CN118255874B

CN118255874B - Natural active collagen and preparation method thereof

Info

Publication number: CN118255874B
Application number: CN202410693055.9A
Authority: CN
Inventors: 刘永进; 袁军林; 郝丽芳; 冯立莎; 张丁木; 孙珍珍; 韩丽伟; 郭雅楠; 衷鸿宾
Original assignee: BEIJING XINKANGCHEN MEDICAL SCIENCE AND TECHNOLOGY DEVELOPMENT CO LTD
Current assignee: BEIJING XINKANGCHEN MEDICAL SCIENCE AND TECHNOLOGY DEVELOPMENT CO LTD
Priority date: 2024-05-31
Filing date: 2024-05-31
Publication date: 2024-08-20
Anticipated expiration: 2044-05-31
Also published as: CN118255874A

Abstract

The invention relates to the technical field of collagen preparation, in particular to natural active collagen and a preparation method thereof, and fibroblasts are obtained through primary extraction or in vitro induction; adding porous microspheres into the heavy suspension of the fibroblast, and sequentially carrying out rolling incubation and suspension culture; adding an activation culture medium into the culture system, and incubating; adding a stimulation culture medium into the culture system, and introducing an incident frequency current when the stimulation culture medium is added each time; regulating the cell culture supernatant to be acidic; adding composite digestive enzyme into the cell culture supernatant to perform digestion reaction; sequentially carrying out dialysis and ultrafiltration treatment on the cell culture supernatant to obtain a collagen solution; and (3) performing freeze drying treatment to obtain collagen. The invention can obtain the collagen with natural activity at the level of milligram per milliliter, and solves the problems of conventional in vitro culture of fibroblasts and low content of the extracted collagen from cell culture supernatant.

Description

Natural active collagen and preparation method thereof

Technical Field

The invention relates to the technical field of collagen preparation, in particular to natural active collagen and a preparation method thereof.

Background

Collagen is the protein with the greatest content and the greatest distribution in animals, widely exists in connective tissues, skin, bones, viscera, tendons, ligaments, sclera and other parts, and is the main component of extracellular matrix, wherein, the type I collagen is most widely applied. Collagen has the advantages of low immunogenicity, degradation controllability, innocuity and innocuity of degradation products, and the like, and contains RGD (composed of arginine, glycine and aspartic acid) structural domains for promoting cell adhesion and growth, and can induce cell migration and stimulate cell proliferation.

The collagen can be applied to aspects such as collagen covers of eye surgery, collagen sponges in wounds and burns, protein-transmitted micropills and tablets, controlled-release gel prescriptions, transdermal drug delivery controlled-release materials, gene delivery nanoparticles, cell culture matrixes and the like in a drug delivery system. And in tissue engineering, can be used for skin substitutes, bone substitutes, artificial blood vessels, valves, artificial dura mater and the like. The collagen sponge is a novel biomedical material, can effectively promote the formation of capillary vessels, accelerate the growth of granulation tissues, serve as a tissue filler at a defect part to guide the regeneration of tissues and promote the rapid healing of various wound surfaces, and has the effects of stopping bleeding, diminishing inflammation, relieving pain, relieving scars, pigmentation and the like. Therefore, collagen is one of the most useful biomaterials, which was approved earlier by FDA and NMPA, because of its superior biocompatibility and safety.

At present, most of the commercially available collagen is extracted from animal tissues such as calf skin, pig skin, bovine Achilles tendon, bovine bone, rat tail, fish scale and the like, and has the defects of animal-derived virus transmission, immunogenicity and the like although the application is relatively wide.

Collagen genes among the allografts are highly conserved, collagen among different individuals is almost completely consistent, and the problem of immunogenicity hardly exists. Therefore, at present, a plurality of scientific research institutions and marketing companies adopt a human collagen gene recombination expression mode to prepare a recombinant collagen product. However, the collagen obtained by the method is only a single polypeptide chain, has no biological activity of biological macromolecules, and has poor anti-degradation performance.

In addition, a small amount of fully-human collagen exists in the collagen sold in the market, but most of the collagen is extracted from peripheral tissues such as umbilical cord and placenta which are abandoned in medical treatment, the yield is low, the problem that raw materials cannot be stably obtained possibly exists, and the requirement of large-scale use cannot be met.

In order to solve the above problems, a method of cell culture has been proposed to extract collagen from cell supernatants.

For example, in patent publication No. CN101299991a, entitled fibroblast activator and fibroblast activation method, collagen synthesis promoter and collagen synthesis promoting method, and skin aging preventive agent and skin aging preventive method, it is proposed to activate fibroblasts with grape sap as an activator to promote collagen secretion by fibroblasts, but the extraction yield of collagen is only nanogram level, which does not have the feasibility of large-scale production.

In the patent with publication number CN106380512A, the invention is named as a preparation method of collagen from human skin cells, a method for preparing collagen by in vitro culture of skin fibroblasts is also proposed, but the method adopted is a laboratory conventional cell culture method, and the yield of collagen prepared by the technology is not clear, and only proved to be feasible in theory.

In the patent with publication number CN115947790A and with the name of three-type collagen composition prepared by fibroblast extracellular matrix, the proliferation and secretion of fibroblasts are promoted by adding active peptide, but the content of collagen finally obtained by the method is still low, and the content of hydroxyproline in the supernatant of a cell culture solution is only 2.5 mug/mL at most.

At present, the production of protein drugs by means of large-scale cell culture fermentation is a very common technology in the industry, but the large-scale production of collagen by using a cell culture fermentation method has not yet made a breakthrough progress in the academia and the industry, and the yield of collagen may be a main reason for limiting the development of the collagen.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide natural active collagen and a preparation method thereof, wherein the method can obtain the collagen with natural activity at the level of milligrams per milliliter, and solves the problems of conventional in vitro culture of fibroblasts and low content of extracted collagen from cell culture supernatant.

In a first aspect, the present invention provides a method for preparing natural active collagen, comprising the steps of:

s1, cell acquisition: obtaining fibroblasts by primary extraction or in vitro induction;

s2, cell inoculation: adding porous microspheres into the heavy suspension of the fibroblast, and sequentially carrying out rolling incubation and suspension culture;

s3, cell activation: adding an activation culture medium into the culture system, and incubating;

s4, sequentially stimulating: adding a stimulation culture medium into the culture system, and introducing an incident frequency current when the stimulation culture medium is added each time;

s5, regulating the pH value: regulating the cell culture supernatant to be acidic;

s6, removing immunogen: adding composite digestive enzyme into the cell culture supernatant to perform digestion reaction;

s7, extracting and purifying: sequentially carrying out dialysis and ultrafiltration treatment on the cell culture supernatant to obtain a collagen solution;

S8, freeze-drying: and (3) performing freeze drying treatment to obtain collagen.

In the step S1, preferably, when a primary extraction mode is selected, extracting tissue-derived functional cells, and performing primary and passage adherence culture until the culture reaches P3-P10 generation;

when the in vitro induction extraction mode is selected, any one of direct induction of stem cells and reprogramming induction of somatic cells can be selected;

The species sources of the functional cells are not strictly limited, and specifically comprise any one or more of human, non-human primate, pig, cow, sheep, horse, donkey, chicken, duck, goose, fish, shrimp and shellfish; and tissue sources also include, but are not limited to, any one or more of skin, muscle, tendon, fascia, fat, umbilical cord, amniotic membrane, placenta, peripheral blood, blood vessels, bone marrow, dental pulp, and bone.

In step S2, the cell inoculation specifically includes: collecting fibroblasts, re-suspending the fibroblasts in a serum-free culture medium, adding porous microspheres into the cell re-suspension, adding the mixed solution of the cell re-suspension and the porous microspheres into a roller bottle, incubating for 2-4 hours in an incubator with 5% CO ₂ at 37 ℃ to ensure that the cells uniformly grow on the surfaces of the microspheres in an adherence manner, and adding the cell suspension into a 3D bioreactor for suspension culture after the incubation is finished; wherein the serum-free medium comprises any one or more of alpha-MEM, DMEM, and 1640; the porous microsphere comprises any one or more of polymethyl methacrylate, polycaprolactone, poly-L-lactic acid, polyhydroxyalkanoate, polyethylene, polystyrene, citric acid polyester, hydroxyapatite and beta-tricalcium phosphate, and the pore diameter of the porous microsphere is 20-1000 mu m, and is preferably 100-500 mu m; the ratio of the cell heavy suspension to the porous microspheres is based on (0.10-1000.00) x 10 ⁵ cells per gram of porous microspheres, and preferably (10.00-100.00) x 10 ⁵ cells per gram of porous microspheres; in addition, the roller bottle can rotate in the incubator at a speed of 10-100rpm/min during the incubation.

Preferably, in step S3, the activating medium is a serum-free medium including an activating agent, the activating agent includes fibroblast activating protein, fibroblast growth factor and ascorbic acid, wherein the concentration of the fibroblast activating protein is 0.1-5000 μg/mL, and preferably is any value between 100-1000 μg/mL, the concentration of the fibroblast growth factor is 1-10 ⁶ U/mL, and preferably is any value between 1000-10000U/mL, and the concentration of the ascorbic acid is 0.1-5000ug/mL, and preferably is any value between 100-1000 μg/mL; whereas the incubation time is between 2 and 12 hours and preferably any value between 4 and 6 hours.

The fibroblast activation protein can activate proliferation of fibroblast, and the added fibroblast growth factor can further promote mass growth of the cell.

In order to further increase the collagen secretion amount of the fibroblast, in the subsequent cell culture process, an inflammatory growth factor or a pro-inflammatory factor is added, and meanwhile, a proper amount of micro-current is introduced into a culture system, so that the collagen secretion yield of the fibroblast is increased in a sequential stimulation mode.

Specifically, in step S4, when the stimulating medium is a serum-free medium to which a stimulating factor is added, the stimulating factor includes any one or more of IL-6, IL-1 alpha, IL-13, IL-17, TNF-alpha, TGF-beta, phytohemagglutinin, concanavalin A, lipopolysaccharide and blue copper peptide, and the concentration of IL-6, IL-1 alpha, IL-13, IL-17, TNF-alpha and TGF-beta is 1-10 ⁶ U/mL, and preferably any value between 1000-10000U/mL, and the concentration of phytohemagglutinin, concanavalin A, lipopolysaccharide and blue copper peptide is 1-10 ⁶ ng/mL, and preferably any value between 100-1000 ng/mL.

When the stimulation medium is a serum-free medium supplemented with a proinflammatory immune cell culture supernatant, the proinflammatory immune cells comprise any one or more of cytotoxic T lymphocytes, T17 cells, M1 type macrophages and neutrophils, and the volume fraction of the proinflammatory immune cell culture supernatant is from 1 to 100%, and preferably any value between 30 and 60%; the frequency of the stimulation medium is 1 time for every 2-3 d.

The output voltage of the radio frequency current is 1-50V, and is preferably any value between 20-30V, the output current is 0.01-50mA, and is preferably any value between 1-10mA, and the frequency is 1-10MHz, and is preferably any value between 3-6 MHz; the time of the entire sequential stimulation treatment is selected to be 7-14d in terms of cell culture cycles.

In step S5, after the cells are cultured for 7-14 days, sampling and observing that the cells are completely covered on the surfaces of the porous microspheres, collecting cell culture supernatant, centrifuging at 1000-10000rpm for 5-10min, removing the cells, the microspheres, fragments and the like, and then adjusting the pH of the cell culture supernatant to 1.5-2.5 by using 2-5M acetic acid solution.

In step S6, the compound digestive enzyme includes any one or more of pepsin, trypsin, papain and ficin, and the mass concentration of the compound digestive enzyme is 0.01-10%, and preferably 1-5%; the digestion reaction is carried out at a temperature of 4-35 ℃ for 1-24 hours.

Step S7, extracting and purifying cell culture supernatant, firstly, adding the supernatant into a dialysis bag, and dialyzing for 2-3d at 4-25 ℃ by taking PBS buffer solution as external liquid; then adding the supernatant into an ultrafiltration system, removing a large amount of water and inorganic salt, and obtaining liquid which is collagen solution with natural activity, wherein the molecular weight cut-off of a dialysis bag in dialysis is 50kDa, and the molecular weight cut-off of an ultrafiltration membrane in ultrafiltration is 50-100kDa.

In the preferred embodiment of the present invention, in step S8, the collagen solution is first frozen at-80 ℃ for 12-48 hours, and then freeze-dried in a freeze dryer for 12-24 hours, thereby obtaining collagen with natural bioactivity.

In the second aspect, the invention also discloses the natural active collagen prepared by the preparation method, and the natural active collagen is also supposed to belong to the protection scope of the invention. The collagen extracted and prepared by the method provided by the invention can be applied to the fields of medical diagnosis, drug development, tissue repair, tissue engineering, medical cosmetology, life nursing, scientific research reagents and the like.

The preparation method of the natural active collagen has at least the following beneficial effects:

On the basis of large-scale continuous flow cell culture, fibroblast activating protein is adopted to activate proliferation of fibroblasts, and fibroblast growth factors are added to promote mass growth of the cells; after the cells are activated, in order to further increase the collagen secretion amount of the fibroblasts, in the subsequent cell culture process, inflammatory growth factors or pro-inflammatory factors are added into a cell culture medium, and a proper amount of microcurrent is introduced into a culture system, so that the collagen secretion amount of the fibroblasts is increased in a sequential stimulation mode; finally, collagen with natural activity at the level of milligrams per milliliter can be obtained through the processes of immunogenicity removal, extraction, purification and the like, so that the level of the protein medicine prepared by the current cell fermentation is reached, and the problems of conventional in vitro culture of fibroblasts and lower content of collagen extracted from cell culture supernatant are solved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the proliferation activity of fibroblasts of each of the examples and comparative examples of the present invention, wherein groups 1 to 3 are examples 1 to 3, and groups 4 to 6 are comparative examples 1 to 3;

FIG. 2 shows the molecular masses of collagen extracted by each of examples and comparative examples of the present invention, wherein A is a collagen standard, B-D is examples 1-3, and F-H is comparative examples 1-3;

FIG. 3 is an SEM photograph of extracted collagen according to various embodiments of the present invention, wherein A-C are the collagens obtained in examples 1-3.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1. Cell harvesting

Extracting skin functional cells in human foreskin tissues discarded in medical treatment, and carrying out primary and passage adherence culture until the culture is carried out for P3-P10 generation to obtain fibroblasts;

2. Cell seeding

Collecting the fibroblast, re-suspending the fibroblast with alpha-MEM serum-free culture medium, adding polymethyl methacrylate porous microsphere with the pore diameter of 100 μm into the cell re-suspension according to the proportion of 10X 10 ⁵ fibroblast per gram of porous microsphere, adding the mixed solution of the cell re-suspension and the porous microsphere into a roller bottle, rotating and incubating in an incubator at 37 ℃ and 5% CO ₂ at a rotating speed of 20rpm/min for 2 hours, and finally adding the cell suspension into a 3D bioreactor for suspension culture;

3. cell activation

Adding an activation culture medium into a culture system, and incubating for 2 hours, wherein the concentration of fibroblast activation protein in the activation culture medium is 100 mug/mL, the concentration of fibroblast growth factor is 1000U/mL, and the concentration of ascorbic acid is 100 mug/mL;

4. sequential stimulation

After the cells are activated, adding a serum-free culture medium with the volume fraction of 30% of T17 cell pro-inflammatory immune cell culture supernatant into a culture system, adding 1 time every 2d, and introducing an incident frequency current when adding the stimulation culture medium each time, wherein the output voltage of the radio frequency current is 20V, the output current is 2mA, the frequency is 1MHz, and the time of the whole sequential stimulation treatment is 7-14d;

5. PH value adjustment

Adjusting the pH of the cell culture supernatant to 2 using a 2-5M acetic acid solution;

6. Immunogen removal

Adding pepsin and trypsin into the cell culture supernatant, and performing digestion reaction for 6 hours at the temperature of 4 ℃, wherein the mass concentration of the pepsin and the trypsin is 1%;

7. Extraction and purification

Adding the supernatant into a dialysis bag with a molecular weight cut-off of 50kDa, and dialyzing at 22 ℃ for 2 days by taking PBS buffer solution as an external solution; then adding the supernatant into an ultrafiltration system with the molecular weight cutoff of 50kDa, removing a large amount of water and inorganic salt, and obtaining liquid which is collagen solution with natural activity;

8. Freeze-drying

And (3) placing the collagen solution into a freeze dryer for freeze treatment for 12 hours at the temperature of minus 80 ℃ and then placing the collagen solution into the freeze dryer for freeze drying for 12 hours to obtain the collagen with natural biological activity.

Example 2

1. Cell harvesting

Extracting functional cells in human foreskin tissues discarded in medical treatment, and carrying out primary and passage adherence culture until the generation of P3-P10 is cultivated to obtain fibroblasts;

2. Cell seeding

Collecting the fibroblast, re-suspending the fibroblast by using a DMEM serum-free culture medium, adding polymethyl methacrylate porous microspheres with the pore diameter of 150 mu m into the cell re-suspension according to the proportion of 50X 10 ⁵ fibroblasts per gram of porous microspheres, adding the mixed solution of the cell re-suspension and the porous microspheres into a roller bottle, rotating and incubating the roller bottle in an incubator at 37 ℃ and 5% CO ₂ at the rotating speed of 50rpm/min for 3 hours, and finally adding the cell suspension into a 3D bioreactor for suspension culture;

3. cell activation

Adding an activation culture medium into the culture system, and incubating for 6 hours, wherein the concentration of fibroblast activation protein in the activation culture medium is 250 mug/mL, the concentration of fibroblast growth factor is 2000U/mL, and the concentration of ascorbic acid is 50 mug/mL;

4. sequential stimulation

After the cells are activated, adding a serum-free culture medium with the concentration of TNF-alpha and canavalin A of 5000U/mL into a culture system, adding 1 time every 2d, and introducing an incident frequency current when adding a stimulation culture medium each time, wherein the output voltage of the radio frequency current is 30V, the output current is 5mA, the frequency is 6MHz, and the time of the whole sequential stimulation treatment is 7-14d;

5. PH value adjustment

6. Immunogen removal

Adding pepsin and papain into the cell culture supernatant, and performing digestion reaction for 12 hours at 15 ℃, wherein the mass concentration of the pepsin and the papain is 2.5%;

7. Extraction and purification

8. Freeze-drying

Example 3

1. Cell harvesting

2. Cell seeding

Collecting the fibroblast, re-suspending the fibroblast with 1640 serum-free culture medium, adding polymethyl methacrylate porous microsphere with the pore diameter of 500 mu m into the cell re-suspension according to the proportion of 100X 10 ⁵ fibroblast per gram of porous microsphere, adding the mixed solution of the cell re-suspension and the porous microsphere into a roller bottle, rotating and incubating in an incubator at 37 ℃ and 5% CO ₂ for 4 hours at the rotating speed of 100rpm/min, and finally adding the cell suspension into a 3D bioreactor for suspension culture;

3. cell activation

Adding an activation culture medium into a culture system, and incubating for 6 hours, wherein the concentration of fibroblast activation protein in the activation culture medium is 1000 mug/mL, the concentration of fibroblast growth factor is 10000U/mL, and the concentration of ascorbic acid is 5000 mug/mL;

4. sequential stimulation

After the cells are activated, adding a serum-free culture medium of IL-6 and TGF-beta with the concentration of 10000U/mL into a culture system, adding 1 time every 2d, and introducing an incident frequency current when adding a stimulation culture medium each time, wherein the output voltage of the radio frequency current is 30V, the output current is 10mA, the frequency is 6MHz, and the time of the whole sequential stimulation treatment is 7-14d;

5. PH value adjustment

6. Immunogen removal

Adding pepsin and trypsin into the cell culture supernatant, and performing digestion reaction for 24 hours at 20 ℃, wherein the mass concentration of the pepsin and the trypsin is 5%;

7. Extraction and purification

Adding the supernatant into a dialysis bag with a molecular weight cut-off of 50kDa, and dialyzing at 22 ℃ for 3 days by taking PBS buffer solution as an external solution; then adding the supernatant into an ultrafiltration system with the molecular weight cutoff of 50kDa, removing a large amount of water and inorganic salt, and obtaining liquid which is collagen solution with natural activity;

8. Freeze-drying

And (3) placing the collagen solution into a freeze dryer for freeze treatment for 24 hours at the temperature of minus 80 ℃ to obtain the collagen with natural biological activity.

Example 4

Extracting functional cells in the dermis of the SPF-grade miniature pig, performing primary and passage adherence culture, and culturing until the generation of P3-P10 to obtain dermis fibroblast;

Other steps and parameters were substantially the same as in example 3.

Example 5

Adopting an in-vitro induction extraction mode, and adopting a chemical reagent induction mode to obtain the pig umbilical cord mesenchymal stem cells, wherein vitamin C phosphate, alkaline fibroblast factors, transforming growth factors and dexamethasone can be added into a culture solution, and the culture is carried out for 7-10 days to obtain the skin fibroblast cells;

Other steps and parameters were substantially the same as in example 3.

Comparative example 1

In the cell activation process, the activation medium added to the cell culture vessel is a serum-free medium to which only ascorbic acid is added;

Other steps and parameters were substantially the same as in example 3.

Comparative example 2

In the sequential stimulation process, the stimulation medium added into the culture system is a serum-free medium added with IL-10;

Other steps and parameters were substantially the same as in example 3.

Comparative example 3

In the sequential stimulation process, an incident frequency circuit is not connected into the culture system;

Other steps and parameters were substantially the same as in example 3.

Test example 1

Evaluation of fibroblast proliferation Activity

The culture of fibroblasts was carried out according to the culture methods in examples 1 to 3 and comparative examples 1 to 3, and cell culture supernatants were aspirated on days 1, 3, 5, and 7, respectively, and absorbance at a wavelength of 450nm was measured in the supernatants by the CCK-8 method, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the proliferation activity of fibroblasts at day 7 in the culture methods of examples 1 to 3 of the present invention was 2 to 3 times that of the culture methods of comparative examples 1 to 3.

Test example 2

Testing of hydroxyproline content in cell culture supernatants

1. Tissue sample preparation

Sucking the cell culture supernatants of each group of examples 1-5 and comparative examples 1-3, and standing at room temperature for use;

2. measurement of hydroxyproline content of samples of each group using Hydroxyproline (HYP) content measurement kit

2.1 Dilution of standard working fluid

Diluting the standard solution into standard solutions of 7.5, 3.75, 1.875, 0.938, 0.469, 0.234 and 0.117 mug/mL by distilled water;

2.2 The procedure for determination of absorbance values for the standard and each set of samples is shown in table 1.

TABLE 1 test procedure

2.3 Drawing of a Standard Curve

Drawing a standard curve by taking the concentration of the standard solution as an x axis and the father A standard (father A _{Standard of}=A_{Standard tube}-A_{Blank pipe}) as a y axis to obtain an equation y=kx+b;

Substituting father a _measurement (fathera measurement=a _{Measuring tube}-A_{Blank pipe}) into the equation yields x (μg/mL).

2.4 Determination of hydroxyproline content

Tissue hydroxyproline content (μg/g) =x×v _{Sample of}÷（W×V_{Sample of}÷V_{Group handle}) =4x++w;

V _{Sample of}: added sample volume, 0.2mL;

V _{Group handle}: tissue extract volume, 4mL;

w: sample mass.

3 Detection results

The hydroxyproline content in each set of extracted cell culture supernatants is shown in table 2.

TABLE 2 hydroxyproline content in cell culture supernatants of each group

As shown in Table 2, the content of hydroxyproline in the cell culture supernatant in examples 1 to 5 of the present invention can be up to 90. Mu.g/ml or more, and the content of collagen in terms of conversion can be up to milligram per milliliter (the content of collagen is 7.46 times that of hydroxyproline), which is remarkably improved as compared with comparative examples 1 to 3.

Test example 3

The molecular weight of the extracted collagen is determined by SDS-PAGE electrophoresis, and the testing method is as follows:

1. sandwich type vertical plate electrophoresis tank

Before installation, the adhesive tape, the glass plate and the groove are clean and dry, and the glass on the adhesive filling surface is not contacted by hands.

2. Preparation of gel plate

Preparation of the separating gel: preparing 20mL of 10% separating gel, uniformly mixing, adding gel liquid into a gap between a long glass plate and a short glass plate by using an elongated head dropper, taking a small amount of distilled water by using a 1mL syringe, slowly injecting the distilled water along the wall of the long glass plate, and sealing the water by using the height of about 3-4 mm; after about 30min, the boundary between the gel and the water seal layer with different refractive indexes shows that the gel is completely polymerized; the distilled water of the water-seal layer was poured off, and the excess water was sucked off with a filter paper strip.

Preparation of concentrated glue: preparing 10mL of 3% concentrated gel, uniformly mixing, adding the concentrated gel above the polymerized separating gel by using a slender head dropper until the distance is about 0.5cm from the upper edge of the short glass plate, and lightly inserting a sample tank template into the concentrated gel to avoid bringing bubbles; gel polymerization is carried out after about 30min, and the gel is placed for 20-30min. After the gel is solidified, carefully pulling out the sample tank template, sucking the excessive water in the sample groove by using a narrow strip of filter paper, pouring Tris-glycine buffer solution with pH of 8.3 into the upper and lower storage tanks, and preparing for sample adding after the short plate is over about 0.5 cm.

3. Sample processing and sample addition

Each standard protein and the protein to be tested are dissolved by a sample dissolving solution with the concentration of 0.5-1mg/mL, heated by boiling water bath for 3 minutes and cooled to room temperature for standby. The treated sample fluid, if stored for a long period of time, should be heated in a boiling water bath for 1 minute before use to eliminate metastable polymerization.

The sample volume is typically 10-15. Mu.L (i.e., 2-10. Mu.g protein). If the sample is thinner, the sample addition volume can be increased.

And adding a sample to the bottom of the gel concave sample groove by using a micro-injector through a buffer solution, and starting electrophoresis when the sample is added into all the concave sample grooves.

4. Electrophoresis

Opening a switch of the direct-current voltage-stabilizing electrophoresis apparatus, and regulating the current to 10mA at the beginning; when the sample enters the separation gel, the current is regulated to 20-30mA; when the blue dye migrates to the bottom, the current is turned back to zero, turning off the power supply. The fixed plate is pulled out, the glass plate is taken out, a piece of glass is gently pried off and removed by a blade, one corner of the rubber plate is cut off to serve as a mark, and the rubber plate is moved into a large culture dish for dyeing.

5. Dyeing and decoloring

Pouring the dyeing liquid into a culture dish, dyeing for about 1h, rinsing with distilled water for several times, and decolorizing with decolorizing liquid until the protein zone is clear, namely measuring the distance between each strip and the top of gel with a ruler.

6. Photographing

As can be seen from FIG. 2, the collagens extracted in examples 1 to 3 and comparative examples 1 to 3 are type I collagens, each comprising 2 α1 chains and 1 α2 chain, and in SDS-PAGE electrophoresis, there are dimers of peptide chains and a small amount of trimers; the difference is that the amount of type I collagen obtained in comparative examples 1 to 3 is significantly different from that obtained in examples 1 to 3.

Test example 4

The collagen sponge surfaces obtained in examples 1 to 3 were subjected to a metal spraying treatment, photographed under a scanning electron microscope, and the microstructures of the collagen proteins of each group were recorded.

As can be seen from FIG. 3, the morphology of the collagen prepared in examples 1-3 of the present invention may be different due to different freeze drying times and different ice crystal sizes.

In conclusion, the invention adopts a biological, chemical and physical crossing method, and by adopting a large-scale cell continuous flow culture technology and a sequential stimulation scheme, the yield of collagen with complete triple helix bioactivity is obviously improved, the collagen with natural activity at the milligram level per milliliter can be obtained, and the prepared collagen contains 2 alpha 1 chains and 1 alpha 2 chains, and the visible peptide chain dimer and a small amount of peptide chain trimer.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. The preparation method of the natural active collagen is characterized by comprising the following steps:

s8, freeze-drying: freeze drying to obtain collagen;

in step S3, the activation medium is a serum-free medium comprising an activator comprising fibroblast activation protein, fibroblast growth factor and ascorbic acid,

Wherein, the concentration of fibroblast activation protein is 0.1-5000ug/mL, the concentration of fibroblast growth factor is 1-10 ⁶ U/mL, and the concentration of ascorbic acid is 0.1-5000ug/mL;

the incubation time is 2-12h;

In the step S4, the stimulating medium is a serum-free medium added with stimulating factors or a serum-free medium added with a culture supernatant of the proinflammatory immune cells,

The stimulating factors comprise any one or more of IL-6, IL-17, TNF-alpha, TGF-beta and canavalin A, wherein the concentration of IL-6, IL-17, TNF-alpha and TGF-beta is 1-10 ⁶ U/mL, and the concentration of canavalin A is 1-10 ⁶ ng/mL;

The proinflammatory immune cells comprise any one or more of cytotoxic T lymphocytes, T17 cells, M1 type macrophages and neutrophils, and the volume fraction of the proinflammatory immune cell culture supernatant is 1-100%;

the frequency of the stimulation culture medium is 1 time for every 2-3 d;

the output voltage of the radio frequency current is 1-50V, the output current is 0.01-50mA, and the frequency is 1-10MHz;

The time of the sequential stimulation treatment is 7-14d.

2. The method according to claim 1, wherein in step S1, the tissue-derived functional cells are extracted during the primary extraction, and primary and passage adherence culture is performed, so as to culture to P3-P10 generation;

The in vitro induction comprises any one of direct induction of stem cells and reprogramming induction of somatic cells;

the species sources of the functional cells comprise any one or more of human beings, non-human primates, pigs, cattle, sheep, horses, donkeys, chickens, ducks, geese, fish, shrimps and shellfish;

The tissue sources include any one or more of skin, muscle, tendon, fascia, fat, umbilical cord, amniotic membrane, placenta, peripheral blood, blood vessels, bone marrow, dental pulp, and bone.

3. The method according to claim 1, wherein in step S2, the cell seeding comprises: collecting fibroblasts, re-suspending the fibroblasts in a serum-free culture medium, adding porous microspheres into the cell re-suspension, adding the mixed solution of the cell re-suspension and the porous microspheres into a roller bottle, incubating for 2-4h in an incubator with 5% CO ₂ at 37 ℃, and finally adding the cell suspension into a 3D bioreactor for suspension culture;

Wherein the serum-free medium comprises any one or more of alpha-MEM, DMEM, and 1640;

The ratio of the cell heavy suspension to the porous microspheres is (0.10-1000.00) x 10 ⁵ cells per gram of porous microspheres;

The porous microsphere comprises any one or more of polymethyl methacrylate, polycaprolactone, poly-L-lactic acid, polyhydroxyalkanoate, polyethylene, polystyrene, citric acid polyester, hydroxyapatite and beta-tricalcium phosphate, and the pore diameter of the porous microsphere is 20-1000 mu m;

During the incubation, the roller bottle rotates in the incubator at a rotation speed of 10-100 rpm/min.

4. The method according to claim 1, wherein in step S5, the pH of the cell culture supernatant is adjusted to 1.5-2.5 using 2-5M acetic acid solution.

5. The preparation method according to claim 1, wherein in step S6, the complex digestive enzyme includes any one or more of pepsin, trypsin, papain and ficin, and the mass concentration of the complex digestive enzyme is 0.01-10%;

The digestion reaction is carried out at a temperature of 4-35 ℃ for 1-24 hours.

6. The method according to claim 1, wherein in step S7, the dialysis is performed at 4-25 ℃ for 2-3d with PBS buffer as an external dialysis solution;

The molecular weight cut-off of the dialysis bag in the dialysis is 50kDa;

The molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration is 50-100kDa.

7. The method according to claim 1, wherein in step S8, the collagen solution is first subjected to freeze-treatment at-80 ℃ for 12-48 hours, and then subjected to freeze-drying in a freeze dryer for 12-24 hours.