CN112602704B - Mesenchymal stem cell factor freeze-drying protective agent and application thereof - Google Patents

Abstract

The invention discloses a protective agent for mesenchymal stem cell factor freeze-drying, which comprises the following raw materials in parts by weight: 1-5 parts of modified wheat gluten, 1-5 parts of ethyl myristate, 0.5-1 part of calcium hydrophosphate, 0.3-0.5 part of furanone and 0.1-0.5 part of tea polyphenol; the preparation process of the modified wheat gluten comprises the following steps: (1) adding wheat gluten into the purified water, adjusting pH to 7-7.5, adding glutamine transaminase, and hydrolyzing at 45-50 deg.C for 1-2 hr; (2) and (2) sterilizing the hydrolysate obtained in the step (1), adding a zymophyte agent into the hydrolysate for fermentation, centrifuging the fermentation liquor, and freeze-drying the fermentation liquor to obtain the modified wheat gluten. The protective agent provided by the invention effectively protects the activity of the cell factor and improves the long-term storage stability of the cell factor product through the synergistic effect of the modified wheat gluten, the ethyl myristate, the calcium hydrogen phosphate, the tea polyphenol and the furanone. The invention also provides the application of the protective agent, and the protective agent is used in the freeze-drying process of the adipose mesenchymal stem cell factor, so that the activity of the cell factor in the freeze-dried powder and the stability of long-term storage are obviously improved.

Description

A kind of protective agent for mesenchymal stem cell factor freeze-drying and its application

technical field

The invention relates to the field of stem cells, in particular to a protective agent for mesenchymal stem cell factor freeze-drying and application thereof.

Background technique

Mesenchymal stem cells (MSCs) are a type of mesenchymal stem cells and ectoderm derived from the prenatal embryonic development stage, and exist in a variety of tissues and organs, such as bone marrow, adipose tissue, muscle tissue, lung, liver, pancreas and synovial membrane, etc. MSCs have multidirectional differentiation ability and paracrine effect. They can differentiate into adipocytes, chondrocytes, and solid cells under the action of different inducing factors; they can also secrete a variety of cytokines, including chemokines, adhesion molecules, etc. , In recent years, studies have shown that various bioactive factors secreted by stem cells can be used as a new cell-free biological regulation mode to promote or complete the signal transduction between cells, so as to respond to changes in the body's physiological or pathological state, thereby affecting the body. The stability of the environment plays an important regulatory role.

Adipose-derived mesenchymal stem cells refer to MSCs existing in adipose tissue, which have the potential of self-renewal and multi-directional differentiation, and can differentiate into osteocytes, chondrocytes and adipocytes. Because adipose tissue is almost all over the body, it has strong plasticity. At present, the composite active factor of adipose-derived mesenchymal stem cells is obtained by culturing mesenchymal stem cells in a specific medium and induction conditions, and purifying and concentrating the supernatant. Liquid cytokines bring great inconvenience to transportation and storage, and also limit the application of cytokines. The prior art discloses the application of cytokines after freeze-drying, and the freeze-dried powder is easily transported and stored. The drying method will cause the inactivation of cytokines. The lyophilized cytokine powder obtained by freeze-drying has poor activity and is unstable in long-term storage, which affects the application effect of cytokine products.

Therefore, in order to ensure the activity of the cytokine freeze-dried powder and preserve the repair and regeneration function of the cytokine to the greatest extent, a protective agent can be added during the freeze-drying process of the mesenchymal stem cell factor to develop an adipose-derived mesenchyme suitable for clinical application and large-scale industrialization. Stem cell compound factor products.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a protective agent for mesenchymal stem cell factor freeze-drying, which can avoid the inactivation of cytokines due to changes in temperature and pressure during the freeze-drying process, and improve the freeze-dried products. cytokine content.

The second purpose of the present invention is to provide an application of a mesenchymal stem cell factor freeze-drying protection agent.

One of the objects of the present invention adopts the following technical scheme to realize:

A protective agent for mesenchymal stem cell factor freeze-drying, comprising the following raw materials in parts by weight: 1-5 parts of modified gluten, 1-5 parts of ethyl myristate, 0.5-1 part of calcium hydrogen phosphate, furanone 0.3-0.5 parts, tea polyphenols 0.1-0.5 parts;

The preparation process of the modified gluten is as follows:

(1) Add gluten to purified water, adjust the pH to 7-7.5, add transglutaminase, and hydrolyze at 45-50°C for 1-2h;

(2) sterilizing the hydrolyzed solution in step (1), adding a fermenting bacterial agent for fermentation, centrifuging the fermented solution and then freeze-drying to obtain modified gluten.

Further, the mass ratio of the gluten and transglutaminase is 1:0.15-0.35.

Further, the fermentation bacterial agent is Lactobacillus plantarum, and the fermentation conditions are fermented at 37° C. for 40 hours.

Further, the concentration of gluten in the purified water in step (1) is 20-25wt%.

The second purpose of the present invention adopts the following technical scheme to realize:

The application of the above-mentioned protective agent for mesenchymal stem cell factor freeze-drying includes the following steps:

(1) Acquisition of adipose-derived mesenchymal stem cells: The isolated adipose tissue is resuspended in a medium for primary culture after enzymatic hydrolysis, and subculture is performed when the cell confluence reaches 80-90%;

(2) Collection of cell culture medium: when the confluence of subcultured cells is 75-85%, it is replaced with serum-free DMEM/F12 medium, starved for 18-24 hours, and the supernatant is collected and filtered to obtain cell culture medium;

(3) passing the cell culture fluid of step (2) through a 40KD ultrafiltration membrane, collecting the dialysate, then passing the dialysate through a 2KD ultrafiltration membrane, collecting the retentate, adding a protective agent to the retentate and freeze-drying to obtain a freeze-dried solution dry powder.

Further, 10-15g of protective agent is added per 100mL of retentate.

Further, in the step (1), the culture medium comprises basal medium DMEM/F12, insulin 25-30 μg/mL in the final concentration of culture medium, human albumin 40-50 μg/mL, glutathione 20- 30 μg/mL, vitamin C 10-15 μg/mL, 2-mercaptoethanol 150-200 μM, aloin 10-15 ng/mL.

Further, the freeze-drying process of step (3) is as follows: pre-freeze at -80°C for 5-8h under normal pressure, put the pre-frozen sample into a freeze dryer at a pressure of 20-40Pa, and a temperature of -40-20°C Freeze dry for 20-25h.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a protective agent for mesenchymal stem cell factor freeze-drying, which comprises modified gluten, ethyl myristate, calcium hydrogen phosphate, tea polyphenols and The synergistic effect of furanones effectively protects the activity of cytokines, avoids the inactivation of cytokines caused by changes in temperature and pressure in the cell culture medium during freezing and drying, and increases the content of active ingredients in cytokine freeze-dried products. The stability of long-term storage provides guarantee for the application of cytokine products.

The present invention also provides the application of the above-mentioned protective agent for mesenchymal stem cell factor freeze-drying, which is used in the freeze-drying process of the adipose-derived mesenchymal stem cell factor, which effectively avoids the effect of the freeze-drying process on the adipose-derived mesenchymal stem cell factor. damage, improve the activity of cytokines in the lyophilized powder and the stability of long-term storage.

Detailed ways

The present invention will be further described below with reference to the specific embodiments. It should be noted that, on the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments.

Example 1

A protective agent for mesenchymal stem cell factor freeze-drying, which is composed of the following raw materials in parts by weight: 1 part of modified gluten, 1 part of ethyl myristate, 0.5 part of calcium hydrogen phosphate, 0.3 part of furanone, and more 0.1 part of phenol;

The preparation process of modified gluten is as follows:

(1) add gluten to purified water, the concentration of gluten is 20wt%, adjust pH to 7 and add transglutaminase, the mass ratio of gluten and transglutaminase is 1:0.15, at 45 ℃ Hydrolysis for 2h;

(2) sterilize the hydrolyzed solution in step (1), add Lactobacillus plantarum and ferment at 37° C. for 40 hours, centrifuge the fermented solution and freeze-dry to obtain modified gluten.

The application of the above-mentioned protective agent for mesenchymal stem cell factor freeze-drying includes the following steps:

(1) Acquisition of adipose-derived mesenchymal stem cells: The isolated adipose tissue was resuspended in the medium after enzymatic hydrolysis, and primary culture was carried out in a 10cm dish at 37°C in an incubator with 5% CO ₂ . Culture medium includes basal medium DMEM/F12, insulin 25 μg/mL, human albumin 40 μg/mL, glutathione 20 μg/mL, vitamin C 10 μg/mL, 2-mercaptoethanol 150 μM, aloe vera at final medium concentration When the cell confluence reaches 80%, add 0.25% trypsin to digest it and transfer it to a culture flask for subculture. The ratio of subculture is 1:3, and the concentration of inoculated cells is 1×10 ⁵ cells/mL;

(2) Collection of cell culture medium: when the confluence of P2 generation cells in subculture is 75%, it is replaced with serum-free DMEM/F12 medium, starved for 18 hours, and the supernatant is collected by centrifugation, and filtered through a 0.22 μm microporous membrane. After obtaining the cell culture medium;

(3) passing the cell culture fluid of step (2) through a 40KD ultrafiltration membrane, collecting the dialysate, then passing the dialysate through a 2KD ultrafiltration membrane, collecting the retentate, and adding a freeze-drying protective agent to the retentate, every 100mL Add 10 g of protective agent to the retentate, pre-freeze at -80 ℃ for 5 hours under normal pressure, put the pre-frozen sample into a freeze dryer at a pressure of 20 Pa, and freeze-dry it at a temperature of -40 to 20 ℃ for 25 hours to obtain the product.

Example 2

A protective agent for mesenchymal stem cell factor freeze-drying, which is composed of the following raw materials in parts by weight: 3 parts of modified gluten, 2 parts of ethyl myristate, 0.8 part of calcium hydrogen phosphate, 0.4 part of furanone, and more 0.3 part of phenol;

The preparation process of modified gluten is as follows:

(1) Add gluten to purified water, the concentration of gluten is 23wt%, adjust the pH to 7.5 and add transglutaminase, the mass ratio of gluten and transglutaminase is 1:0.25, at 48°C Hydrolysis for 1.5h;

(2) sterilize the hydrolyzed solution in step (1), add Lactobacillus plantarum and ferment at 37° C. for 40 hours, centrifuge the fermented solution and freeze-dry to obtain modified gluten.

The application of the above-mentioned protective agent for mesenchymal stem cell factor freeze-drying includes the following steps:

(1) Acquisition of adipose-derived mesenchymal stem cells: The isolated adipose tissue was resuspended in the medium after enzymatic hydrolysis, and primary culture was carried out in a 10cm dish at 37°C in an incubator with 5% CO ₂ . Culture medium includes basal medium DMEM/F12, insulin 28 μg/mL at final medium concentration, human albumin 45 μg/mL, glutathione 25 μg/mL, vitamin C 12 μg/mL, 2-mercaptoethanol 180 μM, aloe vera When the cell confluence reaches 85%, add 0.25% trypsin to digest it and transfer it to a culture flask for subculture. The ratio of subculture is 1:3, and the concentration of inoculated cells is 1×10 ⁵ cells/mL;

(2) Collection of cell culture medium: when the confluence of the subcultured P3 cells was 80%, it was replaced with serum-free DMEM/F12 medium, starved for 20 hours, and the supernatant was collected by centrifugation, and filtered through a 0.22 μm microporous membrane. After obtaining the cell culture medium;

(3) passing the cell culture fluid of step (2) through a 40KD ultrafiltration membrane, collecting the dialysate, then passing the dialysate through a 2KD ultrafiltration membrane, collecting the retentate, and adding a freeze-drying protective agent to the retentate, every 100mL Add 12g of protective agent to the retentate, pre-freeze at -80°C for 7h under normal pressure, put the pre-frozen sample into a freeze dryer at a pressure of 30Pa, and freeze-dry it at -40-20°C for 23h to obtain the product.

Example 3

A protective agent for mesenchymal stem cell factor freeze-drying, which is composed of the following raw materials in parts by weight: 5 parts of modified gluten, 5 parts of ethyl myristate, 1 part of calcium hydrogen phosphate, 0.5 part of furanone, tea polyphenol 0.5 part of phenol;

The preparation process of modified gluten is as follows:

(1) Add gluten in purified water, the concentration of gluten is 25wt%, adjust pH to 7.5 and add transglutaminase, the mass ratio of gluten and transglutaminase is 1:0.35, at 50°C Hydrolysis for 2h;

(2) sterilize the hydrolyzed solution in step (1), add Lactobacillus plantarum and ferment at 37° C. for 40 hours, centrifuge the fermented solution and freeze-dry to obtain modified gluten.

The application of the above-mentioned protective agent for mesenchymal stem cell factor freeze-drying includes the following steps:

(1) Acquisition of adipose-derived mesenchymal stem cells: The isolated adipose tissue was resuspended in the medium after enzymatic hydrolysis, and primary culture was carried out in a 10cm dish at 37°C in an incubator with 5% CO ₂ . Culture medium includes basal medium DMEM/F12, insulin 30 μg/mL, human albumin 50 μg/mL, glutathione 30 μg/mL, vitamin C 15 μg/mL, 2-mercaptoethanol 200 μM, aloe vera at final medium concentration When the cell confluence reaches 90%, add 0.25% trypsin to digest it and transfer it to a culture flask for subculture. The ratio of subculture is 1:3, and the concentration of inoculated cells is 1×10 ⁵ cells/mL;

(2) Collection of cell culture medium: when the subcultured P5 cells are 85% confluent, they are replaced with serum-free DMEM/F12 medium, starved for 24 hours of culture, and the supernatant is collected by centrifugation, filtered through a 0.22 μm microporous membrane obtain cell culture fluid;

(3) passing the cell culture fluid of step (2) through a 40KD ultrafiltration membrane, collecting the dialysate, then passing the dialysate through a 2KD ultrafiltration membrane, collecting the retentate, and adding a freeze-drying protective agent to the retentate, every 100mL Add 15g of protective agent to the retentate, pre-freeze at -80°C for 8h under normal pressure, put the pre-frozen sample into a freeze dryer at a pressure of 40Pa, and freeze-dry at -40-20°C for 20h to obtain the product.

Comparative Example 1

Comparative Example 1 provides a protective agent for mesenchymal stem cell factor freeze-drying, and the difference from Example 1 is that the modified gluten is adjusted to gluten, and the rest are the same as those of Example 1.

Comparative Example 2

Comparative Example 2 provides a protective agent for mesenchymal stem cell factor freeze-drying, which is different from Example 1 in that the modified gluten is omitted, and the rest are the same as Example 1.

Comparative Example 3

Comparative Example 3 provides a protective agent for mesenchymal stem cell factor freeze-drying, and the difference from Example 1 is that ethyl myristate is omitted, and the rest are the same as Example 1.

Comparative Example 4

Comparative Example 4 provides a protective agent for mesenchymal stem cell factor freeze-drying, and the difference from Example 1 is that calcium hydrogen phosphate is omitted, and the rest are the same as Example 1.

Comparative Example 5

Comparative Example 5 provides a protective agent for mesenchymal stem cell factor freeze-drying, and the difference from Example 1 is that calcium hydrogen phosphate is replaced with disodium hydrogen phosphate, and the rest are the same as Example 1.

Comparative Example 6

Comparative Example 6 provides a protective agent for mesenchymal stem cell factor freeze-drying, which is different from Example 1 in that furanone is omitted, and the rest are the same as Example 1.

Comparative Example 7

Comparative Example 7 provides a protective agent for mesenchymal stem cell factor freeze-drying, and the difference from Example 1 is that tea polyphenols are omitted, and the rest are the same as Example 1.

Comparative Example 8

Comparative Example 8 provides a protective agent for mesenchymal stem cell factor freeze-drying, and the difference from Example 1 is that the tea polyphenols are replaced with ascorbic acid, and the rest are the same as those in Example 1.

Select 55 SD rats (male, body weight 200-250g), 5 rats in each group, divided into 11 groups, after anesthetized with 80mg/kg ketamine, the back hair is removed, and the skin of 1.5cm×1.5cm is cut with a scalpel The wound surface was washed with normal saline, and the freeze-dried powders of Examples 1 to 3 and Comparative Examples 1 to 8 were dissolved in water and applied to the wound, and the wound was bandaged with gauze. The dressing was changed once a day, and the average healing time of each group was counted. The results are shown in Table 1.

Table 1

group Average healing time/d Example 1 16 Example 2 15 Example 3 17 Comparative Example 1 twenty three Comparative Example 2 27 Comparative Example 3 25 Comparative Example 4 twenty four Comparative Example 5 twenty two Comparative Example 6 20 Comparative Example 7 twenty three Comparative Example 8 twenty one

It can be seen from Table 1 that the average healing time of the rats using the cytokine freeze-dried powder of Example 1 is shorter than that of Comparative Examples 1 to 8, indicating that the cytokine activity of the product of Example 1 is better. In Comparative Examples 1 to 2, after the modified gluten was replaced with gluten or the modified gluten was omitted, the wound healing time was prolonged, indicating that the activity of cytokines decreased after the composition of the lyophilized protective agent was adjusted. In the ratios 3 to 8, a component in the freeze-drying protective agent is respectively omitted, or after it is replaced with other components, the healing-promoting and effect of the product become poor, indicating that the present invention is achieved by the lyophilization process of the adipose mesenchymal stem cell factor. The protective agent composed of modified gluten, ethyl myristate, calcium hydrogen phosphate, tea polyphenols and furanone is added to effectively avoid the damage to cytokines in the freeze-drying process, and improve the activity of cytokine products after freeze-drying.

Detect the content of fibroblast growth factor (FGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in the lyophilized powder of cytokines in Examples 1 to 3 and Comparative Examples 1 to 8 with ELISA kit respectively, And after 180 days of storage, the test was performed again to observe the changes in the content of the same cytokines. The results are shown in Table 2.

Table 2

As can be seen from Table 2, the cytokine freeze-dried powder in Examples 1 to 3 has little change in content after being stored for 180 days, and the initial concentration of the cytokine freeze-dried powder in Comparative Examples 1 to 8 after freeze-drying is lower than that of the Example 1 to 3, and the content of cytokines decreased significantly after storage for 180 days, indicating that the freeze-drying protective agent of the present invention was added by adding modified gluten, ethyl myristate, calcium hydrogen phosphate, tea polyphenols and furanone, It can not only increase the content of active cytokines after freeze-drying, avoid the damage of cytokine activity caused by temperature and pressure changes in the freeze-drying process, but also help maintain the stability of cytokines, which is conducive to the long-term preservation of cytokine products.

The above-mentioned embodiments are only preferred embodiments of the present invention, and cannot be used to limit the scope of protection of the present invention. Any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of the present invention. Scope of protection claimed.

Claims (8)

1. The protective agent for the mesenchymal stem cell factor freeze-drying is characterized by comprising the following raw materials in parts by weight: 1-5 parts of modified wheat gluten, 1-5 parts of ethyl myristate, 0.5-1 part of calcium hydrophosphate, 0.3-0.5 part of furanone and 0.1-0.5 part of tea polyphenol;

the preparation process of the modified wheat gluten comprises the following steps:

(1) adding wheat gluten into the purified water, adjusting pH to 7-7.5, adding glutamine transaminase, and hydrolyzing at 45-50 deg.C for 1-2 hr;

(2) and (2) sterilizing the hydrolysate obtained in the step (1), adding a zymophyte agent into the hydrolysate for fermentation, centrifuging the fermentation liquor, and freeze-drying the fermentation liquor to obtain the modified wheat gluten.

2. The mesenchymal stem cell factor protective agent for freeze-drying according to claim 1, wherein the mass ratio of the vital gluten to the glutamine transaminase is 1: 0.15-0.35.

3. The mesenchymal stem cell factor protective agent for freeze-drying according to claim 1, wherein the zymophyte agent is lactobacillus plantarum, and the fermentation condition is fermentation at 37 ℃ for 40 h.

4. The mesenchymal stem cell factor protective agent for freeze-drying according to claim 1, wherein the concentration of the wheat gluten in the purified water in the step (1) is 20-25 wt%.

5. The use of the mesenchymal stem cell factor cryoprotectant of any one of claims 1 to 4, comprising the steps of:

(1) obtaining adipose-derived mesenchymal stem cells: carrying out enzymolysis on the adipose tissues obtained by separation, then suspending the adipose tissues in a culture medium for primary culture, and carrying out subculture when the cell fusion degree reaches 80-90%;

(2) collecting cell culture solution: replacing with serum-free DMEM/F12 culture medium when the cell fusion degree in subculture is 75-85%, starving and culturing for 18-24h, collecting supernatant, and filtering to obtain cell culture solution;

(3) and (3) passing the cell culture solution obtained in the step (2) through a 40KD ultrafiltration membrane, collecting dialysate, passing the dialysate through a 2KD ultrafiltration membrane, collecting trapped fluid, adding a protective agent into the trapped fluid, and freeze-drying to obtain freeze-dried powder.

6. The application of the mesenchymal stem cell factor protective agent for freeze-drying according to claim 5, wherein 10-15g of the protective agent is added to every 100mL of the retention solution.

7. The mesenchymal stem cell factor protective agent for freeze-drying according to claim 5, wherein the culture medium in the step (1) comprises a basal medium DMEM/F12, 25-30 μ g/mL of insulin, 40-50 μ g/mL of human serum albumin, 20-30 μ g/mL of glutathione, 10-15 μ g/mL of vitamin C, 200 μm of 2-mercaptoethanol and 10-15ng/mL of aloin.

8. The use of the protectant for lyophilization of mesenchymal stem cell factor according to claim 5, wherein the lyophilization process of step (3) is as follows: pre-freezing at-80 ℃ for 5-8h under normal pressure, putting the pre-frozen sample into a freeze dryer, and freeze-drying at-40-20 ℃ for 20-25h under the pressure of 20-40 Pa.