CN110590932A - Preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder - Google Patents
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Abstract
The invention provides umbilical cord mesenchymal stem cell factor freeze-dried powder and a preparation method thereof.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder.
Background
Mesenchymal Stem Cells (MSCs) secrete a variety of cytokines during stable growth. TGF-beta (transforming growth factor-beta), VEGF (vascular endothelial growth factor), FGF, HGF (liver growth factor), SOD (superoxide dismutase), IL-6 (interleukin-6), EGF (epidermal growth factor), collagen (COL I, COL III), Fibronectin (FN), PDGF (platelet-derived factor) and the like secreted by MSC can effectively prevent oxidative damage, and have significant effects in the aspects of skin aging resistance, wrinkle resistance, skin whitening, wound healing and the like. TGF-beta 1 secreted by MSC plays a role in whitening by inhibiting TYR (TYR) activity, reducing expression of tyrosinase related protein, inhibiting synthesis of melanin and other processes. By collecting the supernatant (MSC-CM) after treating the cultured stem cells, a large amount of cytokines can be obtained.
The MSC-CM in liquid state has short storage time at room temperature, and is not favorable for transportation. The freeze-drying operation is carried out on the freeze-dried powder, and the prepared freeze-dried powder can keep the activity of the cell factors and is convenient to store and transport.
Disclosure of Invention
The invention aims to provide a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, which can quickly prepare umbilical cord mesenchymal stem cell factor freeze-dried powder which can keep activity for a long time and has good anti-aging effect. In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of umbilical cord mesenchymal stem cell factor, optionally, comprises the following steps:
(1) taking Wharton's jelly on the umbilical cord, and performing isolated culture to obtain umbilical cord mesenchymal stem cells, which are marked as P0 generation;
(2) carrying out passage for 3-5 times, namely, replacing a serum-free blank culture medium after P3-P5 generation;
(3) continuously culturing for 24-96 h;
(4) collecting cell supernatant, centrifuging, dialyzing, collecting trapped fluid to obtain umbilical cord mesenchymal stem cell factor, and lyophilizing.
And (3) MSC culture medium for passage in the step (2).
The centrifugation in the step (4) is as follows: centrifuging the supernatant at the temperature of 2-6 ℃ at the rpm of 800-2000/min for 5-15 min, and taking the supernatant.
The dialysis in the step (4) is as follows: and (3) putting the supernatant into a dialysis bag with 3-50 KD, putting the dialysis bag into a PBS solution, dialyzing for 24-48 h at the temperature of 0-6 ℃, and collecting dialysate.
The freeze-drying protective agent is as follows: and adding 0-15 g of mannitol into every 100mL of the umbilical cord mesenchymal stem cell factor.
In some of these embodiments, optionally, the lyophilization process is: (1) adding the sample into a 5ml penicillin bottle, reducing the temperature of the cold hydrazine to-80 ℃ per 1.5ml penicillin bottle (2), and reducing the temperature of a box body to-40 ℃; (3) placing the penicillin bottle semi-pressing plug in a vacuum freeze dryer, and keeping freezing for 4 hours; (4) reducing the pressure to 25 Pa; (5) heating to-20 deg.C at a speed of 0.5 deg.C/min, and maintaining for 16.5 h; (6) reducing the pressure to 5 Pa; (7) heating to 25 deg.C at a speed of 0.5 deg.C/min, maintaining for 6.5h, and lyophilizing; (8) and (5) pressing a piston in the penicillin bottle, and taking out.
The invention also provides umbilical cord mesenchymal stem cell factor freeze-dried powder, which has the following specific technical scheme:
alternatively, it is prepared by the preparation method as described above.
The invention also provides application of the umbilical cord mesenchymal stem cell factor freeze-dried powder, and the specific technical scheme is as follows:
the umbilical cord mesenchymal stem cell factor freeze-dried powder is applied to skin whitening, anti-aging and alopecia prevention.
Based on the technical scheme, the invention has the following beneficial effects:
the umbilical cord mesenchymal stem cell factor is prepared by collecting the umbilical cord mesenchymal stem cell conditioned medium. Wherein, the umbilical cord mesenchymal stem cells are selected to avoid social ethical disputes, and have the advantages of rapid proliferation and low immunological rejection. The umbilical cord mesenchymal stem cell factor freeze-dried powder is prepared from the stem cell factor prepared by culturing the umbilical cord mesenchymal stem cells by the method and a suitable freeze-dried powder protective agent, is favorable for long-term stable storage of the stem cell factor, and has an anti-aging effect.
Drawings
FIG. 1 is a graph of the effect of different concentrations of lyoprotectant on the morphology of lyophilized samples;
FIG. 2 is a DSC of lyophilized samples (MSC-CM + 4% mannitol);
FIG. 3 is a graph showing the effect of each sample group on the moisture content in the skin of mice;
FIG. 4 is a graph of the effect of each sample group on hydroxyproline content in mouse skin;
FIG. 5 is a graph showing the effect of various sample groups on SOD activity in mouse skin;
FIG. 6 is a graph of HE staining of mouse skin;
FIG. 7 is a graph of the effect of each sample on the thickness of the dermis of the skin of a mouse;
FIG. 8 is a graph of MASSON staining of mouse skin;
FIG. 9 is a graph showing the effect of each sample on the collagen content of mouse skin.
Detailed Description
The invention provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, and in order to make the purpose, technical scheme and effect of the invention clearer, the invention is further described in detail with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. The various reagents commonly used in the examples are all commercially available products
The invention will now be described more fully with reference to the following specific examples:
example 1
The embodiment provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, which comprises the following specific steps:
(1) preparation of umbilical cord mesenchymal stem cell factor
Taking Wharton's jelly on the umbilical cord, and performing isolated culture to obtain umbilical cord mesenchymal stem cells, which are recorded as P0 generation. And (4) carrying out passage 5 times, namely after P5 generation and when the cells grow to 75-85%, replacing the serum-free blank culture medium, continuing to culture for 72h, and collecting cell supernatant. Centrifuging the supernatant at 4 deg.C at 1000rpm/min for 5min, collecting supernatant, and filtering with 0.22 μm filter membrane. And (3) putting the supernatant into a dialysis bag with 3KD, putting the dialysis bag into a PBS solution, dialyzing for 36h at the temperature of 4 ℃, and collecting dialysate to obtain the umbilical cord mesenchymal stem cell factor.
(2) Umbilical cord mesenchymal stem cell factor freeze-dried powder
Every 100mL of umbilical cord mesenchymal stem cell factor, 6g of mannitol is added. The lyophilization was performed as follows: (1) adding the sample into a 5ml penicillin bottle, reducing the temperature of the cold hydrazine to-80 ℃ per 1.5ml penicillin bottle (2), and reducing the temperature of a box body to-40 ℃; (3) placing the penicillin bottle semi-pressing plug in a vacuum freeze dryer, and keeping freezing for 4 hours; (4) reducing the pressure to 25 Pa; (5) heating to-20 deg.C at a speed of 0.5 deg.C/min, and maintaining for 16.5 h; (6) reducing the pressure to 5 Pa; (7) heating to 25 deg.C at a speed of 0.5 deg.C/min, maintaining for 6.5h, and lyophilizing; (8) and (5) pressing a piston in the penicillin bottle, and taking out.
Example 2
The embodiment provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, compared with embodiment 1, in the step (2), 0g of mannitol is added to every 100mL of umbilical cord mesenchymal stem cell factor.
The rest of the procedure was the same as in example 1.
Example 3
The embodiment provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, compared with embodiment 1, in the step (2), 0.5g of mannitol is added to every 100mL of umbilical cord mesenchymal stem cell factor.
The rest of the procedure was the same as in example 1.
Example 4
The embodiment provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, compared with embodiment 1, in the step (2), 1g of mannitol is added to every 100mL of umbilical cord mesenchymal stem cell factor.
The rest of the procedure was the same as in example 1.
Example 5
The embodiment provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, compared with embodiment 1, in the step (2), 2g of mannitol is added to every 100mL of umbilical cord mesenchymal stem cell factor.
The rest of the procedure was the same as in example 1.
EXAMPLE 6 evaluation of the Effect of mannitol at various concentrations
The appearance, color and rehydration of the umbilical cord mesenchymal stem cell factor lyophilized powder in examples 1-5 were evaluated according to the criteria in table 1.
TABLE 1 appearance, color and rehydration solubility criteria of lyophilized powder of umbilical cord mesenchymal stem cell factor
As shown in fig. 1 and table 2, when the concentration of mannitol as the cryoprotectant is 0%, 0.5%, 1% and 6%, the lyophilized product is poor in form, delamination occurs, the sample is atrophied and not full, the rehydration time is over 120s, the sample is colorless, clear and transparent after being rehydrated, and the score is low, and is respectively 2, 3 and 4. When the concentration of mannitol is 2% and 6%, the freeze-dried product has good shape, the freeze-dried powder is full and has no layering phenomenon, the time for dissolving the sample in water is relatively short, about 60s, the sample is colorless, clear and transparent after dissolving in water, and the score is high and is respectively 16 and 16.
TABLE 2 results of different mannitol contents on appearance, color, rehydration, and comprehensive evaluation
| Content of mannitol/%) | Appearance and color | Rehydration property | Comprehensive evaluation |
| 0 | 1 | 1 | 2 |
| 0.5 | 1 | 1 | 2 |
| 1 | 2 | 1 | 3 |
| 2 | 8 | 8 | 16 |
| 6 | 8 | 8 | 16 |
。
EXAMPLE 7 eutectic point detection of lyophilized samples
Every 100mL of lyophilized powder of the umbilical cord mesenchymal stem cell factor obtained in example 2 was added with 6g of mannitol. Placing the crucible on a one-tenth-ten-thousandth balance, returning the balance to zero, placing a small amount of sample solution in the crucible, accurately weighing the sample, and placing the crucible at the detection position of a differential scanning calorimeter. Setting detection conditions, gradually cooling the system temperature to-50 ℃, then carrying out temperature programming, heating to 50 ℃, and finishing detection; as a result, as shown in FIG. 2, the eutectic point of the sample was-17 ℃.
Example 8 study on anti-aging effects of umbilical cord mesenchymal stem cell factor and umbilical cord mesenchymal stem cell factor lyophilized powder
ICR mice were randomly divided into five groups: blank group, model group, MSC-CM group, freeze-dried preparation group, and positive drug group, each group contains 8 drugs. Animals were fed with normal feed for one week to acclimatize.
The skin hair on the back of the mouse was shaved by electric hair clipper, the hair was removed by depilatory cream, and a-Gal PBS solution (1000mg/kg) was injected subcutaneously into the back neck of the mouse once a day for 42 days, in a volume of about 200. mu.L, and the weight was weighed once every six days, and the dose was adjusted according to the weight. 24h after the last intervention, the mice were sacrificed by cervical dislocation and the skin of the depilated area of the back of the mice was quickly removed. The preparations used in the experiment are all prepared aseptically, and instruments are all disinfected in advance. (1) Blank group: physiological saline, 100. mu.L/10 g/patient, was injected daily, and then 0.5mL of physiological saline was applied. (2) Model group: 10% D-Gal was injected daily at 100. mu.L/10 g/mouse, and the skin of the back of the mouse was smeared with 0.5mL of physiological saline. (3) MSC-CM group: 10% -Gal was injected daily, 100. mu.L/10 g/mouse, and 0.5mL MSC-CM was smeared on the skin of the back of the mouse. (4) Freeze-drying the sample groups: 10% -Gal is injected daily, 100 mu L of the lyophilized sample is dissolved in 1.5mL of single distilled water, and 0.5mL of the lyophilized solution is smeared on the back skin of the mouse. (5) Positive control group: 10% -Gal was injected daily, 100. mu.L/10 g/mouse, and 0.5mL of 10g/L aminoguanidine hydrochloride (AG) solution was applied to the skin of the back of the mouse. And (3) detecting the water content of the skin by a drying method, and detecting the SOD activity and the hydroxyproline content of the skin by using a kit. HE staining and MASSON staining were performed on skin sections.
The detection results are shown in FIGS. 3 to 9. In the model group, the water content, SOD activity and HYP content of the skin of the mouse are all remarkably reduced. After administration, MSC-CM and lyophilized preparation groups all had an improvement in the damage in the model group. The model group has a very significant reduction in dermal thickness (p <0.001) and a significant reduction in collagen content (p < 0.05). The MSC-CM and lyophilized formulation groups significantly increased the dermal thickness and collagen content of the mice relative to the model group (p < 0.01). Experiments in mice show that the umbilical cord mesenchymal stem cell factor and the umbilical cord mesenchymal stem cell factor freeze-dried powder have a certain anti-aging effect.
Claims (8)
1. A preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder is characterized by comprising the following steps:
(1) taking Wharton's jelly on the umbilical cord, and performing isolated culture to obtain umbilical cord mesenchymal stem cells, which are marked as P0 generation;
(2) carrying out passage for 3-5 times, namely, replacing a serum-free blank culture medium after P3-P5 generation;
(3) continuously culturing for 24h ~ 96 h;
(4) collecting cell supernatant, centrifuging, dialyzing, collecting trapped fluid, and lyophilizing to obtain lyophilized powder.
2. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 1, wherein the MSC culture medium is used for passage in step (2).
3. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 1, wherein the centrifugation condition in step (4) is to centrifuge the supernatant at 800 ~ 2000rpm/min for 5 ~ 15min at 2 ~ 6 ℃ and take the supernatant.
4. The method for preparing the umbilical cord mesenchymal stem cell factor freeze-dried powder according to claim 1, wherein the dialysis in the step (4) comprises placing the supernatant in a dialysis bag with 3-50 KD, placing the dialysis bag in PBS solution, dialyzing for 24 ~ 48h at 0 ~ 6 ℃ and collecting dialysate.
5. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 1, wherein the lyophilization conditions in step (4) are adding a lyophilization protectant, and lyophilizing.
6. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 5, wherein the lyophilized protectant in step (4) is 0 ~ 15g of mannitol per 100mL of umbilical cord mesenchymal stem cell factor.
7. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 6, wherein the process of lyophilizing in step (4) is as follows:
A. the sample is added into a 1 ~ 10 ml penicillin bottle, and a 1 ~ 5 ml/bottle
B. Reducing the temperature of the cold hydrazine to-80 ~ -60 ℃, and reducing the temperature of the box body to-60 ~ -40 ℃;
C. placing the penicillin bottle semi-pressing plug in a vacuum freeze dryer, and keeping freezing for 1 ~ 6 h;
D. reducing the pressure to 10 ~ 50 Pa;
E. heating to-25 ~ -17 deg.C at a speed of 0.5 ~ 5 deg.C/min, and maintaining for 10- ~ 20 h;
F. reducing the pressure to 0 ~ 30 Pa;
G. heating to 0 ~ 25 ℃ at the speed of 0.5 ~ 5 ℃/min, maintaining for 1 ~ 10 h, and finishing freeze-drying;
H. and (5) pressing a piston in the penicillin bottle, and taking out.
8. The application of the umbilical cord mesenchymal stem cell factor freeze-dried powder in the preparation of medicines for whitening skin, resisting aging and losing hair is characterized in that the umbilical cord mesenchymal stem cell factor freeze-dried powder is prepared according to any one of the methods in claims 1 to 7.
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| PCT/CN2020/102209 WO2021077812A1 (en) | 2019-10-21 | 2020-07-15 | Umbilical cord mesenchymal stem cell factor freeze-dried powder, preparation method therefor and application thereof |
| KR1020207024758A KR102279343B1 (en) | 2019-10-21 | 2020-07-15 | Umbilical cord mesenchymal stem cell factor frozen powder, production method and use thereof |
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| CN112640887A (en) * | 2020-12-25 | 2021-04-13 | 武汉睿健医药科技有限公司 | Neural stem cell cryopreservation liquid and application thereof |
| WO2021077812A1 (en) * | 2019-10-21 | 2021-04-29 | 江苏艾洛特医药研究院有限公司 | Umbilical cord mesenchymal stem cell factor freeze-dried powder, preparation method therefor and application thereof |
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| CN114053205A (en) * | 2021-12-08 | 2022-02-18 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of anti-aging facial skin nutrient solution freeze-dried powder, product and application thereof |
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