Method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment

Abstract

The invention discloses a culture method of umbilical cord mesenchymal stem cells for treating cognitive impairment, and belongs to the technical field of cell culture. The culture method specifically comprises the following steps: (1) preparing a special culture medium; (2) umbilical cord pretreatment; and (3) cell culture. The traditional Chinese medicine extract (astragaloside), the B cell depleting agent (rituximab and ibrutinib) and the metabolism regulator (eicosapentaenoic acid) are jointly applied to the UC-MSCs in-vitro culture process, the multiplication capacity, the anti-inflammatory characteristic and the neural restoration function of the UC-MSCs can be synergistically enhanced, and therefore the cognitive disorder treatment efficiency of the UC-MSCs is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically to a method for culturing umbilical cord mesenchymal stem cells for the treatment of cognitive impairment. Background Technology

[0002] Cognitive impairment is a neurological syndrome characterized by a decline in higher brain functions such as learning, memory, and executive function. Its causes include cerebrovascular diseases, neurodegenerative diseases, and perioperative stress. With the increasing aging of the global population, the incidence of cognitive impairment continues to rise, becoming a serious public health problem. Currently, there are no drugs or treatments that can effectively reverse or significantly slow its pathological progression, making the development of new and effective therapies an urgent priority.

[0003] In recent years, cell therapies based on mesenchymal stem cells (MSCs) have brought new hope for the treatment of cognitive impairment. Among them, umbilical cord-derived MSCs (UC-MSCs) are considered a highly promising therapeutic tool due to their abundant source, ethically sound collection methods, strong proliferative capacity, low immunogenicity, and powerful immunomodulatory and tissue repair potential. UC-MSCs can improve cognitive function through multiple mechanisms, including migrating to damaged brain regions, differentiating into neuron-like cells, secreting various neurotrophic factors (such as BDNF and GDNF), inhibiting neuroinflammation, promoting angiogenesis, and synaptic remodeling.

[0004] However, the clinical application of UC-MSCs still faces many technical bottlenecks. UC-MSCs cultured using conventional methods suffer from low survival rates and limited homing efficiency after transplantation. Pathological brain microenvironments (such as persistent neuroinflammation) can severely affect the survival and function of transplanted cells, with a large number of cells undergoing apoptosis or inactivation before reaching the target site. This results in an insufficient number of cells actually reaching the lesion and exerting their effects, thus limiting their function and restricting their therapeutic efficacy.

[0005] Therefore, how to develop a method for culturing umbilical cord mesenchymal stem cells for the treatment of cognitive impairment is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for culturing umbilical cord mesenchymal stem cells for the treatment of cognitive impairment, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for culturing umbilical cord mesenchymal stem cells for the treatment of cognitive impairment specifically includes the following steps:

[0009] (1) Preparation of special culture medium

[0010] Prepare stock solutions of astragaloside A, rituximab, ibrutinib and eicosapentaenoic acid, then add them to the basal medium and dilute to obtain the special medium for later use.

[0011] (2) Umbilical cord pretreatment

[0012] After umbilical cord collection, it is immersed in physiological saline or PBS solution containing penicillin and streptomycin, and then transferred to a sterile culture dish. It is repeatedly rinsed with PBS solution containing penicillin and streptomycin until blood and dirt are washed away. Subsequently, residual antibiotics are thoroughly rinsed with PBS. The outer membrane of the umbilical cord is cut longitudinally, and the two umbilical arteries and one umbilical vein are peeled off and removed. The remaining Wharton's jelly is transferred to another sterile culture dish, washed with PBS, and then cut into small pieces.

[0013] (3) Cell Culture

[0014] The shredded tissue pieces were placed in a T25 cell culture flask, and complete culture medium was added. The flask was then incubated in an incubator. Cells in the logarithmic growth phase were seeded into 24-well plates, the original culture medium was discarded, and a special culture medium was used to continue culturing. This yielded umbilical cord mesenchymal stem cells for the treatment of cognitive impairment.

[0015] Furthermore, in step (1) above, the basal culture medium is low-sugar DMEM medium.

[0016] Furthermore, in step (1) above, the concentration of astragaloside A in the special culture medium is 15 μM, the concentration of rituximab is 1.0 nM, the concentration of ibrutinib is 30 nM, and the concentration of eicosapentaenoic acid is 5.0 μM.

[0017] Furthermore, in step (2) above, the concentration of penicillin in physiological saline or PBS solution is 100 U / mL, and the concentration of streptomycin is 100 μg / mL.

[0018] Furthermore, in step (2) above, the pieces are cut to 1-3mm. 3 It is a paste-like or very small tissue mass.

[0019] Furthermore, in step (3) above, the complete culture medium is low-sugar DMEM medium + 10% FBS + 1% double antibiotics (penicillin, streptomycin).

[0020] Furthermore, in step (3) above, the cultivation conditions are 37°C and 5% CO2.

[0021] Furthermore, in step (3) above, the cell density for inoculation is 5 × 10⁻⁶. 3 cells / mL, time: 24h.

[0022] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention combines traditional Chinese medicine extracts (astragaloside A), B-cell depletion agents (rituximab, ibrutinib), and metabolic regulators (eicosapentaenoic acid) in the in vitro culture process of UC-MSCs, which can synergistically enhance their proliferation capacity, anti-inflammatory properties, and nerve repair function, thereby significantly improving their efficacy in treating cognitive impairment. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1

[0026] The method for culturing umbilical cord mesenchymal stem cells for the treatment of cognitive impairment specifically includes the following steps:

[0027] (1) Preparation of special culture medium

[0028] Prepare stock solutions of astragaloside A, rituximab, ibrutinib, and eicosapentaenoic acid, then add them to basal medium (low-glucose DMEM medium) and dilute to obtain a special medium containing 15 μM of astragalus injection, 1.0 nM of rituximab, 30 nM of ibrutinib, and 5.0 μM of eicosapentaenoic acid for later use.

[0029] (2) Umbilical cord pretreatment

[0030] After umbilical cord collection, the cord was immersed in physiological saline containing 100 U / mL penicillin and 100 μg / mL streptomycin, then transferred to a sterile culture dish and repeatedly rinsed with PBS solution containing penicillin and streptomycin until blood and contaminants were removed. Subsequently, residual antibiotics were thoroughly rinsed with PBS. The outer membrane of the umbilical cord was longitudinally cut, and the two umbilical arteries and one umbilical vein were peeled off and removed. The remaining Wharton's jelly was transferred to another sterile culture dish, washed with PBS, and then cut into 1-3 mm pieces. 3 Paste-like or very small tissue clumps;

[0031] (3) Cell Culture

[0032] The shredded tissue pieces were placed in T25 cell culture flasks, and complete culture medium (low-glucose DMEM medium + 10% FBS + 1% antibiotics) was added. The flasks were then incubated at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were then harvested at a rate of 5 × 10⁻⁶ cells / year. 3 Cells / mL were seeded into 24-well plates. After 24 hours, the original culture medium was discarded and replaced with a special culture medium for continued culture, thus obtaining umbilical cord mesenchymal stem cells for the treatment of cognitive impairment.

[0033] Performance testing

[0034] Experimental process

[0035] Astragaloside A was purchased from Shanghai Yuanye Biotechnology Co., Ltd., rituximab from Xinda Biopharmaceutical (Suzhou) Co., Ltd., ibrutinib from Qilu Pharmaceutical (Hainan) Co., Ltd., and eicosapentaenoic acid (EPA) from Shandong Yuwang Pharmaceutical Co., Ltd.

[0036] Take appropriate amounts of the above solutions to prepare stock solutions, and then add them to the basal culture medium (low-glucose DMEM medium) to dilute to the 12 concentration designs shown in Table 1.

[0037] Table 1. 12 Concentration Design Schemes

[0038]

[0039] Experiment 1: Study on proliferation efficiency

[0040] The umbilical cord was collected from healthy, full-term, vaginally delivered newborns at Peking University Third Hospital (PKU Third Hospital). Immediately after collection, the umbilical cord was immersed in physiological saline containing penicillin (100 U / mL) and streptomycin (100 μg / mL) and transported to the laboratory as quickly as possible at 4°C. In a laminar flow hood, the umbilical cord was transferred to a sterile culture dish and repeatedly rinsed with PBS solution containing penicillin and streptomycin until blood and contaminants were removed. Residual antibiotics were then thoroughly rinsed with PBS. Using sterile ophthalmic scissors and forceps, the outer membrane of the umbilical cord was longitudinally cut, and the two umbilical arteries and one umbilical vein were carefully dissected and removed. The remaining Wharton's jelly was transferred to another sterile culture dish, washed with PBS, and then shredded as finely as possible to 1-3 mm. 3 It is a paste-like or very small tissue mass.

[0041] Evenly spread the chopped tissue pieces into T25 cell culture flasks, add complete culture medium (low-glucose DMEM medium + 10% FBS + 1% antibiotics), and incubate at 37°C in a 5% CO2 incubator. Take cells in the logarithmic growth phase and culture at a rate of 5 × 10⁻⁶ cells / year. 3Cells / mL were seeded into 24-well plates. After 24 hours, the original culture medium was discarded and replaced with 12 sets of experimental culture media. On days 1, 2, and 3 after culture, 10 μL of CCK-8 reagent was added to each well, and after incubation for 2 hours, the absorbance was measured at 450 nm using a microplate reader, and the population doubling time was calculated. The results are shown in Table 2.

[0042] Table 2. OD value and population doubling time on day 3.

[0043] Group Day 3 OD value PDT(h) The shortening ratio relative to the Ctrl group Ranking Ctrl 0.80±0.06 35.2 - 1 0.92±0.08 32.5 7.7％ 9 2 1.25±0.10 28.1 20.2％ 6 3 1.55±0.12** 24.8** 29.5％ 1 4 1.40±0.11 26.5 24.7％ 3 5 0.85±0.07 34.0 3.4％ 10 6 0.78±0.06 36.5 -3.7％ 12 7 1.15±0.09 29.5 16.2％ 7 8 1.00±0.08 31.8 9.7％ 8 9 1.50±0.12** 25.2** 28.4％ 2 10 0.82±0.07 34.8 -1.1％ 11 11 1.30±0.10 27.8 21.0％ 4 12 1.28±0.10 28.0 20.5％ 5

[0044] As shown in Table 2, groups 3, 9, 4, 11 and 12 all showed extremely strong proliferative effects, with a significant increase in PDT effect of more than 20% compared to the Ctrl group. Among them, the PDT effect of groups 3 and 9 was significantly lower than that of the Ctrl group, while groups 6 and 10 showed inhibitory effects.

[0045] Experiment 2: Stem Cell Function Study

[0046] The culture method was the same as in "Experiment 1". After 24 hours of culture, the original culture medium was discarded and replaced with the experimental culture media for groups 3, 9, 4, 11, and 12. After 5 days of culture, the concentrations of BDNF and GDNF in the cell culture supernatant were detected using an ELISA kit. The results are shown in Table 3.

[0047] Table 3. Neurotrophic factor secretion levels

[0048] Group BDNF (pg / mL) Compared to Ctrl change GDNF (pg / mL) Compared to Ctrl change Ctrl 185±15 - 120±10 - 3 345±28** 86.5％ 235±20** 95.8％ 9 330±26** 78.4％ 225±18** 87.5％ 4 278±22 50.3％ 188±15 56.7％ 11 155±12* -16.2％ 105±8* -12.5％ 12 170±14* -8.1％ 110±9* -8.3％

[0049] Table 3 shows that groups 11 and 12 exhibited inhibitory effects on functional indicators, with significantly lower levels of neurotrophic factor secretion compared to the control group. Groups 3 and 9 showed the strongest functional responses, significantly higher than the control group. Although the neurotrophic factor secretion level in group 4 was higher than that in the control group, the increase was not significant.

[0050] Trial 3: In vivo therapeutic effect study

[0051] Healthy male Sprague-Dawley rats, aged 10-12 weeks and weighing 250-300g, were selected. All animals were acclimatized for one week under standard conditions (12 / 12h light / dark cycle, suitable temperature and humidity, free access to water and food). The rats were randomly divided into six groups: blank control group, model group, traditional stem cell control group, experimental group 1 (group 3), experimental group 2 (group 9), and inhibition control group (group 6), with seven rats in each group. Cognitive impairment in rats was induced by intraperitoneal injection of D-galactose.

[0052] Seven days after successful model establishment, cell transplantation was performed via tail vein injection, adjusting the cell density of groups 3, 9, and 6 to 5 × 10⁶ cells / day. 61 mL of cell suspension (i.e., 5 × 10⁶ cells / mL) was slowly injected into each rat. 6 (Number of cells). The control group and the model group were injected with an equal volume of PBS. Injections were repeated for 3 consecutive weeks.

[0053] The Morris water maze test was used weekly to study the effects of different groups on behavioral improvement in rats. The results are shown in Table 4.

[0054] The concentrations of inflammatory factors (TNF-α and IL-1β) in hippocampal tissue homogenates were detected by ELISA after 3 weeks of intervention. The results are shown in Table 5.

[0055] Table 4 Behavioral Improvement Effects

[0056] Group Week 1 Week 2 Week 3 Blank group 42.5±5.2 20.8±3.5 15.2±2.8 Model group 65.3±7.1 60.1±6.8 55.8±6.2 Traditional stem cell control group 62.5±6.8 48.3±5.5 35.5±4.5 Experiment 1 (Group 3) 55.2±6.0** 32.5±4.0** 20.5±3.2** Experiment 2 (Group 9) 56.8±6.2 35.2±4.3 22.8±3.5 Inhibition control group (group 6) 64.1±7.0 58.5±6.5 52.1±5.8

[0057] Table 5 Levels of inflammatory factors

[0058] Group TNF-α (pg / mg) IL-1β (pg / mg) Blank group 14.8±2.0 10.2±1.5 Model group 48.5±6.0 41.3±5.2 Traditional stem cell control group 35.2±4.5 28.5±3.8 Experiment 1 (Group 3) 18.5±2.5** 14.5±2.0** Experiment 2 (Group 9) 20.8±2.8 16.8±2.2 Inhibition control group (group 6) 44.2±5.5 37.8±4.8

[0059] As shown in Tables 4-5, Group 3 performed best among all treatment groups, with rat behavior and inflammatory factor levels closest to the sham-operated group, and its efficacy was significantly better than that of the traditional stem cell group and other experimental groups.

[0060] Experiment 4 and Study of Stem Cell Function in Single Components

[0061] The culture method was the same as in "Experiment 1". After 24 hours of culture, the original culture medium was discarded and replaced with group 3: astragaloside A group (15 μM), rituximab group (1.0 nM), ibrutinib group (30 nM), and eicosapentaenoic acid group (5.0 μM). After 5 days of culture, the concentrations of BDNF and GDNF in the cell culture supernatant were detected using an ELISA kit. The results are shown in Table 6.

[0062] Table 6. Neurotrophic factor secretion levels

[0063]

[0064] As shown in Table 6, the combined formulation (Group 3) of the present invention has a significantly better effect than any single component. The effect of Group 3 is also higher than the theoretical summation effect of a single component. This proves that the components of Group 3 work together through a synergistic effect.

[0065] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for culturing umbilical cord mesenchymal stem cells for the treatment of cognitive impairment, characterized in that, Specifically, the following steps are included: (1) Preparation of special culture medium Prepare stock solutions of astragaloside A, rituximab, ibrutinib and eicosapentaenoic acid, then add them to the basal medium and dilute to obtain the special medium for later use. (2) Umbilical cord pretreatment After umbilical cord collection, it is immersed in physiological saline or PBS solution containing penicillin and streptomycin, and then transferred to a sterile culture dish. It is repeatedly rinsed with PBS solution containing penicillin and streptomycin until blood and dirt are washed away. Subsequently, residual antibiotics are thoroughly rinsed with PBS. The outer membrane of the umbilical cord is cut longitudinally, and the two umbilical arteries and one umbilical vein are peeled off and removed. The remaining Wharton's jelly is transferred to another sterile culture dish, washed with PBS, and then cut into small pieces. (3) Cell Culture The shredded tissue pieces were placed in a T25 cell culture flask, and complete culture medium was added. The flask was then incubated in an incubator. Cells in the logarithmic growth phase were then seeded into 24-well plates. The original culture medium was discarded and replaced with a special culture medium for further culture. This yielded the umbilical cord mesenchymal stem cells used to treat cognitive impairment.

2. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (1), the basic culture medium is low-sugar DMEM medium.

3. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (1), the concentration of astragaloside A in the special culture medium is 15 μM, the concentration of rituximab is 1.0 nM, the concentration of ibrutinib is 30 nM, and the concentration of eicosapentaenoic acid is 5.0 μM.

4. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (2), the concentration of penicillin in the saline or PBS solution is 100 U / mL and the concentration of streptomycin is 100 μg / mL.

5. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (2), the material is cut into pieces of 1-3mm. 3 It is a paste-like or very small tissue mass.

6. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (3), the complete culture medium is low sugar DMEM medium + 10% FBS + 1% antibiotics.

7. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (3), the culture conditions are 37°C and 5% CO2.

8. The method for culturing umbilical cord mesenchymal stem cells for treating cognitive impairment according to claim 1, characterized in that, In step (3), the cell density for seeding is 5 × 10⁻⁶. 3 cells / mL, time: 24h.

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