Preparation method of human mesenchymal stem cell preparation

Abstract

According to the preparation method of the human mesenchymal stem cell preparation provided by the invention, a compound electrolyte solution containing human serum albumin is used as a digestion stop solution and a rinsing solution to be applied to middle and downstream preparation processes of the cell preparation, so that residues of heterologous proteins in the preparation are reduced, the preparation process is simplified, and the recovery rate and motility rate of cells are improved; the invention provides a preparation scheme of the mesenchymal stem cell preparation with clinical transformation guidance, high preparation quality stability, safety and high efficiency, and is beneficial to large-scale industrial preparation and application of products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a highly efficient method for preparing human mesenchymal stem cell preparations. Background Technology

[0002] Mesenchymal stem cells (MSCs) have shown great promise in regenerative medicine and immunotherapy. A key step in the preparation of MSC formulations is the digestion of adherent MSCs with digestive enzymes, which hydrolyzes adhesion proteins to detach the cells from the culture vessel surface and obtain a cell suspension.

[0003] In the preparation of cell preparations, existing techniques often use a culture medium containing 10%-20% fetal bovine serum (FBS) to neutralize digestive enzymes and terminate nitrification. The principle is that FBS contains various protease inhibitors (such as α-1-antitrypsin), which can rapidly inactivate digestive enzymes. However, this method has the drawback of introducing a large amount of foreign proteins, especially bovine serum albumin (BSA). These foreign proteins adsorb onto the cell surface and are difficult to completely remove even after multiple washes. When the cell product is reinfused into the patient, the residual foreign proteins may trigger unnecessary immune responses (such as allergies, fever, etc.) and may even affect the therapeutic effect (Reference: Pittenger MF, Discher DE, Péault BM, et al. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJRegen Med. 2019;4:22.). Other preparation techniques use solutions such as PBS / physiological saline / compound electrolytes to dilute and terminate digestion, but solution dilution-terminated digestion can affect cell viability and washing effectiveness. To reduce the residue of foreign proteins, at least 3-4 washes are required during the formulation process. This can lead to low cell recovery rates during pilot-scale production, which is a problem for industrialization.

[0004] Therefore, there is an urgent need to develop a new formulation preparation method that can efficiently reduce the digestive activity of digestive enzymes, avoid the introduction of heterologous proteins, and protect cell viability and function, so as to facilitate the large-scale production of mesenchymal stem cell preparations and promote the clinical translational application of mesenchymal stem cells. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new method for preparing human mesenchymal stem cell preparations.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a human mesenchymal stem cell preparation, characterized by comprising the following steps:

[0008] A method for preparing a human mesenchymal stem cell preparation, characterized by comprising the following steps:

[0009] (1) Resuspend the mesenchymal stem cells in culture medium and seed them into culture flasks;

[0010] (2) After culturing for 72-96 hours, discard the culture supernatant in the culture flask, and wash twice with DPBS. Each time, the amount of DPBS used should not be less than 50 μl / cm. 2 ;

[0011] (3) Discard the DPBS, add TrypLE digestion solution, and digest for 5 min;

[0012] (4) Add the stop solution, mix by pipetting, and centrifuge the cell suspension. The stop solution is a compound electrolyte solution containing 0.5% human serum albumin. The amount of stop solution added is 2.5-10 times the volume of TrypLE digestion solution.

[0013] (5) Discard the supernatant, add rinsing solution to suspend the cells, centrifuge, and repeat the rinsing process twice; the rinsing solution is a compound electrolyte solution containing 0.05%-0.5% human serum albumin, and the amount of rinsing solution added is 3-10 times the volume of TrypLE digestion solution;

[0014] (6) Discard the supernatant and suspend the cells in a compound electrolyte solution containing 0.5% human serum albumin to prepare a cell preparation.

[0015] Preferably, the amount of the termination solution added in step (4) is 2.5-5 times the volume of the TrypLE digestion solution.

[0016] Preferably, the rinsing solution in step (5) is a compound electrolyte solution containing 0.05%-0.1% human serum albumin, and the amount of rinsing solution added is 3-5 times the volume of TrypLE digestion solution.

[0017] In step (6), the corresponding amount of cell protection solution is added to prepare the cell preparation according to the target density of the cell preparation.

[0018] The compound electrolyte solution described above is prepared by combining human serum albumin (HA) injection and compound electrolyte injection.

[0019] On the other hand, the present invention provides a human mesenchymal stem cell preparation obtained by the methods described above.

[0020] In another aspect, the present invention provides the use of human mesenchymal stem cell preparations obtained by the methods described above in medicaments for treating immune and nervous system-related diseases.

[0021] Preferably, the immune and nervous system-related diseases are selected from Alzheimer's disease, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, and graft-versus-host disease.

[0022] This invention utilizes a compound electrolyte solution containing human serum albumin (HA) as both the cell digestion termination solution and the cell washing solution after cell digestion, offering the following significant advantages: 1) Source quality control: Eliminates the residue of foreign proteins such as BSA at the source, reducing the risk of immunogenicity in the product. 2) Simplified process: HA itself is a commonly used clinical cell protectant and does not need to be removed like BSA, thus simplifying the process steps in scale-up production, reducing the number of washing cycles, improving cell recovery rate, and reducing the risk of cell damage during processing. 3) Clear chemical composition and good batch-to-batch consistency: FBS is a complex biological product with significant batch-to-batch variations; while human serum albumin injection and compound electrolyte injection are both pharmaceuticals prepared according to strict industrial standards, with clear composition, controllable purity, and minimal batch-to-batch variations. 4) Environmental stability: Provides cells with a human-derived, physiologically compatible environment with clearly defined components, contributing to the stable and controllable functional state of cells during the preparation process.

[0023] This patent provides a clinically translational, safe, and efficient mesenchymal stem cell preparation method, facilitating large-scale product preparation and application. Compared with existing formulation technologies, this invention successfully achieves animal-free process and simplifies the workflow. This not only improves the safety and quality stability of the final cell preparation but also increases cell recovery rate and activity, solving a key bottleneck in the transformation of traditional cell preparation processes from "laboratory" to "industrialization." The preparation obtained by the method of this invention has good therapeutic effects on immune and nervous system-related diseases, especially Alzheimer's disease. Attached Figure Description

[0024] Figure 1 Figure 2 shows the BSA residue results in the comparison of different stop solutions in Example 2.

[0025] Figure 2 Figure 2 shows the residual results of TrypLE in comparison of different stop solutions in Example 2.

[0026] Figure 3 Figure 3 shows the BSA residue results in comparison of different washing cycles in Example 3.

[0027] Figure 4 Figure 3 shows the TrypLE residue results in comparison of different washing cycles in Example 3.

[0028] Figure 5 Figure 3 shows the cell recovery rate results in comparison of different washing cycles in Example 3.

[0029] Figure 6 Figure 4 shows the cell recovery rate results under pilot-scale production with different washing cycles in Example 4.

[0030] Figure 7 This is a trajectory diagram of the fifth day of the water maze learning latency in APP / PS1 mice after MSC administration in Example 6.

[0031] Figure 8 The figure shows the effect of MSC administration on the time (A) and number of crossings (B) of the first crossing in the exploratory phase of the water maze experiment in APP / PS1 mice in Example 6.

[0032] Figure 9 The figure shows the effect of MSC administration on the latency (A) and number of errors (B) of APP / PS1 mice in Example 6.

[0033] Figure 10 This is a graph showing the effect of MSC administration on the spontaneous activity of APP / PS1 mice in Example 6.

[0034] Figure 11 Scanning images of neurons and nerve fibers in the hippocampus of APP / PS1 mice in Example 6.

[0035] Figure 12 Scanning images of neurons and nerve fibers in the Cu and M1 regions of APP / PS1 mice in Example 6.

[0036] Figure 13 This is a blood vessel scan image of the CA1 region in APP / PS1 mice from Example 6.

[0037] Figure 14 This is a vascular scan of the olfactory bulb region of APP / PS1 mice in Example 6.

[0038] Figure 15 This is a blood vessel scan image of the M2 region in APP / PS1 mice from Example 6.

[0039] Figure 16 The graph shows the effect of MSCs preparation secretion on the survival rate of human APP / SKN cells in Example 7.

[0040] Figure 17 Figure 8 shows the effect of MSCs preparation secretion on the survival rate of primary rat cortical neurons. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments and comparative examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments and comparative examples are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0042] Example 1: Effects of different stop solutions on cell viability and stability in MSCs formulation preparation methods

[0043] Materials and Methods:

[0044] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; Human serum albumin: Sichuan Yuanda Shuyang; Compound electrolyte injection, physiological saline injection: Shijiazhuang No.4 Pharmaceutical.

[0045] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo.

[0046] Preparation method:

[0047] 1) Resuscitate the frozen mesenchymal stem cells, resuspend them in the culture medium, and seed them into T182 culture flasks at an appropriate density;

[0048] 2) After culturing for 96 hours, discard the culture supernatant in the culture flask and wash twice with 10 ml of DPBS;

[0049] 3) Discard the DPBS, add 3 ml of TrypLE digestion solution to each T182 culture flask, and digest for 5 min;

[0050] 4) Add 15 ml of different digestion termination solutions respectively, mix well by pipetting, and then centrifuge the cell suspension;

[0051] 5) Discard the supernatant, add DPBS (10ml / T182) to suspend the cells, centrifuge, and repeat this process twice;

[0052] 6) After centrifugation, discard the supernatant, add appropriate amounts of different group preservation solutions to dilute the cells, obtaining a density of 1.0 × 10⁶ cells / year. 6 Cell preparations per ml.

[0053] 7) Examine the cell viability and preservation stability of different groups.

[0054] The different grouping situations are as follows:

[0055]

[0056] Experimental results: Cell viability and preservation stability are shown in Table 1.

[0057] Table 1. Cell viability and preservation stability results of different termination solutions

[0058]

[0059] Data shows that under the conditions of group D (cell termination solution and preservation solution), the cell viability was the highest and the stability was the best, followed by group B, while the viability and stability of groups A and C were both poor.

[0060] Experimental summary: The cell termination solution and preservation solution containing 0.5% human serum albumin-compound electrolyte injection can ensure cell viability and stability, making it the optimal choice for the formulation preparation process.

[0061] Example 2: Effect of different stop solutions on formulation residues in MSCs formulation preparation methods

[0062] Materials and Methods:

[0063] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; Human serum albumin: Sichuan Yuanda Shuyang; Compound electrolyte injection: Shijiazhuang No.4 Pharmaceutical.

[0064] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo.

[0065] Preparation method:

[0066] 1) Resuscitate frozen mesenchymal stem cells, resuspend them in culture medium, and then incubate at 6000 cells / cm³. 2 Inoculate the culture flasks at the appropriate density;

[0067] 2) After culturing for 96 hours, discard the culture supernatant in the culture flask and wash twice with 10 ml of DPBS;

[0068] 3) Discard the DPBS, add 3 ml of TrypLE digestion solution to each T182 culture flask, and digest for 5 min;

[0069] 4) Add 15 ml of different digestion termination solutions respectively, mix well by pipetting, and then centrifuge the cell suspension;

[0070] 5) Discard the supernatant, add 0.05% human serum albumin-compound electrolyte solution (10ml / T182) to suspend the cells, centrifuge, and repeat this process twice;

[0071] 6) After centrifugation, collect the final wash buffer and use ELISA to detect the concentration of bovine serum albumin (BSA residue) in the solution; discard the remaining supernatant, add 0.5% human serum albumin-electrolyte solution (5 ml of resuspension per T182 bottle), mix the cells by pipetting, count the cells, and dilute the cells with 0.5% human serum albumin-electrolyte solution to obtain a density of 0.5 × 10⁻⁶ cells / mL. 6 Cell preparations were prepared using ELISA to detect the concentration of TrypLE in the preparation (referred to as TrypLE residue).

[0072] Step 4: Termination solution grouping as follows

[0073]

[0074] Test results: BSA residue results are shown in [link to test results]. Figure 1 See Table 2 for TrypLE residue results. Figure 2 And Table 3.

[0075]

[0076]

[0077] The data show that: 1) The BSA residue in the stop solution group A (0.5% human white-compound electrolyte solution) was significantly lower than that in the stop solution group B (10% FBS complete culture medium) and the stop solution group C (compound electrolyte injection); 2) The TrypLE residue in the stop solution group A was significantly lower than that in the stop solution group C.

[0078] Experimental Summary: Using 0.5% human serum albumin-compound electrolyte solution as the stop solution can significantly reduce BSA residue and TrypLE residue, making it the optimal choice for the formulation preparation process.

[0079] Example 3: Effect of different washing times on formulation residues and recovery rate in MSCs formulation preparation method

[0080] Materials and Methods:

[0081] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; Human serum albumin: Sichuan Yuanda Shuyang; Compound electrolyte injection: Shijiazhuang No.4 Pharmaceutical.

[0082] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo.

[0083] Preparation method:

[0084] 1) Resuscitate frozen mesenchymal stem cells, resuspend them in culture medium, and then incubate at 6000 cells / cm³. 2 Inoculate the culture flasks at the appropriate density;

[0085] 2) After culturing for 96 hours, discard the culture supernatant in the culture flask and wash twice with 10 ml of DPBS;

[0086] 3) Discard the DPBS, add 3 ml of TrypLE digestion solution to each T182 culture flask, and digest for 5 min;

[0087] 4) Add 15 ml of 0.5% human serum albumin-compound electrolyte solution, mix well by pipetting, count the cells to obtain the total number of cells, and centrifuge the cell suspension;

[0088] 5) Discard the supernatant, add 0.05% human serum albumin-compound electrolyte solution (10ml / T182) to suspend the cells, centrifuge, and wash the cells a different number of times for different groups; take the supernatant of the last wash for different groups and use ELISA to detect the residual amount of BSA.

[0089] 6) For different groups, after washing, discard the remaining supernatant, add 0.5% human serum albumin-compound electrolyte solution (5ml / T182), mix the cells by pipetting, count the cells, and obtain the total number of cells in the cell preparation; dilute the cells with 0.5% human serum albumin-compound electrolyte solution to obtain a density of 0.5×10⁻⁶ cells / mL. 6 Cell formulation per ml. TrypLE residue in the formulation was detected using ELISA.

[0090] The number of washes in step 5) is grouped as follows:

[0091]

[0092] Test results: BSA residue results are shown in [link to test results]. Figure 3 See Table 4 for TrypLE residue results. Figure 4 And Table 5, cell recovery rates are shown in [Table 5]. Figure 5 .

[0093] Data shows that washing twice can control the residual levels of BSA and TrypLE while ensuring cell recovery rate. Reducing the number of washes to one wash can improve the cell recovery rate, but the residual levels of BSA and TrypLE will increase. Increasing the number of washes to three washes can reduce the residual levels of BSA and TrypLE, but will reduce the cell recovery rate.

[0094] Experimental summary: Washing twice is the optimal choice for the formulation preparation process.

[0095] Example 4: Effect of different washing cycles on cell recovery rate at pilot scale

[0096] Materials and Methods:

[0097] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; Human serum albumin: Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd. (Shuyang); Compound electrolyte injection: Shijiazhuang No.4 Pharmaceutical Co., Ltd.

[0098] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo.

[0099] Preparation method:

[0100] 1) Resuscitate frozen mesenchymal stem cells, resuspend them in culture medium, and then incubate at 6000 cells / cm³. 2 Multiple T875 culture flasks were inoculated at a density of [insert density here];

[0101] 2) After culturing for 96 hours, discard the culture supernatant in the culture flasks and wash each flask twice with 60 ml of DPBS.

[0102] 3) Discard the DPBS, add 20 ml of TrypLE digestion solution to each T875 culture flask, and digest for 5 min;

[0103] 4) Add 45ml of 0.5% human serum albumin-compound electrolyte solution, mix well by pipetting, count the cells, calculate the total number of harvested cells, and then centrifuge the cell suspension.

[0104] 5) Discard the supernatant, add 0.05% human serum albumin-compound electrolyte solution (60ml / T875) to suspend the cells, centrifuge, and the number of washing times varies for different groups.

[0105] 6) For different groups, after washing, discard the remaining supernatant, add an appropriate amount of 0.5% human serum albumin-compound electrolyte solution, mix the cells by pipetting, count them, and calculate the total number of cells for the harvested formulation. Recovery rate data for three batches under pilot-scale harvesting conditions.

[0106] In step 5), the washing solution is grouped as follows:

[0107]

[0108] Data shows that at the pilot-scale level, washing twice can significantly improve cell recovery rate, reduce cell loss, and ensure cell yield.

[0109] Experimental Summary: At the pilot-scale level, this patented method for preparing cell preparations can solve the cell loss caused by process scale-up and simplify the process scale-up.

[0110] Example 5 Preparation of MSCs formulation

[0111] Materials and Methods:

[0112] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; Human serum albumin: Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd. (Shuyang); Compound electrolyte injection: Shijiazhuang No.4 Pharmaceutical Co., Ltd.

[0113] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo.

[0114] Preparation method:

[0115] 1) Resuscitate the frozen mesenchymal stem cells, resuspend them in culture medium, and incubate at 6000 cells / cm³. 2 Inoculate the culture flasks at the appropriate density;

[0116] 2) After culturing for 96 hours, discard the culture supernatant in the culture flask and wash twice with 10 ml of DPBS;

[0117] 3) Discard the DPBS, add 3 ml of TrypLE digestion solution to each T182 culture flask, and digest for 5 min;

[0118] 4) Add 15 ml of 0.5% human serum albumin-compound electrolyte solution, mix well by pipetting, and then centrifuge the cell suspension;

[0119] 5) Discard the supernatant, add 0.05% human serum albumin-compound electrolyte solution (10ml / T182) to suspend the cells, centrifuge, and repeat this process twice.

[0120] 6) Discard the supernatant, add 0.5% human serum albumin-compound electrolyte solution (5 ml of resuspension per T182 bottle), mix well by pipetting, count the cells, and dilute the cells with 0.5% human serum albumin-compound electrolyte solution to obtain a density of 1×10⁻⁶ cells / mL. 6 Cell preparations per ml.

[0121]

[0122] Example 6: Pharmacodynamic study of MSCs formulation in the treatment of Alzheimer's disease (AD)

[0123] The MSCs preparation obtained in Example 5 was used for efficacy studies of Alzheimer's disease (AD).

[0124] Materials and Methods:

[0125] Reagents:

[0126]

[0127]

[0128] Research plan:

[0129] Sixteen 8-month-old APP / PS1 transgenic mice and eight C57BJ wild-type mice were used in the experiment, divided into three groups: WT, Model, and UC-MSC (MSC preparation obtained in Example 4), with eight mice in each group (see Table 5). After the animals were observed to adapt to the environment, four animals in each of the Model and UC-MSC groups were injected intravenously with 40 μL of AAV2 / PHPEB1 (20 μL of purchased AAV2 / PHPEB diluted with 140 μL of physiological saline) for brain imaging. One week before drug administration, the neuron-specific promoter hSyn and blood-brain barrier-crossing virus were injected intravenously to specifically label mature neurons at a concentration of hsyn: 3.05E+12 vg / ml EGFP, with 100 μL injected into each mouse.

[0130] The UC-MSC group was administered a dose of 3×10⁻⁶. 7 The average animal weight was approximately 30 g, and the administration volume was 0.15 ml (5 ml / kg). The UC-MSC group was administered UC-MSC suspension (6 × 10⁻⁶ cells / kg). 6 (cells / ml), administered via tail vein injection, with each animal receiving 9 × 10⁹ cells / ml. 5For the Model and WT groups, blank solvent was injected via tail vein, with the same volume as above. The drug was administered once a week for 8 weeks.

[0131]

[0132] Eight weeks after drug administration, behavioral experiments were conducted. The water maze test was used to assess the learning and memory abilities of the mice, the platform jump test to assess their memory abilities, and the spontaneous activity test to observe differences between groups. After completing the behavioral experiments, samples were collected for subsequent mechanistic studies. Mice used for brain imaging were anesthetized with 3% isoflurane on the day of sample collection. The mice were fixed on a dissection table, and the thoracic cavity was opened to expose the heart using surgical instruments (large scissors and forceps). 100 μL of Dylight 594 vascular dye was injected into the left ventricle, and after 1.5 minutes, PBS was perfused until the liver turned white. Then, the liver was fixed with 4% PFA. The mouse tail would twitch and gradually stiffen before the brain was collected. The brain was embedded in LR-white resin, and high-precision ultrastructural imaging of the whole brain was performed using an fMOST5000 instrument. The results were analyzed. Immunohistochemical staining was performed on samples from the remaining groups of mice to observe the condition of Aβ plaques in the brain.

[0133] Results of preclinical pharmacodynamic studies of AD:

[0134] Quantitative data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using SPSS 21.0 software. One-way ANOVA was conducted with homogeneous variances. LSD tests were used for pairwise comparisons when significant differences were found. The significance level was set at α = 0.05, and p ≤ 0.05 was considered statistically significant.

[0135] The effect of UC-MSCs on the water maze in APP / PS1 mice: The learning latency of APP / PS1 transgenic mice (Model) was significantly longer than that of wild-type mice (WT). Treatment with UC-MSCs showed a trend towards shorter learning latency, suggesting that UC-MSCs can improve the spatial learning ability of APP / PS1 mice (see...). Figure 7 The first crossing time in APP / PS1 transgenic mice was significantly longer than that in wild-type mice, while the number of crossings was significantly lower. Treatment with UC-MSCs significantly shortened the first crossing time and showed a trend towards increasing the number of crossings, suggesting that UC-MSCs can improve the memory ability of APP / PS1 mice (see...). Figure 8The learning latency, first crossing time during the exploration phase, and number of crossings during the exploration phase were significantly different in the Model group compared to the WT group, indicating that the learning and memory abilities of APP / PS1 transgenic mice were decreased compared to wild-type mice. The learning latency in the UC-MSC group showed an improving trend compared to the Model group, and the first crossing time during the exploration phase in the UC-MSC group was significantly different compared to the Model group, indicating that 8 weeks of tail vein injection of UC-MSCs improved the learning and memory abilities of APP / PS1 transgenic mice.

[0136] In the step-down test, the APP / PS1 transgenic mice had a significantly shorter step-down latency and a significantly higher number of errors than wild-type mice. Treatment with UC-MSCs prolonged the step-down latency and reduced the number of errors, suggesting that UC-MSCs can improve the learning and memory abilities of APP / PS1 mice (see...). Figure 9 The latency and number of errors in the Model group were significantly different from those in the WT group, indicating that the learning and memory abilities of APP / PS1 transgenic mice were lower than those of wild-type mice. The latency and number of errors in the UC-MSC group showed an improving trend compared with the Model group, indicating that UC-MSC injection into the tail vein for 8 weeks had a trend of improving the learning and memory abilities of APP / PS1 transgenic mice.

[0137] UC-MSCs had no significant effect on spontaneous activity in APP / PS1 transgenic mice (see [link to article]). Figure 10 There was no significant difference in the number of spontaneous activities between the Model group and the WT group, and there was no significant difference in the number of spontaneous activities between the UC-MSC group and the Model group. This indicates that UC-MSC injection via tail vein for 8 weeks had no effect on the spontaneous activity ability of APP / PS1 transgenic mice.

[0138] In the study of the mechanism of action of UC-MSCs, brain imaging was performed using fMOST technology for microscopic imaging and 3D reconstruction. The number and diameter of neurons in the CCu and M1 brain regions were statistically analyzed; the average diameter, total length, and total volume of nerve fibers in the CCu region; and the average diameter, total length, and total volume of blood vessels in the olfactory bulb, M2, and CA1 brain regions. The number of MSC neurons in the CCu group was twice that of the Model group, and the number of MSC neurons in the cortical M1 was also greater than that in the Model group, although the neuron diameters were similar. This indicates that stem cell therapy has a protective or neuronal regeneration effect. The average diameter of nerve fibers in MSCs in the CCu group was 1.73 μm, larger than that in the Model group; the total length of nerve fibers was 2.7 times that of the Model group; and the total volume of nerve fibers was 3.8 times that of the Model group. This indicates that stem cell therapy can repair nerve damage and protect nerve fibers. The average blood vessel diameter in the olfactory bulb and cortical M2 region of the MSC group was smaller than that in the Model group, while the total blood vessel length was 1.6 times that of the Model group. Comprehensive analysis suggests that stem cell therapy promoted extensive capillary angiogenesis, thus lowering the overall average blood vessel diameter. The results showed that MSC stem cell therapy had a protective or angiogenic effect on neurons in multiple brain regions, repairing nerve damage and protecting nerve fibers. Another finding was a significant increase in the total length of blood vessels in some brain regions, while their diameter and volume decreased, indicating that MSCs can promote extensive capillary angiogenesis, especially in densely vascularized brain regions (see...). Figure 11 , 12 13, 14, 15).

[0139] Summary of pharmacodynamic studies: The MSC preparation prepared in this invention significantly improves the learning and memory abilities of APP / PS1 mice after administration. It can improve learning and memory abilities by promoting the regeneration of neurons and nerve fibers in the hippocampus of mice, and improve brain perfusion and energy supply by promoting the regeneration of brain capillaries in mice, thus treating Alzheimer's disease (AD).

[0140] Example 7: Effect of MSC preparation secretion on APP / SKN cell viability in OGD / R model

[0141] The MSCs preparation obtained in Example 4 was used to study the effect of secretions on the viability of APP / SKN cells in the OGD / R model.

[0142] Materials and Methods:

[0143] Reagents: Low-glucose medium: EallBio; DMEM / F12 medium: Gibco; DMEM medium: Gibco; FBS: Gibco; CCK-8: APEXBIO. APP / SKN: APP transgenic SK-N-SH human nerve cells, a classic in vitro cell model for AD research, provided by Peking Union Medical College Hospital.

[0144] Instruments: Cell culture incubator: Thermo; Biosafety cabinet: Thermo; Microplate reader: BIO-TEK.

[0145] Protocol for studying APP / SKN cell viability in the OGD / R model:

[0146] Stem cell secretion fluid: at 3×10 4 / cm 2 The MSCs preparation obtained in Example 4 was seeded into 6-well plates with 1 mL of culture medium per well and cultured for 48 hours. The supernatant was then collected, centrifuged at 800 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter. The filtrate was collected as the supernatant for mesenchymal stem cell culture. Stem cell blank culture medium: 1 mL of stem cell culture medium was added to each well of a 6-well plate and placed in a cell culture incubator for 48 hours. The collected medium was then filtered through a 0.22 μm filter, and the filtrate was used as the stem cell blank culture medium. Both the stem cell blank culture medium and the stem cell secretion fluid were prepared fresh for use.

[0147] The APP / SKN modeling drug administration components were: OGD / R group, OGD / R + 5% stem cell blank culture medium, OGD / R + 10% stem cell blank culture medium, OGD / R + 25% stem cell blank culture medium, OGD / R + 5% stem cell secretion fluid, OGD / R + 10% stem cell secretion fluid, and OGD / R + 25% stem cell secretion fluid.

[0148] Cell culture: APP / SKN cells were seeded into 96-well plates at a rate of 2 × 10⁵ cells / mL, 200 μL / well; incubated for 16 hours in a cell culture incubator (37℃, 5% CO₂); old culture medium was discarded, and low-glucose medium, stem cell blank medium, and stem cell secretion solution were added sequentially to each group to a final volume of 200 μL / well; the cells were then incubated in a tri-gas incubator for 8 hours under low-glucose and low-oxygen conditions (tri-gas incubator: 5% CO₂, 1% O₂, 94% Nitrogen); old culture medium was discarded, and new culture medium, stem cell blank medium, and stem cell secretion solution were added sequentially to each group to a final volume of 200 μL / well; the cells were then incubated for another 16 hours. After 16 hours, 20 μL of CCK8 solution was added to each well of each group, and the cells were incubated for 3 hours. Cell viability was measured using a microplate reader at 450 nm.

[0149] Results of APP / SKN cell viability study in OGD / R model: Effect of MSC preparation secretion for 24 hours on APP / SKN cell viability in OGD / R model: Compared with the control group, the viability of APP / SKN cells in the model group was significantly reduced; compared with the model group, there was no significant change in cell viability in the 5% MSC blank medium, 10% MSC blank medium, and 25% MSC blank medium groups; compared with 5%, 10%, and 25% blank medium, the neuronal cell activity in the 5%, 10%, and 25% MSC secretion groups was significantly increased. Figure 16 Data showed that under conditions simulating the local environment of Alzheimer's disease (OGD / R), the secretion fluid of MSC preparations could significantly enhance the cell viability of APP / SKN.

[0150] The results showed that, for the APP / SKN cell model, under glucose-oxygen deprivation conditions, the MSC preparation obtained in this invention had a significant neuroprotective effect, which was positively correlated with the dose.

[0151] Example 8: Effect of MSCs preparation secretion on the viability of primary neurons in rats in the OGD / R model.

[0152] The MSCs preparation obtained in Example 4 was used to study the effect of secretions on the viability of primary neurons in rats in the OGD / R model.

[0153] Materials and Methods:

[0154] Reagents: Low-glucose medium: EallBio; DMEM / F12 medium: Gibco; DMEM medium: Gibco; FBS: Gibco; CCK-8: APEXBIO.

[0155] Instruments: Cell culture incubator: Thermo; Biosafety cabinet: Thermo; Microplate reader: BIO-TEK.

[0156] Protocol for studying the viability of primary rat neurons in the OGD / R model:

[0157] Stem cell secretion fluid: at 3×10 4 / cm 2The MSCs preparation obtained in Example 4 was seeded into 6-well plates with 1 mL of culture medium per well and cultured for 48 hours. The supernatant was then collected, centrifuged at 800 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter. The filtrate was collected as the supernatant for mesenchymal stem cell culture. Stem cell blank culture medium: 1 mL of stem cell culture medium was added to each well of a 6-well plate and placed in a cell culture incubator for 48 hours. The collected medium was then filtered through a 0.22 μm filter, and the filtrate was used as the stem cell blank culture medium. Both the stem cell blank culture medium and the stem cell secretion fluid were prepared fresh for use.

[0158] The drug administration components for modeling were: OGD / R group, OGD / R + 5% stem cell blank culture medium, OGD / R + 10% stem cell blank culture medium, OGD / R + 25% stem cell blank culture medium, OGD / R + 5% stem cell secretion fluid, OGD / R + 10% stem cell secretion fluid, and OGD / R + 25% stem cell secretion fluid.

[0159] Cell culture: Primary cortical neurons were obtained by dissecting the cerebral cortex of Wistar rats (fetal rats) and cultured at a rate of 3 × 10⁻⁶ cells / year. 5 Cells were inoculated at a density of 200 μL / well in 96-well plates after preparing the inoculation medium. The plates were then incubated for 16 hours at 37°C (5% CO2). The old medium was discarded, and low-glucose medium, stem cell blank medium, and stem cell secretion solution were added sequentially to each well according to the group, bringing the total volume to 200 μL / well. The plates were then incubated in a tri-gas incubator (5% CO2, 1% Oxygen, 94% Nitrogen) for 3 hours. The old medium was discarded, and new medium, stem cell blank medium, and stem cell secretion solution were added sequentially to each well according to the group, bringing the total volume to 200 μL / well. The plates were then continued to be cultured for 4 days. Afterward, 20 μL of CCK8 solution was added to each well of each group, and the plates were incubated for 3 hours. Cell viability was measured using a microplate reader at 450 nm.

[0160] Results of primary cortical neuronal viability study in OGD / R model: Effect of MSCs secretion for 4 days on the viability of primary cortical neurons in the OGD / R model: Compared with the control group, cell viability in the model group was significantly reduced; compared with the model group, cell viability in the 5% MSC blank medium, 10% MSC blank medium, and 25% MSC blank medium groups was significantly increased; compared with 5%, 10%, and 25% blank medium, the activity of primary cortical neurons in the 5%, 10%, and 25% MSC secretion groups was significantly increased, approximately twice that of the blank medium group. Figure 17Data showed that under conditions simulating the local environment of Alzheimer's disease (OGD / R), the secretion fluid of MSC preparations could significantly enhance the viability of primary cortical neurons.

[0161] The results showed that the MSC preparation obtained in this invention had a significant neuroprotective effect on rat primary cortical nerve cells under glucose-oxygen deprivation conditions, and this effect was positively correlated with the dosage.

Claims

1. A method for preparing a human mesenchymal stem cell preparation, characterized in that, Includes the following steps: (1) Resuspend the mesenchymal stem cells in culture medium and seed them into culture flasks; (2) After culturing for 72-96 hours, discard the culture supernatant in the culture flask, and wash twice with DPBS. Each time, the amount of DPBS used should not be less than 50 μl / cm. 2 ; (3) Discard the DPBS, add TrypLE digestion solution, and digest for 5 min; (4) Add the stop solution, mix by pipetting, and centrifuge the cell suspension. The stop solution is a compound electrolyte solution containing 0.5% human serum albumin. The amount of stop solution added is 2.5-10 times the volume of TrypLE digestion solution. (5) Discard the supernatant, add rinsing solution to suspend the cells, centrifuge, and repeat the rinsing process twice; the rinsing solution is a compound electrolyte solution containing 0.05%-0.5% human serum albumin, and the amount of rinsing solution added is 3-10 times the volume of TrypLE digestion solution; (6) Discard the supernatant and suspend the cells in a compound electrolyte solution containing 0.5% human serum albumin to prepare a cell preparation.

2. The method according to claim 1, characterized in that: In step (4), the amount of stop solution added is 2.5-5 times the volume of TrypLE digest solution.

3. The method according to claim 1, characterized in that: The rinsing solution mentioned in step (5) is a compound electrolyte solution containing 0.05%-0.1% human serum albumin, and the amount of rinsing solution added is 3-5 times the volume of TrypLE digestion solution.

4. A human mesenchymal stem cell preparation obtained by the method according to any one of claims 1-3.

5. Use of the human mesenchymal stem cell preparation obtained by the method of any one of claims 1-3 in the preparation of a medicament for treating immune and nervous system-related diseases.

6. The use according to claim 5, characterized in that, The immune and nervous system-related diseases are selected from Alzheimer's disease, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, and graft-versus-host disease.

Images