KR102279343B1 - Umbilical cord mesenchymal stem cell factor frozen powder, production method and use thereof - Google Patents

Abstract

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

Description

Umbilical cord mesenchymal stem cell factor frozen powder, production method and use thereof

The present invention relates to the field of biotechnology, and more particularly, to umbilical cord mesenchymal stem cell factor frozen powder, preparation method, and utilization thereof.

During stable growth, mesenchymal stem cells (MSCs) secrete various cell factors. TGF-β (transformation growth factor-β), VEGF (vascular endothelial growth factor), FGF, HGF (hepatic 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 in other aspects. TGF-β1 secreted from MSC reduces the expression of tyrosinase-related proteins through inhibition of TYR (TYR) activity and plays a whitening role in the process, such as 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 and culture of stem cells.

Liquid MSC-CM has a short shelf life at room temperature and is unfavorable to transportation. By making it into a frozen powder through freezing immobilization 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 for preparing frozen umbilical cord mesenchymal stem cell factor frozen powder, which can preserve activity for a long period of time, and to rapidly produce frozen umbilical cord mesenchymal stem cell factor frozen powder having good anti-aging efficacy.

Preferably, the steps below:

(1) Take Wharton's flesh from the umbilical cord, obtain umbilical cord mesenchymal stem cells through in vitro culture, and mark as P0,

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

(3) continuously incubated for 24 h to 96 h,

(4) collection of the cell supernatant, centrifugation, and dialysis, collecting the residual liquid, obtaining umbilical cord mesenchymal stem cell factors, and freeze-drying;

It provides a method for producing umbilical cord mesenchymal stem cell factors comprising the steps.

For the passage in step (2), MSC medium is used.

Centrifugation in step (4): The supernatant is centrifuged for 5-15 min at 800-2000 rpm/min under 2-6°C conditions, and the supernatant is collected.

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

Freeze-drying conditions of step (4): A freeze-drying protective agent is added and lyophilized to obtain.

The freeze-drying protectant: 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 examples, the preferred freeze-drying process is: (1) putting the sample in a 5 ml vial bottle, making it 1.5 ml/bottle, (2) lowering the cold hydrazine temperature to -80°C, and lowering the temperature of the chamber to - Lower the temperature to 40℃, (3) seal the vial and put it in a vacuum freeze dryer, keep the frozen state for 4 hours, (4) lower the pressure to 25 Pa, (5) lower the temperature to -20 at a rate of 0.5℃/min ℃, hold 16.5 h, (6) lower the pressure to 5 Pa, (7) raise the temperature to 25 ℃ at a rate of 0.5 ℃ / min, hold for 6.5 h to complete lyophilization, (8) Close the stopper of the Bayer bottle and take it out.

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

Preferably, it is obtained by manufacturing by the above manufacturing method.

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

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

The present invention obtains the umbilical cord mesenchymal stem cell factor through the umbilical cord mesenchymal stem cell conditioned medium. Among them, due to the use of umbilical cord mesenchymal stem cells, social ethics disputes can be avoided, the proliferation is fast, and the immune exclusion response is small. The stem cell factor obtained by culturing umbilical cord mesenchymal stem cells by the method of the present invention and the freeze-dried powder of the umbilical cord mesenchymal stem cell factor prepared with a suitable freeze-drying protectant are beneficial for long-term and stable preservation of the stem cell factor, and anti-aging have efficacy

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of each concentration of lyophilized protective agent on lyophilized sample form.
Figure 2 is a DSC detection diagram of a lyophilized sample (MSC-CM + 6% mannitol).
3 is a diagram showing the effect of each sample group on the moisture content in the skin of a mouse.
Figure 4 is a diagram showing the effect of each sample group on the hydroxyproline content in the skin of mice.
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.
9 is a diagram showing the effect of each sample on the collagen content of mouse skin.

The present invention provides a method for preparing a freeze-dried powder of umbilical cord mesenchymal stem cell factor. In order to make the task, technical means and effect of the present invention more distinct, the present invention will be described in more detail with reference to Examples below. do it with As will be appreciated, the embodiments described herein are merely illustrative of the present invention and not limiting of the present invention. Various reagents used in the examples are all commercially available products.

Hereinafter, the present invention will be fully described with reference to specific examples.

Example 1

This embodiment provides a method for preparing a freeze-dried powder of umbilical cord mesenchymal stem cell factor, and specifically consists of the following steps.

(1) Production of umbilical cord mesenchymal stem cell factors

Take Wharton's flesh from the umbilical cord, obtain umbilical cord mesenchymal stem cells through in vitro culture, and mark as P0. After passage 5, that is, after P5, when the cells have grown to 75-85%, change to a serum-free blank medium, continue culturing for 72 h, and collect the supernatant. The supernatant was centrifuged for 5 min at 1000 rpm/min under 4°C conditions, and the supernatant was collected and filtered through a 0.22 μm filter. The supernatant is placed in a 3 KD dialysis bag, the dialysis bag is placed in a PBS solution, and the dialysis solution is dialyzed for 36 h under 4°C conditions.

(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 vial bottle, make 1.5 ml/bottle, (2) lower the cold hydrazine temperature to -80°C, lower the temperature of the chamber to -40°C, (3) loosen the vial bottle sealed and put in a vacuum freeze dryer, kept frozen for 4 h, (4) lowered to 25 Pa, (5) raised to -20 °C at a rate of 0.5 °C/min, maintained for 16.5 h, (6) Reduce the pressure to 5 Pa, (7) raise the temperature to 25°C at a rate of 0.5°C/min, hold for 6.5 h, complete freeze-drying, (8) stopper the vial and take it out.

Example 2

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

Other than that, it is the same as Example 1.

Example 3

This example provides a method for preparing the umbilical cord 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 cord mesenchymal stem cell factor per 100 ml during step (2).

Other than that, it is the same as Example 1.

Example 4

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

Other than that, it is the same as Example 1.

Example 5

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

Other than that, it is the same as Example 1.

Example 6

Evaluating the effect of different mannitol concentrations

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

Appearance, color, and rehydration standard of lyophilized powder for umbilical cord mesenchymal stem cells score Exterior Color rehydration/s 0 to 2 atrophy++ Separation, the color difference between the top and bottom is remarkable+++
Separation and color difference between top and bottom are remarkable++ >120 3 to 5 atrophy++ 90 to 120 6 to 8 atrophy+ No significant color difference between the top and bottom 60 to 90 9 to 10 satiation Uniformity, no color difference 0 to 60

As shown in Figure 1 and Table 2, when the freeze-drying protective agent mannitol concentration is 0%, 0.5%, 1%, the shape of the freeze-dried product is bad, a layering phenomenon appears, the sample is atrophied, the satiety is low, and the , the rehydration time is all 120 s or more, after the sample is rehydrated, it is colorless, clarified, transparent, and the score is low, scoring 2, 2, and 3, respectively. When the mannitol concentration is 2% and 6%, the shape of the freeze-dried product is good, the lyophilized powder has good satiety, there is no segregation, the rehydration time of the sample is short, about 60s, and after rehydration of the sample, it is colorless , clarification, and transparent, and the score is high, with 16 and 16 points, respectively.

Comprehensive evaluation of appearance, color, and rehydration properties of different mannitol contents 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

Eutectic point detection of freeze-dried samples

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

Example 8

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

ICR mice were randomly divided into 5 groups of 8 mice each: a blank group, a model group, MSC-CM, a freeze-dried oxalic drug group, and a positive drug group. Feed the animals regular food to acclimatize to the environment for a week.

After shaving the mouse's belly hair with a barricade, and thoroughly treating the hair with a depilatory agent, a PBS solution of D-Gal (1000 mg/kg) was injected subcutaneously once daily for 42 d continuously into the back of the mouse. Weigh the weight once every 6 days, and adjust the dosage according to the body weight. 24 h after the finish treatment, the neck is cut and killed, and the skin of the bald area of the dorsal part of the mouse is taken immediately. All materials used during the experiment are prepared under aseptic conditions, and all devices have been sterilized in advance. (1) Blank group: 100 μl/10 g/animal of physiological saline is injected daily, and 0.5 ml of physiological saline is applied. (2) Model group: 10% D-Gal is injected every day at 100 μl/10 g/mouse, and 0.5 ml of physiological saline is applied to the dorsal skin of mice. (3) MSC-CM group: 100 μl/10 g/mouse of 10% D-Gal is injected every day, and 0.5 ml of MSC-CM is applied to the dorsal skin of mice. (4) Lyophilized sample group: 10% D-Gal is injected daily at 100 μl/10 g/mouse, 1.5 ml of primary distilled water is added to the freeze-dried sample to dissolve, and 0.5 ml of lyophilized on the dorsal skin of mice Apply the solution. (5) Positive control group: 10% D-Gal is injected daily at 100 μl/10 g/mouse, and 0.5 ml of 10 g/L aminoguanidine hydrochloride (AG) solution is applied to the dorsal skin of mice. The moisture content of the skin is measured by a drying method, and the SOD activity and hydroxyproline content of the skin are measured with a reagent kit. HE staining and Masson staining of the skin sections are carried out.

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

Claims (10)

(1) taking Wharton's flesh from the umbilical cord, obtaining umbilical mesenchymal stem cells (MSC) through in vitro culture, and labeling as P0;
(2) changing to serum-free blank medium when the umbilical cord mesenchymal stem cells grow by 75% to 85% after the 3rd to 5th passage using MSC medium, ie, P3 to P5;
(3) continuously incubating for 72 hours; and
(4) taking the lyophilized powder through the following i) to iii) processes
i) Process: centrifuging the cell supernatant at a speed of 1000 rpm/min for 5 minutes under the condition of 4 degrees Celsius to take the supernatant, and filtering with a 0.22 micrometer filter;
ii) Procedure: putting the supernatant into a 3 KD dialysis bag, putting the dialysis bag in PBS solution, and dialysis under the condition of 4 degrees Celsius for 36 hours to collect the dialysate;
iii) process: freeze-drying the collected dialysate to obtain a freeze-dried powder
including,
The freeze-drying process of step iii) belonging to step (4) is
A. Put the sample in a 5 ml vial bottle, make 1.5 ml/bottle,
B. Reduce the temperature of cold hydrazine to -80 degrees Celsius, and lower the temperature of the chamber to -40 degrees Celsius;
C. Loosely close the vial bottle and put it in a vacuum freeze dryer to keep it frozen for 4 hours,
D. Reduce the pressure to 25 Pa,
E. Raise the temperature to -20 °C at a rate of 0.5 °C/min, hold for 16.5 hours,
F. Reduce the pressure to 5 Pa,
G. Raise the temperature to 25°C at a rate of 0.5°C/min and hold for 6.5h to complete lyophilization,
H. Closing and pulling out the Bayer bottle
including,
The freezing condition of step iii) in step (4) is obtained by freeze-drying by adding a freeze-drying protective agent, and the freeze-drying protective agent is to put 6 g of mannitol in the umbilical cord mesenchymal stem cell factor per 100 ml,
The eutectic melting point of the freeze-dried powder prepared through the steps (1) to (4) is -17 degrees Celsius. delete delete delete delete delete delete delete delete delete

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