Number of mice that died and survival rate after irradiation treatment

[0041] Based on the survival rate of mice, the survival rate of the stem cell preparation group 1 was significantly higher than that of the other groups.

[0042] The serum concentrations of cytokines TNF-α and IL-6 on day 7 after radiation treatment were as follows: Figure 4 and Figure 5 As shown. From Figure 4 and Figure 5 The results showed that stem cell preparations 1#, 2# and 3# all had the effect of preventing radiation-induced lung injury, among which stem cell preparation 1# had the best effect in reducing pro-inflammatory factors.

Claims

1. A method for preparing a stem cell preparation, comprising: 1) Isolate and primary culture bone marrow mesenchymal stem cells; 2) Passage the bone marrow mesenchymal stem cells, and add lactose-N-neohexose and epinephrine to the bone marrow mesenchymal stem cell culture medium during P5 and / or P6 passage culture; and 3) Collect the bone marrow mesenchymal stem cells and suspend them in physiological saline containing vitamin C, glucose and trehalose.

2. The method of claim 1, wherein the concentration of lactose-N-neohexose in the bone marrow mesenchymal stem cell culture medium is 2-20 μM.

3. The method of claim 2, wherein the concentration of lactose-N-neohexose in the bone marrow mesenchymal stem cell culture medium is 2-10 μM.

4. The method of claim 1, wherein the concentration of adrenaline in the bone marrow mesenchymal stem cell culture medium is 1-5 μM.

5. The method of claim 1, wherein the concentration of bone marrow mesenchymal stem cells in the stem cell preparation is 5 x 10⁻⁶. 6 per mL.

6. The stem cell preparation prepared by the method according to any one of claims 1-5.

7. The use of the stem cell preparation of claim 6 in the preparation of a medicament for the prevention and / or treatment of lung injury in a subject, wherein the lung injury is LPS or radiation-induced lung injury, and the bone marrow mesenchymal stem cells included in the stem cell preparation are or are derived from the subject's autologous stem cells.

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