Umbilical cord mesenchymal stem cells, preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to umbilical cord mesenchymal stem cells and a preparation method and application thereof. The preparation method takes umbilical cord Wharton's jelly as raw material, and obtains high-purity umbilical cord mesenchymal stem cells through cleaning treatment, tissue block preparation, serum-free culture and liquid change subculture. The culture conditions and operation parameters are strictly controlled in the preparation process, the MSC serum-free culture medium and trypsin substitutes are used, so that the cell activity and purity can be guaranteed. The umbilical cord mesenchymal stem cells have no obvious toxicity and tumorigenic risk under a safe dose, and have no damage to organs in long-term use. The umbilical cord mesenchymal stem cells can significantly improve the motor dysfunction after cerebral ischemia, reduce the volume of cerebral ischemic injury, protect the integrity of the blood-brain barrier, reduce the neuroinflammatory response, and play a multi-dimensional role in brain tissue protection and repair, thereby providing a new effective means for the treatment of ischemic stroke, and having important clinical transformation value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to umbilical cord mesenchymal stem cells, their preparation methods, and applications. Background Technology

[0002] Stroke, commonly known as cerebrovascular accident, is a sudden-onset cerebrovascular disease characterized by stenosis, occlusion, or rupture of cerebral arteries due to various triggers. This disease has an extremely high disability rate, imposing a heavy medical burden and economic pressure on patients' families and society. Currently, commonly used clinical treatments include thrombolytic therapy, interventional surgery, and hematoma evacuation and decompression. While these methods can achieve vascular recanalization and blood flow restoration, the neuronal necrosis in the central area of ​​the lesion is often difficult to eradicate completely.

[0003] Traditional medical theory holds that human neurons lack regenerative capabilities, and existing drugs cannot revive necrotic neurons or restore nerve function. Finding a life form that can induce the formation of new nerve cells at the site of injury while simultaneously promoting the repair of damaged neurons would offer a potential cure for ischemic stroke. Stem cells, as a group of undifferentiated or primitive cells with self-renewal, high proliferation, and multi-directional differentiation potential, are considered to be the closest living organism to meeting these regenerative requirements to date.

[0004] Mesenchymal stem cells (MSCs) are important members of the stem cell family. They can be induced to differentiate into various tissue cells, including nerve, muscle, cardiomyocytes, liver, endothelial cells, and pancreatic cells. Even after continuous passage culture and cryopreservation, they maintain their multi-lineage differentiation potential and show no significant rejection reaction during allogeneic transplantation, making them an ideal cell source for the regeneration and repair of diseased and damaged tissues. In contrast, the research and clinical application of embryonic stem cells have long been stagnant due to ethical constraints in Western societies. The discovery of the transdifferentiation capacity of mesenchymal stem cells breaks the limitation that stem cells for clinical treatment can only be derived from embryos or fertilized eggs, providing a completely new approach to stem cell therapy and representing a revolutionary development in the field of stem cell research.

[0005] Given the shortcomings of existing technologies, there is an urgent need for a method to prepare umbilical cord mesenchymal stem cells that is stable, highly pure, and has a proven safety and efficacy for the treatment of ischemic stroke, so as to provide technical support for clinical translation. Summary of the Invention

[0006] Based on the above technical background, the main objective of this invention is to provide umbilical cord mesenchymal stem cells, their preparation methods and applications, in order to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] The first aspect of this invention is to provide a method for preparing umbilical cord mesenchymal stem cells, the method comprising the following steps: Step 1: Cut the cleaned umbilical cord into small segments, remove the umbilical vein and umbilical artery from the small segments, tear off the Wharton's glue, cut it into tissue blocks, add the tissue blocks to the culture medium, mix well, and culture in a culture flask. Step 2: Collect the old culture medium and non-adherent tissue blocks from Step 1, add them to centrifuge tubes and centrifuge. After centrifugation, discard the supernatant. Then add serum-free MSC culture medium to the centrifuge tubes and mix it with Wharton's gum in the centrifuge tubes. Transfer the suspension to the original culture flasks and continue culturing. Change the medium intermittently during the culturing process until passage. Step 3: Add physiological saline to the culture flask of umbilical cord mesenchymal stem cells cultured in Step 2, wash twice, then add trypsin substitute to wet the bottom of the culture flask. After observing the cells becoming round under a microscope, stop digestion with complete culture medium, then centrifuge, discard the supernatant, resuspend the cell pellet in MSC serum-free culture medium, and then perform multiple passage cultures to obtain purified mesenchymal stem cells.

[0009] The steps described above are described in detail below.

[0010] In step 1, the size of the tissue block is 1–3 mm. 3 .

[0011] The culture medium used was serum-free MSC medium.

[0012] The cultivation conditions are as follows: the cultivation temperature is 35-38℃, and the CO2 concentration is 4.0-6.0%.

[0013] Preferably, the cultivation conditions are: a cultivation temperature of 37°C and a CO2 concentration of 5.0%.

[0014] In step 2, the tissue fluid is changed after 7 days of culture.

[0015] The centrifugation conditions are as follows: the centrifugation temperature is 18-22℃, the centrifugation speed is 900-1200 g for 3-7 min, and the rise and fall are set to 9 / 9.

[0016] Preferably, the centrifugation conditions are: a centrifugation temperature of 20°C, a centrifugation speed of 1000 g for 5 minutes, and a rise / fall ratio of 9 / 9.

[0017] The culture conditions are as follows: the culture temperature is 35-38℃, the CO2 concentration is 4.0-6.0%, and the medium is changed every 5-7 days until subculture is carried out.

[0018] Preferably, the culture conditions are: a culture temperature of 37°C, a CO2 concentration of 5.0%, and medium changes every 6 days until subculture is performed.

[0019] In step 3, umbilical cord mesenchymal stem cells are taken and observed under a microscope. If cells are observed to adhere to the wall and grow, appearing spindle-shaped under the microscope, and the degree of confluence reaches 80%, or if the cells around the local tissue block (P0 generation) reach 80%, then passage processing can be performed.

[0020] Wash with saline solution 2-5 times.

[0021] Preferably, the patient is rinsed twice with saline solution.

[0022] The centrifugation conditions are as follows: centrifugation at 17–23°C and 250–350 g for 5–8 minutes, with the acceleration / deceleration rate set to 7 / 6.

[0023] Preferably, the centrifugation conditions are: centrifugation at 20°C and 300 g for 6 minutes, with the acceleration / deceleration speed set to 7 / 6.

[0024] The conditions for the subculture were: a culture temperature of 35–38°C and a CO2 concentration of 4.0–6.0%.

[0025] Preferably, the subculture conditions are: a culture temperature of 37°C and a CO2 concentration of 4.0–6.0%.

[0026] The number of subcultures is 3 to 5 times, preferably 4 times.

[0027] A second aspect of the present invention is to provide umbilical cord mesenchymal stem cells prepared by the preparation method described in the first aspect of the present invention.

[0028] A third aspect of the present invention is to provide the use of the umbilical cord mesenchymal stem cells described in the second aspect of the present invention in the preparation of a medicament for treating or improving cerebral ischemia injury.

[0029] The active ingredient of the drug is umbilical cord mesenchymal stem cells prepared by the preparation method described in the second aspect of the present invention.

[0030] The beneficial effects of this invention are as follows: (1) This invention uses Wharton's jelly from umbilical cord as raw material to isolate, clean, culture, and passage multiple times to prepare mesenchymal stem cells. Serum-free culture medium and trypsin substitutes are used throughout the process, and key conditions such as culture temperature, CO2 concentration, and centrifugation parameters are strictly controlled, effectively avoiding batch variations caused by serum components and the risk of exogenous contamination. The preparation method described in this invention can obtain high-purity, high-activity umbilical cord mesenchymal stem cells. After four passages, the cell purity meets the requirements for clinical application, providing a core guarantee for subsequent treatment effects. This preparation method has strong reproducibility, enabling standardized and large-scale culture and production, meeting the needs of batch applications, and is more suitable for industrial transformation compared to traditional stem cell preparation methods.

[0031] (2) The umbilical cord mesenchymal stem cells prepared in this invention can effectively improve motor dysfunction in mice with cerebral ischemia, significantly increase the grip strength of mice after ischemia, prolong the rotarod time, and increase the driving distance in the mining experiment; at the same time, the umbilical cord mesenchymal stem cells can significantly reduce the volume of cerebral ischemia injury and alleviate ischemic damage to brain tissue; they can significantly reduce EB exudation of the blood-brain barrier (BBB), protect the integrity of the blood-brain barrier, and block the key pathological links of cerebral ischemia injury; at the same time, they can effectively reduce the infiltration of macrophages in the brain and alleviate the neuroinflammatory response. The umbilical cord mesenchymal stem cells can exert therapeutic effects from four dimensions: functional repair, tissue protection, barrier maintenance, and anti-inflammatory regulation, which solves the technical bottleneck that existing thrombolysis and interventional surgery cannot reverse neuronal necrosis and is difficult to achieve neurological function reconstruction.

[0032] (3) The median lethal dose (LD50) of umbilical cord mesenchymal stem cells prepared in this invention 50 The value is 1.129 × 10 8 Units / kg, at 0.6×10 6 2.61 × 10 7 At doses of 2 × 10⁻⁶ cells / kg and below, mice showed no death or symptoms of poisoning. During a long-term feeding period of 80 days, mice in all dose groups maintained normal weight, diet, and mental state. The quality and structure of the heart, liver, spleen, lungs, kidneys, and brain tissues were normal, and HE staining results showed no pathological changes in any organ. Further tumorigenicity tests demonstrated that even at doses of 2 × 10⁻⁶ cells / kg... 6 4×10 6 At a dose of one mouse per mouse, no tumor growth was observed at the injection site in nude mice, completely avoiding the tumorigenic risk in the clinical application of stem cells, and clarifying the safe dosage range, providing safety data support for clinical translation.

[0033] (4) The Wharton's jelly from the umbilical cord is used as the source of stem cells in this invention. The umbilical cord is a waste product after the delivery of newborns, which is convenient and non-invasive to obtain, and the raw material supply is stable and the cost is relatively low. The prepared stem cells have been identified as having the typical characteristics of mesenchymal stem cells, and there is no rejection reaction when transplanted from an allogeneic body. They can not only be used for the treatment of ischemic stroke, but also provide a new candidate solution for cell therapy of neurological injury diseases. They have extremely high clinical translational value and market application prospects in the biomedical field. Attached Figure Description

[0034] Figure 1 A schematic diagram showing the effect of UMSC reinfusion on mouse body weight is shown. Figure 2 HE staining images of the hearts of mice in each group are shown; Figure 3 The images show HE staining photographs of the livers of mice in each group; Figure 4 HE staining images of the spleens of mice in each group are shown; Figure 5 HE staining images of the lungs of mice in each group are shown; Figure 6 HE-stained images of brain tissue from each group of mice are shown. Figure 7 HE-stained images of kidney tissue from each group of mice are shown. Figure 8 The images show photographs of mice in the positive control groups A and B of Experiment Example 1; Figure 9 Photographs of mice in UMSC A group, UMSC B group and saline group in Experiment Example 1 are shown; Figure 10 The results of grip strength tests in mice after brain ischemia-reperfusion injury are shown. Figure 11 The results of the rotarod test in mice after cerebral ischemia-reperfusion injury are shown. Figure 12 The results of the regional driving distance test in mice after brain ischemia injury are shown. Figure 13 The image shows TTC staining and brain ischemia injury in the ischemic brain tissue of mice in Experiment Example 2. Figure 14 The image shows the EB exudation in the mouse brain tissue in Experiment Example 2; Figure 15 The diagram shows the activation of microglia and neutrophil infiltration in the mouse brain tissue of Experiment Example 2. Detailed Implementation

[0035] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0036] Example The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention are commercially available.

[0037] Example 1 This embodiment provides a method for preparing umbilical cord mesenchymal stem cells, the method comprising the following steps: 1. Wharton's jelly separation of the umbilical cord 1.1 Umbilical Cord Cleaning and Treatment: Prepare three 150 mm sterile petri dishes. Using sterile forceps, remove the umbilical cord and transfer it to the petri dishes, immersing it in 0.9% saline solution. Using two sterile straight-tipped forceps, gently scrape the umbilical cord three times from one end to the other to remove blood, mucus, and intravascular clots from the surface. Then transfer the umbilical cord to Petri dish #2 and wash again to thoroughly remove blood. Transfer the umbilical cord to Petri dish #3, and using new sterile scissors, cut off both ends of the washed umbilical cord, cutting the remaining portion into 3 cm segments, removing any blood clots and stagnant blood. Transfer the umbilical cord segments to Petri dish #4 and wash again to further remove blood clots and stagnant blood. Then transfer the umbilical cord to Petri dish #5 and wash again to thoroughly remove any blood clots and stagnant blood within the umbilical cord segments. Place the cleaned umbilical cord segments in Petri dish #6.

[0038] 1.2. Tissue Block Preparation: Using new forceps, unfold the umbilical cord, place the amniotic membrane at the bottom, tear off the umbilical vein, fully expose the umbilical artery, remove the umbilical artery, peel off Wharton's jelly, and transfer it to a 25 mL beaker. Using new sterile scissors, cut the weighed jelly into 1-3 mm pieces. 3 The organizational block.

[0039] 1.3 Washing with Wharton's Glue: (1) Take a 50 mL centrifuge tube, use a micro-spoon to transfer the chopped gluten tissue pieces into the 50 mL centrifuge tube, add MSC serum-free medium, mix well, and then use a pipette to mix the gluten. Dispense the gluten evenly into 6 T75 culture flasks for tissue culture, 0.5 g of gluten into each flask. Then add culture medium to each T75 flask to make the final volume of culture medium 8.0 mL / flask. (2) Place the culture flasks horizontally so that the tissue pieces are evenly distributed on the bottom of the flask. Then stack the T75 flasks together and place them in a CO2 incubator for incubation. Culture conditions: 37.0℃, CO2 concentration of 5.0%.

[0040] 2. Umbilical cord Wharton's jelly rehydration and fluid replacement On day 7 of culture, a complete medium change was performed on the Wharton's glial: Using a pipette, all old culture medium and non-adherent tissue pieces were transferred to new centrifuge tubes. Centrifugation was performed at 20°C, 1000 g for 5 min, with the acceleration / deceleration rate set to 9 / 9. The supernatant was discarded using a 25 mL pipette, and an appropriate amount of serum-free MSC medium was added to the centrifuge tube using a new 25 mL pipette. 2 mL of serum-free MSC medium was added to each original culture flask using a 10 mL pipette (with the tip removed). The Wharton's glial tissue from the centrifuge tube was then mixed thoroughly with the complete medium (i.e., serum-free MSC medium) and evenly aliquoted into the original culture flasks, resulting in a final culture volume of 8 mL per flask. The culture flasks were placed horizontally to ensure the tissue pieces were evenly distributed across the bottom. The T75 flasks were then stacked together and placed in a CO2 incubator for incubation. Culture conditions: 37°C, CO2 concentration 5.0%. After the initial medium change for the Wharton's glial tissue, the medium was changed every 6 days until subculturing.

[0041] 3. Passaging of umbilical cord mesenchymal stem cells Take cultured umbilical cord mesenchymal stem cells and observe them under an inverted microscope. If the cells adhere to the culture vessel, appear spindle-shaped under the microscope, and show 80% confluence, or if the cells around a local tissue block (P0 generation) reach 80%, they are ready for passage. Remove the complete culture medium and trypsin substitute from the refrigerator and allow them to return to room temperature. Collect the old culture medium from each culture flask. Add 0.9% physiological saline to each culture flask to wash the bottom, then discard the saline and wash twice. Add 3.0 mL of T75 trypsin substitute to each culture flask. Gently shake the stacked culture flasks to allow the trypsin substitute to wet the bottom of the flasks. Time for 2 minutes, then place the culture flasks under an inverted microscope to observe the cell state. Once the cells become rounded, stop the digestion with 6.0 mL / T75 of the collected complete culture medium. Subsequently, centrifuge at 20℃, 300 g for 6 min (setting the rise and fall speed to 7 / 6), discard the supernatant, and resuspend the cell pellet in an appropriate amount of serum-free culture medium. Take 20 μL of the cell suspension for cell counting. Based on the calculation results, according to 6000 / cm 2 The culture medium was aliquoted into culture flasks at the desired passage density, with a final culture volume of 20 mL / T175. After mixing well, the flasks were placed horizontally in a CO2 incubator. Culture conditions: 37℃, CO2 concentration 5.0%. Cells were cultured for 3–4 days until 80% confluence was achieved, at which point the cells could be passaged again.

[0042] 4. Obtaining umbilical cord mesenchymal stem cell preparations Following step 3, four passages were performed to obtain relatively purified MSCs (umbilical cord mesenchymal stem cells). Flow cytometry was used to detect the expression of CD73, CD90, CD105, HLA-DR, CD14, CD19, CD34, and CD45 in the cells. The results are shown in Table 1. The cultured MSCs were then identified. After 21 days of continuous culture, their ability to differentiate into osteogenic, adipogenic, and chondrogenic cells was assessed.

[0043] 5. The effects of the obtained high-purity umbilical cord mesenchymal stem cells on acute cerebral ischemia in mice were evaluated in terms of both safety and efficacy.

[0044] Example 2 Umbilical cord mesenchymal stem cells were prepared in a manner similar to that in Example 1, except that: the culture flask was placed under an inverted microscope to observe the cell state. Once the cells became rounded, digestion was terminated with 6.0 mL / T75 of the collected complete culture medium. Subsequently, the cells were centrifuged at 20°C and 250 g for 8 min, with the centrifugation speed set to 7 / 6, and the supernatant was discarded.

[0045] Example 3 Umbilical cord mesenchymal stem cells were prepared in a manner similar to that in Example 1, except that: the culture flask was placed under an inverted microscope to observe the cell state. Once the cells became rounded, digestion was terminated with 6.0 mL / T75 of the collected complete culture medium. Subsequently, the cells were centrifuged at 23°C and 350 g for 5 min, with the centrifugation speed set to 7 / 6, and the supernatant was discarded.

[0046] The expression of CD73, CD90, CD105, HLA-DR, CD14, CD19, CD34, and CD45 in the umbilical cord mesenchymal stem cells prepared in Examples 1, 2, and 3 was detected by flow cytometry. The test results are shown in Table 1.

[0047] Table 1

[0048] As can be seen from Table 1, the umbilical cord mesenchymal stem cells prepared in Examples 1, 2 and 3 all have high activity and purity, indicating that high-purity and high-activity umbilical cord mesenchymal stem cells can be obtained by the preparation method described in this invention.

[0049] Example 4 Acute toxicity test Male BALB / c mice, weighing 22-24g, were randomly divided into 9.6 groups of umbilical cord mesenchymal stem cells (UMSCs). 10 6 4.8 10 6 2.4 106 1.2 10 6 0.6 10 6 The saline group consisted of 10 mice per group. Each group received 120 μL of the corresponding fluid (umbilical cord mesenchymal stem cell group received different concentrations of fluid prepared in Example 1 via the tail vein, 9.6 μL). 10 6 4.8 10 6 2.4 10 6 1.2 10 6 0.6 10 6 The stem cell suspension of the mice was used (the saline group was reinfused with saline), and the cell mortality of each group of mice within 24 hours after reinfusion was observed, as shown in Table 2.

[0050] Table 2. Observation of poisoning in mice during acute toxicity test (mortality rate within 24 hours)

[0051] As can be seen from Table 2, 9.6 10 6 4.8 10 6 2.4 10 6 and 1.2 10 6 In some or all of the mice in the group that underwent UMSC reinfusion, symptoms of poisoning appeared, such as decreased spontaneous activity, lethargy or abnormal agitation, and convulsions. A few mice died from asphyxiation, convulsions, and respiratory depression. No abnormalities were observed in the remaining groups. Symptoms subsided within 3-6 hours, and surviving mice recovered. In the acute toxicity test, the time to death from poisoning gradually shortened with increasing cell infusion volume. The cause of death may be local infarction caused by the large number of cells infused into the body.

[0052] The median lethal dose (LD50) of UMSC in mice was calculated using SPSS software to be 1.129 g / mL. 10 8 The 95% confidence interval (CI) for the sample size is 0.792-1.601 per kg. 10 8 per kg.

[0053] Example 5 Long-term toxicity test Male BALB / c mice, weighing 22-24g, were randomly divided into 4.8 groups for umbilical cord mesenchymal stem cell (UMSC) transplantation. 10 6 (40 pieces), 2.4 10 6 (30 pieces), 1.2 10 6 (15 pieces), 0.6 10 6 The mice were divided into two groups: a group of 10 mice in the saline group and a group of 10 mice in the saline group. Each mouse received 120 μL of fluid. The surviving mice were fed for 80 days, and 10 mice from each group were randomly selected to observe changes in body weight and general condition.

[0054] (1) Effects of UMSC reinfusion on mouse body weight changes Balb / c male mice, 22-24 g, were administered 120 μL of fluid of varying concentrations via tail vein infusion (4.8 μL). 10 6 2.4 10 6 1.2 10 6 0.6 10 6 Each group returned 4.8. 10 6 2.4 10 6 1.2 10 6 0.6 10 6 Mice were housed in a clean-grade animal room with a stem cell suspension, kept at a constant temperature (22±2℃) and alternating day and night (7:00–19:00), with free access to water and food. After 80 days, the weight changes of the mice in each group were compared. Figure 1 As shown.

[0055] from Figure 1 As can be seen, different concentrations of stem cell reinfusion had no effect on mouse body weight, and there was no statistically significant difference.

[0056] All surviving mice in all groups had normal diets, good mental state, normal body size, agile movement, and quick reactions, exhibiting moderate resistance when grasped. Their fur was glossy and smooth. Their eyes were bright and alert. Urination was normal, and their stool was yellow and not dry. Gross observation revealed no abnormalities on the surface of any of the mice's organs. The quality assessment of the heart, liver, spleen, lungs, kidneys, and brain is shown in Table 3.

[0057] Table 3. Organ weight (g) in long-term toxicity tests

[0058] As can be seen from Table 3, the quality of the heart, liver, spleen, lungs, kidneys and brain of mice in each group was not significantly different, indicating that different concentrations of stem cell reinfusion had little effect on the heart, liver, spleen, lungs, kidneys and brain of mice.

[0059] (2) Histopathological examination: Heart, liver, spleen, lung, kidney, and brain tissues from each group of mice were paraffin-embedded, and serial sections (one section per 1 mm) were prepared for HE staining. The HE staining results for the heart are shown below. Figure 2 As shown, the HE staining results of the liver are as follows: Figure 3 As shown, the HE staining results of the spleen are as follows: Figure 4 As shown, the HE staining results of the lungs are as follows: Figure 5 As shown, the HE staining results of brain tissue are as follows: Figure 6 As shown, the HE staining results of the kidney tissue are as follows: Figure 7 As shown.

[0060] from Figures 2-7 It can be seen that no structural changes were found in any of the organs.

[0061] Experimental Example Experimental Example 1: Tumorigenicity Experiment Male BALB / c nude mice, weighing 22-24g, were divided into 5 groups of 6 mice each, including: a positive control (colon cancer hct116 cells) (1... 10 6 Group A, positive control (1.5) 10 6 Group B, UMSC (2 10 6 Group A, UMSC (4 10 6 In Group B and the saline group, each mouse received a subcutaneous injection of 100 μL of the corresponding fluid into its left hind limb. The positive control group mice were injected with hct116 colon cancer cells, while Group A mice received an injection concentration of 1 μL. 10 6 Group B received an injection concentration of 1.5%. 10 6 The UMSC group was injected with umbilical cord mesenchymal stem cells prepared in Example 1, wherein the concentration injected in group A was 2... 10 6 The concentration injected into group B was 4. 10 6 The photos of the mice after injection are shown below. Figure 8 and Figure 9 As shown.

[0062] from Figure 8It can be seen that in the positive control group, small nodules grew at all injection sites in nude mice after injection, and the nodules gradually increased in size over time. In both positive control groups A and B, tumors began to form in nude mice 5-7 days after hct116 cell injection. In the positive control group, tumors of varying sizes grew at all injection sites, and their size gradually increased over time; some nude mice showed hemorrhage and necrosis of the tumors. After 35 days, the experimental animals in positive control groups A and B were euthanized by cervical dislocation under anesthesia, and tumorigenesis was detected at the injection sites.

[0063] from Figure 9 As can be seen from the data, no tumor growth was observed at the injection sites in groups A, B, and the saline group in the UMSC. Seventy days after injection, the experimental animals were euthanized by cervical dislocation under anesthesia, and tumorigenesis was detected at the injection sites.

[0064] Experiment Example 2: Validity Test Male Balb / c mice, weighing 22-24g, were randomly divided into a Control group and a UMSC treatment group. A pMCAL (brain ischemia-associated encephalopathy) model was established in all mice using electrocautery.

[0065] (1) Effects of UMSC treatment on the behavior of mice after cerebral ischemia injury UMSC (umbilical cord mesenchymal stem cells prepared in Example 1, with a concentration of 0.6%) was tested using gripping, rotarod, and mining field experiments. 10 6 The effects of treatment on the behavior of mice after brain ischemia-reperfusion injury. Grasp strength test results are as follows: Figure 10 As shown, the test results of the rotating rod are as follows: Figure 11 As shown, the mine experiment is as follows Figure 12 As shown.

[0066] from Figure 10 , Figure 11 and Figure 12 The results showed that, compared with the Contro group, in the grip strength test, the grip strength of mice with 5 days of ischemia was significantly improved under UMSC treatment intervention (P5d < 0.05); in the rotarod test, the time spent on the rod was significantly increased in mice with 3 and 5 days of ischemia under UMSC treatment intervention (P3d < 0.05, P5d < 0.05); and in the mine test, the distance traveled within the area was significantly increased in mice with 1 day of ischemia under UMSC treatment intervention (P1d < 0.05). These results indicate that umbilical cord mesenchymal stem cell (UMSC) treatment can effectively improve motor dysfunction in mice with cerebral ischemia.

[0067] (2) HUMSC treatment can reduce cerebral ischemia-reperfusion injury. A pMCAL mouse model was established. Brain tissue was collected on days 1, 3, and 5, and TTC staining was used to assess brain damage. The results are as follows: Figure 13 As shown, Figure 13 The top image shows TTC staining of ischemic brain tissue, and the bottom image shows the extent of ischemic brain injury. P < 0.05.

[0068] Figure 13 The results showed that, compared with the control group, UMSC (0.6) 10 6 The volume of cerebral ischemia-reperfusion injury was significantly reduced in the treatment group (P3d < 0.05, P5d < 0.05). These results indicate that the umbilical cord mesenchymal stem cells (UMSCs) prepared in this invention can alleviate cerebral ischemia-reperfusion injury.

[0069] (3) UMSC therapy can protect the integrity of the blood-brain barrier during cerebral ischemia. Blood-brain barrier (BBB) ​​dysfunction is one of the key pathological mechanisms of ischemic brain injury. This invention uses the EB assay to detect changes in BBB integrity after ischemic brain injury. A pMCAL mouse model was established, and brain tissue was harvested on days 1, 3, and 5. Two hours before harvesting, 0.1 mL of 4% EB (ethidium bromide) was injected via the tail vein. EB exudation in the brain tissue was observed, and the integrity of the blood-brain barrier was assessed. The results are as follows: Figure 14 As shown, Figure 14 The top image shows an image of EB staining in ischemic brain tissue, and the bottom image shows the EB exudation in ischemic brain tissue. P < 0.05 P < 0.0001.

[0070] Figure 14 The test results showed that, compared with the control group, UMSC (0.6) 10 6 The treatment group showed a significant reduction in EB exudation (P1d < 0.05, P3d < 0.0001), indicating that the umbilical cord mesenchymal stem cells (UMSC) prepared in this invention can reduce EB exudation and protect the integrity of the brain screen.

[0071] (4) HUMSC treatment can reduce neuroinflammatory response during cerebral ischemia. A pMCAL mouse model was established. On days 1, 3, and 5, samples from the Control group and HUMSC (0.6 mg / L) were collected. 10 6 Brain tissue from the treatment group. Flow cytometry was used to detect microglia activation and neutrophil infiltration. Results are as follows: Figure 15 As shown.

[0072] Figure 15The test results showed that, compared with the Control group, the HUMSC treatment group had significantly reduced macrophage infiltration in the brain (P3d<0.05, P5d<0.05), while there was no significant difference in neutrophil infiltration. These results indicate that the umbilical cord mesenchymal stem cells prepared in this invention can reduce and inhibit macrophage infiltration during cerebral ischemia.

[0073] In summary, the above results indicate that the umbilical cord mesenchymal stem cells can be transferred to the ischemic brain injury area in mice and participate in brain tissue protection and repair. Specifically, the umbilical cord mesenchymal stem cells can alleviate ischemic brain injury and protect the integrity of the blood-brain barrier during ischemia. At the same time, the umbilical cord mesenchymal stem cells prepared by this invention can reduce the neuroinflammatory response during ischemic brain injury.

[0074] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing umbilical cord mesenchymal stem cells, characterized in that, The preparation method includes the following steps: Step 1: Cut the cleaned umbilical cord into small segments, remove the umbilical vein and umbilical artery from the small segments, tear off the Wharton's glue, cut it into tissue blocks, add the tissue blocks to the culture medium, mix well, and culture in a culture flask. Step 2: Collect the old culture medium and non-adherent tissue blocks from Step 1, add them to centrifuge tubes and centrifuge. After centrifugation, discard the supernatant. Then add serum-free MSC culture medium to the centrifuge tubes and mix with the Wharton's jelly tissue blocks in the centrifuge tubes. Transfer the suspension to the original culture flasks and continue culturing. Change the medium intermittently during the culturing process until passage. Step 3: Add physiological saline to the culture flask of umbilical cord mesenchymal stem cells cultured in Step 2, wash twice, then add trypsin substitute to wet the bottom of the culture flask. After observing the cells becoming round under a microscope, stop digestion with complete culture medium, then centrifuge, discard the supernatant, resuspend the cell pellet in MSC serum-free culture medium, and then perform multiple passage cultures to obtain purified mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that, In step 1, The size of the tissue block is 1–3 mm. 3 ; The culture medium is MSC serum-free medium.

3. The preparation method according to claim 1, characterized in that, In step 1, The cultivation conditions are as follows: the cultivation temperature is 35-38℃, and the CO2 concentration is 4.0-6.0%.

4. The preparation method according to claim 1, characterized in that, In step 2, The centrifugation conditions are as follows: the centrifugation temperature is 18-22℃, the centrifugation speed is 900-1200g for 3-7 minutes, and the rise and fall are set to 9 / 9.

5. The preparation method according to claim 1, characterized in that, In step 2, The culture conditions are as follows: the culture temperature is 35-38℃, the CO2 concentration is 4.0-6.0%, and the medium is changed every 5-7 days until subculture is carried out.

6. The preparation method according to claim 1, characterized in that, In step 3, Wash with saline solution 2-5 times.

7. The preparation method according to claim 1, characterized in that, In step 3, The centrifugation conditions are as follows: centrifugation at 17–23°C and 250–350g for 5–8 minutes, with the acceleration and deceleration speeds set to 7 / 6.

8. The preparation method according to claim 1, characterized in that, In step 3, The conditions for the subculture were: a culture temperature of 35–38°C and a CO2 concentration of 4.0–6.0%.

9. An umbilical cord mesenchymal stem cell prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the umbilical cord mesenchymal stem cells according to claim 9 in the preparation of a medicament for treating or improving cerebral ischemia injury.

Images