KR20200128526A - Umbilical cord mesenchymal stem cell factor frozen powder, manufacturing method and application thereof - Google Patents

Abstract

The present invention provides umbilical mesenchymal stem cell factor frozen powder, a manufacturing method, and its utilization, and at the same time collecting umbilical mesenchymal stem cell condition medium by this method, mannitol is used as a freeze-dried protective agent, and a non-dried method is used. Umbilical cord mesenchymal stem cell factor frozen powder prepared by lyophilization is beneficial for long-term and stable preservation and has good anti-aging effect.

Description

Umbilical cord mesenchymal stem cell factor frozen powder, manufacturing method and application thereof

The present invention relates to the field of biotechnology, and in particular, to a frozen umbilical cord mesenchymal stem cell factor powder, a manufacturing method, and application thereof.

Mesenchymal stem cells (MSCs) secrete various cellular factors during stable growth. TGF-β (transformed growth factor-β), VEGF (vascular endothelial growth factor), FGF, HGF (liver growth factor), SOD (peroxide dismutase), IL-6 (interleukin-6), secreted from MSC EGF (epidermal growth factor), collagen (COL I, COL III), fibronectin (FN), PDGF (platelet derived factor), etc. can effectively prevent oxidative damage, prevent skin aging, prevent wrinkles, skin whitening, wound union It has a remarkable effect on the back side. TGF-β1 secreted from MSC plays a whitening role in the process, such as suppressing the activity of TYR (TYR), reducing the expression of thyrosinase-related proteins and inhibiting the synthesis of melanin. A large amount of cell factors can be obtained from the supernatant (MSC-CM) that has undergone the collection treatment culture of stem cells.

Liquid MSC-CM has a short storage period at room temperature and is unfavorable for transportation. By making it into frozen powder through frozen inflammation treatment, the activity of cell factors can be preserved, and storage and transportation are convenient.

An object of the present invention is to provide a method of manufacturing umbilical mesenchymal stem cell factor frozen powder, to quickly produce umbilical mesenchymal stem cell factor frozen powder having long-term activity and good anti-aging effect.

Preferably, the steps below:

(1) Take Wotton meat from the umbilical cord, obtain umbilical mesenchymal stem cells through in vitro culture, and mark it as P0,

(2) After passage 3 ~ 5, that is, after P3 ~ P5, it is changed to serum-free blank medium,

(3) Continue culturing for 24 h to 96 h,

(4) After collecting the cell supernatant, centrifugation, and dialysis, the residual solution is collected, and umbilical mesenchymal stem cell factor is obtained and freeze-dried.

It provides a method for manufacturing umbilical mesenchymal stem cell factor comprising the steps.

The passage during step (2) uses MSC medium.

Centrifugation during step (4): The supernatant is centrifuged for 5 to 15 min at 800 to 2000 rpm/min under the conditions of 2 to 6°C to take the supernatant.

Dialysis in Step (4): Put the supernatant in a 3-50 KD dialysis bag, put the dialysis bag in a PBS solution, and dialyzate for 24 ~ 48 h under 0 ~ 6°C conditions, and then collect the dialysis solution.

Freeze-drying conditions of step (4): A freeze-dried protectant is added and freeze-dried to obtain.

The lyophilized protective agent: 0 to 15 g of mannitol is added to the umbilical cord mesenchymal stem cell factor per 100 ml. For example, add 0.5 to 6 g of mannitol.

Among them, in some embodiments, the preferred lyophilization process: (1) put the sample in a 5 ml Bayer bottle to make 1.5 ml/bottle, (2) lower the cold hydrazine temperature to -80°C, and reduce the temperature of the chamber to- Lower it to 40℃, (3) close the Bayer bottle loosely and put it in a vacuum freeze dryer, keep it frozen for 4h, (4) reduce the pressure to 25 Pa, and (5) reduce the temperature to -20 at a rate of 0.5℃/min. Raise it to °C, hold it for 16.5 h, (6) lower the pressure to 5 Pa, (7) raise the temperature to 25 °C at a rate of 0.5 °C/min, hold it for 6.5 h, and complete freeze-drying, (8) Stop the Bayer bottle and take it out.

The present invention provides a umbilical cord mesenchymal stem cell factor frozen powder, and specific technical means are as follows.

Preferably, it is produced by the above production method.

The present invention provides the utilization of the umbilical cord mesenchymal stem cell factor frozen powder, and specific technical means are as follows.

Umbilical cord mesenchymal stem cell factor frozen powder is used for skin whitening, anti-aging, and hair loss prevention.

The present invention obtains umbilical mesenchymal stem cell factors through umbilical mesenchymal stem cell condition medium. Among them, by using umbilical mesenchymal stem cells, social ethics disputes can be avoided, proliferation is fast, and immune rejection reactions are small. The stem cell factor obtained by culturing umbilical mesenchymal stem cells by the above method of the present invention and the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared as a suitable freeze-dried protectant are beneficial for long-term and stable preservation of stem cell factors, and anti-aging Has efficacy.

Figure 1 is a diagram showing the effect of each concentration of lyophilized protective agent on the lyophilized sample form.
Figure 2 is a DSC detection diagram of a lyophilized sample (MSC-CM + 6% mannitol).
Figure 3 is a diagram showing the effect of each sample group on the moisture content in the mouse skin.
Figure 4 is a diagram showing the effect of each sample group on the hydroxyproline content in mouse skin.
Fig. 5 is a diagram showing the effect of each sample group on SOD activity in mouse skin.
Figure 6 is a mouse skin HE staining diagram.
Figure 7 is a diagram showing the effect of each sample on the dermal thickness of the mouse skin.
Figure 8 is a mouse skin MASSON staining diagram.
Figure 9 is a diagram showing the effect of each sample on the collagen content of the mouse skin.

The present invention provides a method for preparing umbilical mesenchymal stem cell factor freeze-dried powder, in order to make the subject, technical means, and effects of the present invention more clear, the present invention will be described in more detail with reference to Examples below. To As can be seen, the embodiments described herein are for illustrative purposes only and are not intended to limit the invention. All of the various reagents used in the examples are commercially available products.

Hereinafter, the present invention will be described in full with reference to specific embodiments.

Example 1

In this embodiment, a method of manufacturing umbilical mesenchymal stem cell factor freeze-dried powder is provided, and specifically consists of the following steps.

(1) Preparation of umbilical mesenchymal stem cell factor

Wharton's salinity is taken from the umbilical cord, and umbilical mesenchymal stem cells are obtained through in vitro culture and labeled as P0. After passage 5, that is, after P5, when the cells grow 75 to 85%, change to a serum-free blank medium, and continue culture for 72 h, and then the supernatant is collected. The supernatant was centrifuged for 5 min at 1000 rpm/min under the condition of 4°C, and the supernatant was taken and filtered through a 0.22 μm filter. The supernatant was placed in a 3 KD dialysis bag, and the dialysis bag was put in a PBS solution, dialyzed for 36 h under 4°C conditions, and the dialysis solution was collected to obtain umbilical mesenchymal stem cell factor.

(2) Umbilical cord mesenchymal stem cell factor freeze-dried powder

Add 6 g of mannitol to umbilical cord mesenchymal stem cell factor per 100 ml, and freeze-dry according to the steps below. (1) Put the sample in a 5 ml Bayer bottle, make it 1.5 ml/bottle, (2) lower the cold hydrazine temperature to -80°C, reduce the temperature of the chamber to -40°C, (3) loosen the Bayer bottle Block and put it in a vacuum freeze dryer, keep the frozen state for 4h, (4) lower the pressure to 25 Pa, (5) raise the temperature to -20°C at a rate of 0.5°C/min, and maintain it for 16.5 h, (6) The pressure was lowered to 5 Pa, (7) the temperature was raised to 25° C. at a rate of 0.5° C./min, maintained for 6.5 h, to complete freeze-drying, and (8) the Bayer bottle was capped and taken out.

Example 2

This example provides a method of preparing umbilical mesenchymal stem cell factor freeze-dried powder, which is different from Example 1 in that 0 g of mannitol is added to the umbilical mesenchymal stem cell factor per 100 ml during step (2).

Others are the same as in Example 1.

Example 3

This example provides a method of preparing umbilical mesenchymal stem cell factor freeze-dried powder, which is different from Example 1 in that 0.5 g of mannitol is added to the umbilical mesenchymal stem cell factor per 100 ml during step (2).

Others are the same as in Example 1.

Example 4

This example provides a method for preparing umbilical mesenchymal stem cell factor freeze-dried powder, which is different from Example 1 in that 1 g of mannitol is added to the umbilical mesenchymal stem cell factor per 100 ml during step (2).

Others are the same as in Example 1.

Example 5

This example provides a method of preparing umbilical mesenchymal stem cell factor freeze-dried powder, which is different from Example 1 in that 2 g of mannitol is added to the umbilical mesenchymal stem cell factor per 100 ml during step (2).

Others are the same as in Example 1.

Example 6

Effect evaluation according to different mannitol concentration

The appearance, color, and rehydration properties of the umbilical mesenchymal stem cell factor lyophilized powder in Examples 1 to 5 were evaluated according to the standards in Table 1.

Standards for appearance, color, and rehydration of umbilical mesenchymal stem cells or freeze-dried powder score Exterior Color Rehydration/s 0 to 2 Atrophy++ Separation, upper and lower color difference is remarkable+++
Separation, upper and lower color difference is remarkable++ >120 3 to 5 Atrophy++ 90 to 120 6 to 8 Atrophy+ There is no significant difference in top and bottom color 60 to 90 9-10 satiation Uniform, no color difference 0 to 60

As shown in Figure 1 and Table 2, when the concentration of the freeze-dried protectant mannitol is 0%, 0.5%, and 1%, the shape of the freeze-dried product is poor, a layering phenomenon appears, the sample is contracted, and the satiety is low. , The rehydration time was all 120 s or more, and after the sample was rehydrated, it was colorless, clarified, and transparent, and the score was low, and 2, 2, and 3 points were respectively. When the concentration of mannitol is 2% and 6%, the shape of the freeze-dried product is good, the lyophilized powder is satisfactory, there is no delamination phenomenon, and the rehydration time of the sample is short, so it is about 60 s, and colorless after sample rehydration , Clarity, transparency, high scores, 16 points and 16 points respectively.

Results of comprehensive evaluation of appearance, color, and rehydration for different mannitol content Mannitol content/% Appearance and color Rehydration Comprehensive evaluation 0 One One 2 0.5 One One 2 One 2 One 3 2 8 8 16 6 8 8 16

Example 7

Detection of the eutectic point of freeze-dried samples

6 g of mannitol was added to the umbilical cord mesenchymal stem cell factor freeze-dried powder obtained in Example 2 per 100 ml. Place the crucible on a hundred thousandths of a balance, reset the balance, put a small amount of sample solution into the crucible, accurately measure the weight of the sample, and place the crucible on the detection point of the differential scanning calorimeter. The detection conditions are set, and the system temperature is gradually raised to -50℃ to complete the detection. As shown in Table 2, the eutectic point of the sample is -17℃.

Example 8

A study on the anti-aging efficacy of umbilical mesenchymal stem cell factor and umbilical mesenchymal stem cell factor freeze-dried powder

ICR mice were randomly divided into 5 groups of 8 mice each into a blank group, a model group, MSC-CM, a freeze-dried compound group, and a positive drug group. They feed on normal animal feed and adapt to the environment for a week.

After shaving the abdominal hair of the mouse with barikang, and cleaning the hair with a depilatory agent, a PBS solution of D-Gal (1000mg/kg) was continuously injected into the back of the mouse once a day for 42d, and the injection volume was approximately 200µl. And, weigh once every 6 days, and adjust the dosage according to the body weight. After 24 h of finishing treatment, the neck is cut and killed, and the skin of the hair loss area of the mouse's abdomen is quickly taken. All of the materials used during the experiment are prepared in a sterile state, and all the devices have been sterilized in advance. (1) Blank group: physiological saline is injected every day at a rate of 100 µl/10 g/mouse, and 0.5 ml of physiological saline is applied. (2) Model group: D-Gal of 10% was injected every day at a rate of 100 µl/10 g/mouse, and 0.5 ml of physiological saline was applied to the back of the mouse. (3) MSC-CM group: Every day, 10% D-Gal is injected at a rate of 100 µl/10 g/mouse, and 0.5 ml of MSC-CM is applied to the mouse abdominal skin. (4) Freeze-dried sample group: 100 µl/10 g/mouse of 10% D-Gal was injected daily, 1.5 ml of primary distilled water was added to the lyophilized sample to dissolve, and 0.5 ml of freeze-dried on the skin of mice Apply the solution. (5) Positive control group: 100 µl/10 g/mouse of 10% D-Gal was injected daily, and 0.5 ml of 10 g/L aminoguanidine hydrochloride (AG) solution was applied to the back skin of mice. The moisture content of the skin is measured by the drying method, and the SOD activity of the skin and the content of hydroxyproline are measured with a reagent kit. Conduct HE salting and MASSON staining on the skin section.

The measurement results are shown in Figures 3 to 9. In the model group, skin moisture content, SOD activity, and HYP content in mice were all significantly decreased. After drug administration, both MSC-CM and lyophilized formulation groups had an improvement effect on the damage of the model group. The dermal thickness of the model group was significantly decreased (p<0.001), and the collagen content was significantly decreased (p<0.05). Compared to the model group, MSC-CM and lyophilized formulation group significantly increased the dermal thickness and collagen content of mice (p<0.01). According to in vivo experiments in mice, it was found that both umbilical mesenchymal stem cell factor and umbilical mesenchymal stem cell factor freeze-dried powder had certain anti-aging effects.

Claims (10)

(1) taking Wotton meat from the umbilical cord, obtaining umbilical mesenchymal stem cells (MSC) through in vitro culture, and marking as P0;
(2) 3rd to 5th passage, that is, after P3 to P5, changing to a serum-free blank medium;
(3) continuously incubating for 24 h to 96 h; And
(4) collecting the cell supernatant, centrifuging, dialysis, collecting the residual liquid, and freeze-drying to obtain a lyophilized powder
Umbilical cord mesenchymal stem cell factor manufacturing method of freeze-dried powder comprising a. The method of claim 1,
The passage in step (2) is a method of manufacturing umbilical mesenchymal stem cell factor freeze-dried powder, characterized in that the MSC medium is used. The method of claim 1,
Centrifugation during step (4) is a method of manufacturing umbilical mesenchymal stem cell factor freeze-dried powder, characterized in that the supernatant is centrifuged for 5 to 15 min at 800 to 2000 rpm/min under conditions of 2 to 6°C. The method of claim 1,
Dialysis in step (4) is characterized in that the supernatant is placed in a 3-50 KD dialysis bag, and the dialysis bag is placed in a PBS solution, and the dialysis solution is collected after dialysis for 24 to 48 h under 0 ~ 6°C conditions. Umbilical mesenchymal stem cell factor freeze-dried powder manufacturing method. The method of claim 1,
The lyophilization condition of step (4) is a method of manufacturing umbilical cord mesenchymal stem cell factor freeze-dried powder, characterized in that obtained by lyophilizing by adding a lyophilized protective agent. The method of claim 5,
The lyophilized protective agent of step (4) is a method of manufacturing umbilical mesenchymal stem cell factor freeze-dried powder, characterized in that 0 to 15 g of mannitol is added to the umbilical mesenchymal stem cell factor per 100 ml. The method of claim 6,
The method of manufacturing umbilical mesenchymal stem cell factor freeze-dried powder, characterized in that 0.56 g of mannitol is added to the umbilical mesenchymal stem cell factor per 100 ml of the freeze-dried protective agent in step (4). The method of claim 5,
The lyophilization process of step (4) is
A. Put the sample in a 1-10 ml Bayer bottle, and make 1-5 ml/bottle,
B. Lower the cold hydrazine temperature to -80 ~ -60℃ and the temperature of the chamber to -60 ~ -40℃,
C. Cover the Bayer bottle loosely and put it in a vacuum freeze dryer, and keep freezing for 1 to 6 h,
D. Reduce the pressure to 10 ~ 50 Pa,
E. Raise the temperature to -25 to -17℃ at a rate of 0.5 to 5℃/min, and maintain it for 10 to 20 h,
F. Reduce the pressure to 0 ~ 30 Pa,
G. Raise the temperature to 0 to 25°C at a rate of 0.5 to 5°C/min and maintain 1 to 10 h to complete freeze-drying,
H. Method of manufacturing umbilical mesenchymal stem cell factor freeze-dried powder, characterized in that the cap of Bayer's bottle is blocked and taken out. Use of umbilical mesenchymal stem cell factor freeze-dried powder obtained by the manufacturing method according to any one of claims 1 to 8 during skin whitening, anti-aging, and hair loss drug production. Umbilical cord mesenchymal stem cell factor freeze-dried powder obtained by the production method according to any one of claims 1 to 8.

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