MSC exosome recombinant type 3 humanized collagen

Abstract

The invention discloses an MSC exosome recombinant type III humanized collagen which comprises the following components in percentage by volume: 70%-90% of recombinant type III humanized collagen liquid, 5%-15% of MSC exosome, 1%-8% of trehalose, 0.5%-4% of carbomer and 0.5%-5% of a humectant, and the recombinant type III humanized collagen liquid, the MSC exosome, the trehalose, the carbomer and the humectant are prepared from the following components in percentage by volume: 70%-90% of the recombinant type III humanized collagen liquid, 5%-15% of the MSC exosome, 1%-8% of the trehalose, 0.5%-4% of the carbomer and 0.5%-5% of the humectant. The sum of the proportions of all the components is 100%. According to the MSC exosome recombinant three-type humanized collagen, the repair and regeneration capacity of the collagen can be enhanced by combining the MSC exosome with the recombinant three-type humanized collagen, and bioactive molecules in the exosome can promote growth and repair of cells, so that the effect of the collagen is improved; when the product is smeared on the skin surface needing to be repaired, the repairing and regeneration speed of skin tissue can be increased, the repairing effect is improved, and the time period needed by repairing is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen, in particular to MSC exosome recombinant type III humanized collagen. Background Art

[0002] Recombinant humanized type III collagen is a collagen produced through bioengineering technology. It simulates the structure and function of type III collagen naturally existing in the human body. The advantage of recombinant humanized type III collagen is that it has good hydrophilicity and can firmly bind a large number of water molecules, which helps to maintain skin elasticity and tissue metabolism. It also has high activity and high compatibility and will not cause rejection and allergic reactions. At the same time, recombinant humanized type III collagen is widely used in the field of skin care. It can tighten the skin, anti-aging, repair the skin barrier, moisturize and fade scars.

[0003] MSC exosomes are small vesicles secreted by MSC cells. They contain a variety of bioactive molecules, such as proteins, lipids and nucleic acids. These molecules can regulate intercellular communication and affect various biological processes such as cell proliferation, differentiation, migration and apoptosis.

[0004] In today's skin care industry or skin treatment institutions, recombinant humanized type III collagen is usually used in drugs or skin care products to repair the skin. Although the addition of recombinant humanized type III collagen can tighten the skin, anti-aging, repair the skin barrier, moisturize and reduce scars, this repair method takes a long time to show up, and the skin repair effect is not very good. For this reason, we proposed a MSC exosome recombinant humanized type III collagen. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the deficiencies raised by the above background technology, the present invention provides MSC exosomes recombinant type III humanized collagen, which solves the problems raised by the above background technology.

[0007] (II) Technical solution

[0008] To achieve the above object, the present invention provides the following technical solution: MSC exosome recombinant type III humanized collagen, comprising the following components: recombinant type III humanized collagen solution, MSC exosomes, trehalose, carbomer and moisturizer, wherein the volume fraction of each component is recombinant type III humanized collagen solution (70%-90%), MSC exosomes (5%-15%), trehalose (1%-8%), carbomer (0.5%-4%) and moisturizer (0.5%-5%), and the total ratio of each component is 100%;

[0009] In the above scheme, trehalose has excellent properties of maintaining cell viability and biomacromolecule activity, and trehalose can maintain cell activity and form a special protective film on the cell surface;

[0010] Carbomer is a very important rheology regulator that can produce a good thickening effect at a lower dosage.

[0011] Preferably, in terms of volume percentage, the proportions of the components are as follows: 80% recombinant type III humanized collagen solution, 10% MSC exosomes, 5% trehalose, 2% carbomer and 3% moisturizer, and the total proportion of the components is 100%.

[0012] Preferably, the moisturizing agent is any one or both of butylene glycol and glycerol.

[0013] Preferably, the method for preparing MSC exosomes comprises the following steps:

[0014] S1. Inoculating mesenchymal stem cells into a first culture medium for a first expansion culture. When the cells are fused to about 75%-85%, transferring them to a second culture medium for a second expansion culture. After the second expansion culture of the cells is completed, collecting the supernatant to obtain a supernatant containing exosomes.

[0015] S2, first centrifuging the supernatant obtained in step 1 for the first time, and collecting the supernatant after the first centrifugation, then centrifuging the supernatant after the first centrifugation for the second time, and collecting the supernatant after the second centrifugation, and finally centrifuging the supernatant after the second centrifugation for the third time, and collecting the supernatant after the third centrifugation;

[0016] S3. Mix the supernatant after the third centrifugation in step 2 with the extraction reagent, and centrifuge, collect and precipitate the extract to obtain MSC exosomes.

[0017] Preferably, in step 1, the mesenchymal stem cells are derived from human umbilical cord blood.

[0018] Preferably, in step 1, the first culture medium is a low-sugar DMEM culture medium, and the second culture medium is a serum-free culture medium.

[0019] Preferably, in step 2, the supernatant is centrifuged three times for about 30 minutes each.

[0020] Preferably, the method for preparing the MSC exosome recombinant type III humanized collagen comprises the following steps:

[0021] (1) Put the corresponding volume fraction of recombinant type III humanized collagen solution into a mixing container, and slowly add MSC exosomes to allow the two to be fully mixed;

[0022] (2) adding trehalose and a moisturizing agent to the mixed solution of step (1) in sequence, stirring the mixed solution, and heating and keeping the mixture warm for a period of time after the stirring is completed;

[0023] (3) Cooling the liquid after the heat preservation in step (2) and slowly adding carbomer so that the liquid gradually becomes viscous, thereby obtaining an MSC exosome recombinant type III humanized collagen product.

[0024] Preferably, in step (2), the mixed solution is stirred using a magnetic stirrer.

[0025] Preferably, in step (3), the liquid needs to be filtered after becoming viscous.

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the present invention provides MSC exosomes recombinant type III humanized collagen, which has the following beneficial effects:

[0028] The MSC exosome recombinant type III humanized collagen can enhance the repair and regeneration ability of collagen by combining MSC exosomes with recombinant type III humanized collagen. This is because the bioactive molecules in the exosomes can promote cell growth and repair, thereby improving the effect of collagen. When the product is applied to the skin surface that needs repair, it can speed up the repair and regeneration of skin tissue, which not only improves the repair effect but also shortens the time period required for repair. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment 1:

[0031] MSC exosome recombinant type III humanized collagen, including the following ingredients: recombinant type III humanized collagen solution, MSC exosomes, trehalose, carbomer and moisturizer (the moisturizer is any one or two of butylene glycol and glycerol), and the proportion of each ingredient is as follows in terms of volume fraction percentage: 85% of recombinant type III humanized collagen solution, 5% of MSC exosomes, 5% of trehalose, 2% of carbomer and 3% of moisturizer, and the total proportion of each ingredient is 100%;

[0032] The method for preparing MSC exosomes comprises the following steps:

[0033] S1. Inoculate mesenchymal stem cells derived from human umbilical cord blood into a low-sugar DMEM medium for the first expansion culture. When the cells are fused to about 75%-85%, transfer them to a serum-free medium for the second expansion culture. After the second expansion culture of the cells is completed, collect the supernatant to obtain the supernatant containing exosomes;

[0034] S2, first centrifuging the supernatant obtained in step 1 for about 30 minutes, and collecting the supernatant after the first centrifugation, then centrifuging the supernatant after the first centrifugation for about 30 minutes, and collecting the supernatant after the second centrifugation, and finally centrifuging the supernatant after the second centrifugation for a third time for about 30 minutes, and collecting the supernatant after the third centrifugation;

[0035] S3. Mix the supernatant after the third centrifugation in step 2 with the extraction reagent, and centrifuge, collect and precipitate the extract to obtain MSC exosomes.

[0036] The preparation method of MSC exosome recombinant type III humanized collagen product comprises the following steps:

[0037] (1) Put the corresponding volume fraction of recombinant type III humanized collagen solution into a mixing container, and slowly add MSC exosomes to allow the two to be fully mixed;

[0038] (2) adding trehalose and a moisturizing agent to the mixed solution of step (1) in sequence, and stirring the mixed solution with a magnetic stirrer. After the stirring is completed, heating and keeping the mixed solution warm for a period of time;

[0039] (3) Cooling the liquid after the heat preservation in step (2), and slowly adding carbomer to make the liquid gradually become viscous, and then filtering it to obtain the MSC exosome recombinant type III humanized collagen product.

[0040] Embodiment 2:

[0041] MSC exosome recombinant type III humanized collagen, including the following ingredients: recombinant type III humanized collagen solution, MSC exosomes, trehalose, carbomer and moisturizer (the moisturizer is any one or two of butylene glycol and glycerol), and the proportion of each ingredient is as follows in terms of volume fraction percentage: 80% of recombinant type III humanized collagen solution, 10% of MSC exosomes, 5% of trehalose, 2% of carbomer and 3% of moisturizer, and the total proportion of each ingredient is 100%;

[0042] The method for preparing MSC exosomes comprises the following steps:

[0043] S1. Inoculate mesenchymal stem cells derived from human umbilical cord blood into a low-sugar DMEM medium for the first expansion culture. When the cells are fused to about 75%-85%, transfer them to a serum-free medium for the second expansion culture. After the second expansion culture of the cells is completed, collect the supernatant to obtain the supernatant containing exosomes;

[0044] S2, first centrifuging the supernatant obtained in step 1 for about 30 minutes, and collecting the supernatant after the first centrifugation, then centrifuging the supernatant after the first centrifugation for about 30 minutes, and collecting the supernatant after the second centrifugation, and finally centrifuging the supernatant after the second centrifugation for a third time for about 30 minutes, and collecting the supernatant after the third centrifugation;

[0045] S3. Mix the supernatant after the third centrifugation in step 2 with the extraction reagent, and centrifuge, collect and precipitate the extract to obtain MSC exosomes.

[0046] The preparation method of MSC exosome recombinant type III humanized collagen product comprises the following steps:

[0047] (1) Put the corresponding volume fraction of recombinant type III humanized collagen solution into a mixing container, and slowly add MSC exosomes to allow the two to be fully mixed;

[0048] (2) adding trehalose and a moisturizing agent to the mixed solution of step (1) in sequence, and stirring the mixed solution with a magnetic stirrer. After the stirring is completed, heating and keeping the mixed solution warm for a period of time;

[0049] (3) Cooling the liquid after the heat preservation in step (2), and slowly adding carbomer to make the liquid gradually become viscous, and then filtering it to obtain the MSC exosome recombinant type III humanized collagen product.

[0050] Embodiment three:

[0051] MSC exosome recombinant type III humanized collagen, including the following ingredients: recombinant type III humanized collagen solution, MSC exosomes, trehalose, carbomer and moisturizer (the moisturizer is any one or two of butylene glycol and glycerol), and the proportion of each ingredient is as follows in terms of volume fraction percentage: recombinant type III humanized collagen solution 75%, MSC exosomes 15%, trehalose 5%, carbomer 2% and moisturizer 3%, and the total proportion of each ingredient is 100%;

[0052] The method for preparing MSC exosomes comprises the following steps:

[0053] S1. Inoculate mesenchymal stem cells derived from human umbilical cord blood into a low-sugar DMEM medium for the first expansion culture. When the cells are fused to about 75%-85%, transfer them to a serum-free medium for the second expansion culture. After the second expansion culture of the cells is completed, collect the supernatant to obtain the supernatant containing exosomes;

[0054] S2, first centrifuging the supernatant obtained in step 1 for about 30 minutes, and collecting the supernatant after the first centrifugation, then centrifuging the supernatant after the first centrifugation for about 30 minutes, and collecting the supernatant after the second centrifugation, and finally centrifuging the supernatant after the second centrifugation for a third time for about 30 minutes, and collecting the supernatant after the third centrifugation;

[0055] S3. Mix the supernatant after the third centrifugation in step 2 with the extraction reagent, and centrifuge, collect and precipitate the extract to obtain MSC exosomes.

[0056] The preparation method of MSC exosome recombinant type III humanized collagen product comprises the following steps:

[0057] (1) Put the corresponding volume fraction of recombinant type III humanized collagen solution into a mixing container, and slowly add MSC exosomes to allow the two to be fully mixed;

[0058] (2) adding trehalose and a moisturizing agent to the mixed solution of step (1) in sequence, and stirring the mixed solution with a magnetic stirrer. After the stirring is completed, heating and keeping the mixed solution warm for a period of time;

[0059] (3) Cooling the liquid after the heat preservation in step (2), and slowly adding carbomer to make the liquid gradually become viscous, and then filtering it to obtain the MSC exosome recombinant type III humanized collagen product.

[0060] By applying the MSC exosome recombinant type III humanized collagen products prepared by the methods of the above-mentioned Examples 1, 2 and 3 to people whose skin needs to be repaired, and comparing the use of the products for a period of time, it can be seen that the MSC exosome recombinant type III humanized collagen product prepared by the method of Example 2 has a short skin repair cycle and the best repair effect, and the comparison results of Examples 1, 2 and 3 are shown in the following table:

[0061] Skin repair cycle Skin repair effect Embodiment 1 Longer better Embodiment 2 shortest most Embodiment 3 Shorter better

[0062] In the above scheme, it should be noted that: in terms of volume fraction percentage, the ratio of each component is as follows: 80% recombinant type III humanized collagen solution, 10% MSC exosomes, 5% trehalose, 2% carbomer and 3% moisturizer, and the total ratio of each component is 100%. The prepared MSC exosome recombinant type III humanized collagen product has a short repair cycle for the skin and the best repair effect.

[0063] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. MSC exosomes recombinant humanized type III collagen, characterized in that: The invention comprises the following ingredients: recombinant humanized type III collagen solution, MSC exosomes, trehalose, carbomer and moisturizer, wherein the volume fraction of each ingredient is recombinant humanized type III collagen solution (70%-90%), MSC exosomes (5%-15%), trehalose (1%-8%), carbomer (0.5%-4%) and moisturizer (0.5%-5%), and the total proportion of each ingredient is 100%.

2. The MSC exosome recombinant type III humanized collagen according to claim 1, characterized in that: In terms of volume percentage, the proportions of the components are as follows: 80% recombinant type III humanized collagen solution, 10% MSC exosomes, 5% trehalose, 2% carbomer and 3% moisturizer, and the total proportion of the components is 100%.

3. The MSC exosome recombinant type III humanized collagen according to claim 1, characterized in that: The moisturizing agent is any one or two of butylene glycol and glycerol.

4. The MSC exosome recombinant type III humanized collagen according to claim 1, characterized in that: The method for preparing MSC exosomes comprises the following steps: S1. Inoculating mesenchymal stem cells into a first culture medium for a first expansion culture. When the cell fusion reaches about 75%-85%, transferring them to a second culture medium for a second expansion culture. After the second expansion culture of the cells is completed, collecting the supernatant to obtain a supernatant containing exosomes. S2, first centrifuging the supernatant obtained in step 1 for the first time, and collecting the supernatant after the first centrifugation, then centrifuging the supernatant after the first centrifugation for the second time, and collecting the supernatant after the second centrifugation, and finally centrifuging the supernatant after the second centrifugation for the third time, and collecting the supernatant after the third centrifugation; S3. Mix the supernatant after the third centrifugation in step 2 with the extraction reagent, and centrifuge, collect and precipitate the extract to obtain MSC exosomes.

5. The MSC exosome recombinant type III humanized collagen according to claim 4, characterized in that: In step 1, the mesenchymal stem cells are derived from human umbilical cord blood.

6. The MSC exosome recombinant type III humanized collagen according to claim 4, characterized in that: In step 1, the first culture medium is a low-glucose DMEM culture medium, and the second culture medium is a serum-free culture medium.

7. The MSC exosome recombinant type III humanized collagen according to claim 4, characterized in that: In step 2, the supernatant is centrifuged three times for about 30 minutes each.

8. The MSC exosome recombinant type III humanized collagen according to claim 1, characterized in that: The method for preparing MSC exosome recombinant type III humanized collagen comprises the following steps: (1) Place the corresponding volume fraction of recombinant type III humanized collagen solution into a mixing container and slowly add MSC exosomes to allow the two to be fully mixed; (2) adding trehalose and a moisturizing agent to the mixed solution of step (1) in sequence, and stirring the mixed solution. After the stirring is completed, heating and keeping the mixed solution warm for a period of time; (3) Cooling the liquid kept warm in step (2) and slowly adding carbomer so that the liquid gradually becomes viscous, thereby obtaining a MSC exosome recombinant type III humanized collagen product.

9. The MSC exosome recombinant type III humanized collagen according to claim 8, characterized in that: In step (2), the mixed liquid is stirred using a magnetic stirrer.

10. The MSC exosome recombinant type III humanized collagen according to claim 8, characterized in that: In step (3), the liquid needs to be filtered after it becomes viscous.