Semi-biosynthetic fur, and method for preparing the same

Abstract

The application discloses a semi-biosynthetic fur and a preparation method thereof, and the preparation method of the semi-biosynthetic fur comprises S1) obtaining of hair, S2) preparation of a hair bundle, S3) integration and culture of a tissue sheath, S4) synthesis of epidermal cells and S5) combination of fur. In one aspect, the required pinna and down are obtained from animals in a painless and sustainable manner by combing the hair and are composed into a hair bundle, and in the other aspect, stem cells are extracted from the blood of animals and induced pluripotent stem cells are cultivated to differentiate into required cells and tissues to form an artificial skin layer, finally, the hair bundle can be embedded into the artificial skin layer by means of biological bonding and semi-biosynthetic fur is formed, the prepared semi-biosynthetic fur is almost the same as natural fur in performance bionics and the like, compared with traditional artificial fur, the semi-biosynthetic fur is extremely small in environmental ecological pollution, and the semi-biosynthetic fur can reduce animal killing and maintain the rights and interests of employees in the original industry such as animal breeders.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fur, in particular to the technical field of semi-biosynthetic fur and a preparation method thereof.

BACKGROUND

[0002] Natural fur is made of animal fur, which has good warmth retention and beautiful appearance. Among them, fox, mink, raccoon, rabbit, cow and sheep are the main sources of natural fur raw materials. In recent years, under the initiative of animal protectionists, artificial fur is becoming more and more popular. Although there is no conclusion on which is more environmentally friendly between natural fur and artificial fur, compared with natural fur which costs tens of thousands of dollars, artificial fur is more economically acceptable to consumers due to its lower price.

[0003] The traditional preparation method of artificial fur is to weave synthetic fibers such as acrylic, modified acrylic and chlorofiber to form plush fabric. For example, the preparation process of a fox fur-like fabric disclosed in the patent CN111041701A and the preparation method of a artificial fur carpet disclosed in the patent CN111101300B. Although with the upgrading of technology, the texture of artificial fur is becoming more and more realistic, and it can be mistaken for real at a distance, but there is still some difference in appearance as a whole compared with natural fur, and the warmth retention is often not as good as natural fur.

[0004] In addition, because the prices of different natural fur raw materials are quite different, some manufacturers choose to process the fur with lower raw material prices to make artificial fur that can imitate fur with higher raw material prices, so as to greatly reduce the price of artificial fur while ensuring the realistic texture of artificial fur. For example, the goat fur processing technology disclosed in the patent CN105803132A. However, this method still needs to skin and take fur from animals, which is not only cruel in obtaining raw materials, but also may be affected by sales due to opposition from animal protectionists.

SUMMARY

[0005] The purpose of the present application is to solve the problems in the prior art and to provide a semi-biosynthetic fur and a preparation method thereof. The semi-biosynthetic fur prepared by the method has almost the same performance as natural fur in terms of bionics, and has little environmental ecological pollution compared with traditional artificial fur, and can reduce animal slaughter and protect the rights and interests of employees in the original industry such as animal breeders.

[0006] To achieve the above-mentioned purpose, the present application realizes the following technical solutions:

[0007] The preparation method of semi-biosynthetic fur comprises the following steps:

[0008] S1) Hair acquisition: Acquiring raw hair mixture from an animal, and removing impurities from the raw hair mixture to obtain hair;

[0009] S2) Preparation of hair bundle: Sorting the hair obtained in step S1) and obtaining guard hair and under-hair, respectively, and mixing the guard hair and the under-hair in a ratio according to the distribution ratio of the guard hair and the under-hair of the animal itself to obtain a hair bundle;

[0010] S3) Integration and culture of connective tissue sheath (CTS): Uniformly arranging the hair bundle obtained in step S2) on connective tissue sheath cells according to the number of guard hairs per unit area of the animal itself, and culturing until the cells and the hair bundle are integrated;

[0011] S4) Synthesis of epidermal cells: Extracting stem cells from the blood of the animal and reprogramming the stem cells to a pluripotent state, and using the induced pluripotent stem cells to directionally synthesize various types of keratinocytes and fibrocytes constituting the epidermis and / or dermis of the animal;

[0012] S5) Combination of fur: According to the structure of the epidermis and / or dermis of the animal itself, the various types of keratinocytes and fibrocytes obtained in step S4) are sequentially grown on the connective tissue sheath loaded with the hair bundle.

[0013] As preferred, in the step S1), the raw hair mixture is collected from the animal by combing in the molting season, and the raw hair mixture is soaked in an alkaline solution having a pH of 8 to 10 and a temperature of 35 to 45°C for 0.5 to 1.5 hours to remove mixed fat and hair roots.

[0014] As preferred, in the step S3), fibrocytes cultured in vitro are used as a source of connective tissue sheath cells.

[0015] As preferred, in the step S3), DMEM / F-12 medium to which 8 to 12% fetal bovine serum and 0.5 to 1.5% antibiotic-antimycotic agent are added is used as a medium for culturing the connective tissue sheath cells loaded with the hair bundle, and the connective tissue sheath cells loaded with the hair bundle are cultured in a culture environment having a temperature of 36.5 to 37.5°C and a CO2 concentration of 4.5 to 5.5% for 15 to 25 days to allow the connective tissue sheath cells and the hair bundle to interact, while maintaining the cell density of the connective tissue sheath cells at 0.5 x 10 5 ~ 1.5 x 10 5 cm 2 .

[0016] As preferred, the step S4) is specifically as follows:

[0017] S41) isolation of peripheral blood mononuclear cells (PBMCs): peripheral blood mononuclear cells are isolated from the blood of an animal;

[0018] S42) preliminary proliferation of peripheral blood mononuclear cells: peripheral blood mononuclear cells obtained in step S41) are cultured to form a monolayer of adherent cells and to elute non-adherent cells;

[0019] S43) directional culture of peripheral blood mononuclear cells: the monolayer of adherent cells obtained in step S42) is seeded onto a culture medium and subjected to electroporation after addition of reprogramming vectors;

[0020] S44) expansion of peripheral blood mononuclear cells: colonies similar to artificial induced pluripotent stem cells (iPSCs) are manually selected and expanded in a culture medium and periodically subcultured.

[0021] As preferred, in the step S42), StemPro 34SFM medium is used as the culture medium for peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are cultured in a culture environment with a temperature of 36.5-37.5°C and a CO2 concentration of 4.5-5.5%, and non-adherent cells are eluted and the culture medium is replaced periodically using phosphate buffered physiological saline.

[0022] As preferred, in the step S43), the culture medium is StemPro 34SFM medium, the reprogramming vector is Epi5 Episomal reprogramming vector, and P2 Primary Cell Kit is used for electroporation.

[0023] As preferred, in the step S44), the culture medium is mTeSR+ medium, subculture is performed every 4-6 days using GCDR, and the expansion subculture environment is maintained at a temperature of 36.5-37.5°C, a CO2 concentration of 4.5-5.5%, and an O2 concentration of 18-22%.

[0024] As preferred, the method further comprises the following step S6):

[0025] S6) post-processing of the fur: the fur is subjected to nitration treatment.

[0026] The semi-biosynthetic fur is prepared by the preparation method.

[0027] The beneficial effects of the present application are as follows:

[0028] The present application is characterized in that, on one hand, the required pin hair and down are obtained from the animal in a painless and sustainable manner by combing and are composed into a hair bundle, and on the other hand, stem cells are extracted from the blood of the animal and induced to differentiate into required cells and tissues to form an artificial skin layer, finally the hair bundle is embedded in the artificial skin layer formed by bio-adhesion to form a semi-biosynthetic fur, the semi-biosynthetic fur prepared by the present application is almost the same as natural fur in terms of performance and bionics, and has little environmental ecological pollution compared with traditional artificial fur, and can reduce animal slaughter and protect the rights and interests of employees in the original industry such as animal breeders.

[0029] The features and advantages of the present application will be described in detail with reference to the embodiments in conjunction with the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a physical diagram of a hair bundle; DETAILED DESCRIPTION

[0031] Example 1:

[0032] Taking imitation mink fur as an example, the semi-biosynthetic fur is prepared according to the following steps:

[0033] S1) Obtaining hair:

[0034] Since most fur animals are mammals, they have the characteristics of keratin layer change and shedding (molting), so their hair can be combed and collected, thereby changing the value of the skin animal into sustainable and painless use. Specifically, in the molting season (summer), the raw hair mixture (i.e. wool utilization) is collected from the anesthetized mink by combing, and the raw hair mixture is soaked in an alkaline solution with a pH of 9 and a temperature of 40°C for 1 h to remove the mixed fat and hair foot.

[0035] S2) Preparation of hair bundle:

[0036] The hair obtained in step S1) is sorted by manual sorting to obtain pin hair and down, and the pin hair and down are arranged and mixed into a bundle in a proportion according to the distribution ratio of pin hair and down of mink to obtain a hair bundle. Since the distribution ratio of pin hair and down of mink is usually 1:60-70, the pin hair and down are arranged and mixed into a bundle in a proportion of 1:65 to obtain a hair bundle. The physical diagram of the hair bundle containing different numbers of hairs is shown in Figure 1 .

[0037] S3) Integration and culture of tissue sheath:

[0038] The hair bundles obtained in step S2) are arranged uniformly on the tissue sheath cells according to the number of needle hairs per unit area of the mink and are cultured until the cells are integrated with the hair bundles. Since the number of needle hairs per unit area of the mink is 479-540 hairs / cm 2 , the hair bundles are arranged uniformly on the tissue sheath cells at a distribution density of 500 hairs / cm 2 , and are used as the skin cell culture medium. Specifically, a cell culture dish with a diameter of 90 mm is selected and the surface of the cell culture dish is ensured to be clean and sterile. DMEM / F-12 medium added with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic is added, and the tissue sheath cells are added to the medium with a cell density of 1 x 10 5 / cm 2 . The hair bundles are carefully placed on the tissue sheath cells in the cell culture dish using a micromanipulation instrument. Finally, the tissue sheath cells loaded with the hair bundles are cultured in a culture environment with a temperature of 37°C and a CO2 concentration of 5% for 21 days, and the tissue sheath cells are allowed to interact with the hair bundles. In this process, fibroblasts cultured in vitro are used as the source of the tissue sheath cells. During the culture, the medium can be replaced every 3-4 days to maintain nutrient supply, and the growth state of the cells and the integration of the hair bundles are observed periodically using a microscope. If the density of the tissue sheath cells increases due to expansion, the excess tissue sheath cells can be divided into new culture dishes to maintain an appropriate cell density. In addition, after the culture is completed, the interaction between the cells and the hair bundles can be evaluated by cell analysis, microscopic observation, and staining.

[0039] S4) Synthesis of epidermal cells:

[0040] Stem cells are extracted from the blood of the mink and reprogrammed to restore the pluripotent state (iPSC induced pluripotent stem cells). The induced pluripotent stem cells are used to direct the synthesis of the epidermal layer and / or the dermal layer of the mink to form various types of keratinocytes and fibroblasts. The specific steps are as follows:

[0041] S41) Isolation of peripheral blood mononuclear cells:

[0042] The blood of the mink under general anesthesia is aseptically aspirated from the spine and gently shaken. Peripheral blood mononuclear cells are isolated from 50 ml of donor blood using a SepMate tube and transferred to a 50 ml conical centrifuge tube. 5 ml of RPMI 1640 medium is added to the conical centrifuge tube containing the PBMCs and gently shaken.

[0043] S42) Primary proliferation of peripheral blood mononuclear cells:

[0044] PBMCs were transferred into 175 cm 2 vented flasks, cultured in StemPro 34 SFM medium in an incubator under standard conditions (5% CO 2 and 37°C), the medium was removed after 24 h of culture and washed with phosphate buffered saline (PBS) to remove non-adherent cells, while the medium was replaced every 3 days until the adherent cells formed a monolayer, after which the adherent cells were treated with trypsin-EDTA and washed with PBS, then counted and distributed into new 175 cm 6 vented flasks at a density of 2 x 10 2 cells per flask and incubated under the same conditions (5% CO 2 and 37°C), and the above steps were repeated as needed several times to obtain more PBMCs;

[0045] S43) Directional culture of peripheral blood mononuclear cells:

[0046] PBMCs were inoculated into 6-well plates at a cell number of 1 x 10 6 per well, 1 ml of StemPro 34 SFM medium was added to each well, and the PBMCs were electroporated using a P2 Primary Cell Kit, while the medium was supplemented daily;

[0047] S44) Expansion of peripheral blood mononuclear cells:

[0048] On the 9th day after electroporation, colonies similar to artificial induced pluripotent stem cells were manually selected and expanded in mTeSR+ medium, while the cells were passaged every 4-6 days using GCDR (STEMCELL Technologies), and the entire process was maintained at 37°C, 5% CO2, and 20% O2.

[0049] S5) Combination of fur:

[0050] According to the structure of the epidermis and / or dermis of the mink itself, the various types of keratinocytes obtained in step S4) were sequentially grown on the tissue sheath loaded with hair bundles. In addition, since the real mink fur will remove a large amount of fat in the hypo-dermis during application, the structures such as fat and blood vessels can be screened out during directional synthesis. The main synthetic cell types, thickness, and arrangement direction (from outside to inside) of the epidermis of the mink itself are as follows:

[0051] ① Stratum corneum - corneocytes, 10 layers of flat, non-nucleated cells, 20 μm thick;

[0052] ② Stratum lucidum - stratum lucidum cells, relatively flat cells, 10 μm thick;

[0053] ③ Stratum granulosum - granular cells, flat cells, 0.45 μm thick;

[0054] ④ Stratum spinosum - spinous cells, 0.15 μm thick;

[0055] ⑤ Stratum basale - basal cells, active stem cells, 0.075 μm thick.

[0056] S6) Post-treatment of the fur:

[0057] Depending on the specific growth development, after waiting for a clear cell stratification, semi-biosynthetic fur is obtained using nitration process. Since the nitration process is a common process in the fur field, it will not be described again.

[0058] Example Two, Hair Tenacity Test:

[0059] The integrated and cultured hair bundle of about 1 cm in length of Example One is taken out and used as a sample, and then a slow stretching force is simulated (the stretching speed is set to 1 mm / s), and the strain (the ratio of the stretching length to the initial length) and the stress (the applied stretching force divided by the initial cross-sectional area) during stretching are recorded, as well as the tensile strength, maximum stretching force and breaking strength of the hair bundle during stretching.

[0060] In the stretching experiment, the recorded data are as follows:

[0061] Initial length: 1 cm;

[0062] Maximum stretching length: 1.8 cm;

[0063] Maximum applied force: 20 N;

[0064] The initial cross-sectional area is 1 mm 2 ;

[0065] Strain = (maximum stretching length - initial length) / initial length = (1.8 cm - 1 cm) / 1 cm = 0.8;

[0066] Stress = maximum applied force / initial cross-sectional area = 20 N / 1 mm 2 = 20 N / mm 2 = 20 MPa.

[0067] Example Three, Hair Shearing Test:

[0068] The integrated and cultivated hair bundle of about 1 cm in length of Example 1 was taken out as a sample and sheared under simulated moderate shearing stress (shearing speed was set at 2 mm / s), and the shearing force applied and the response of the hair bundle were recorded, and whether the hair was broken or displaced was observed.

[0069] During shearing, the recorded data were as follows:

[0070] The maximum shearing force applied was 10 N;

[0071] The initial cross-sectional area was 1 mm 2 ;

[0072] The shearing stress = the maximum shearing force applied / the initial cross-sectional area = 10 N / 1 mm 2 = 10 N / mm 2 = 10 MPa.

[0073] Example Four, Hair Bending Test:

[0074] The integrated and cultivated hair bundle of about 1 cm in length of Example 1 was taken out as a sample and bent under simulated small curvature (the curvature radius of the bending test equipment was set at 5 mm), and the strain and stress of the hair bundle under a given curvature were recorded, and whether the hair was broken or deformed was observed.

[0075] During the bending test, the bending test equipment with a curvature radius of 5 mm was used. The recorded data were as follows:

[0076] The strain under 5 mm curvature: 0.05 (indicating 5% elongation);

[0077] The initial cross-sectional area was 1 mm 2 ;

[0078] The bending force applied was 10 N;

[0079] According to this strain, the stress under 5 mm curvature was calculated, and the bending stress = the bending force applied / the initial cross-sectional area = 10 N / 1 mm 2 = 10 N / mm 2 = 10 MPa.

[0080] Example Five, Humidity Test of Hair:

[0081] The integrated and cultivated hair bundle of about 1 cm in length of Example 1 was taken out as a sample, and the sample was placed in an environment with a relative humidity of 80% to simulate high humidity conditions, and the changes (including deformation and color change, etc.) of the hair bundle in the humid environment were observed.

[0082] In the humidity test, the integrated and conditioned hair bundles were placed in an 80% relative humidity environment. The observed phenomena in the humidity environment were that the hair bundles slightly deformed and the color of the hair became slightly darker. These observations suggest that the hair can absorb water under high humidity conditions, resulting in slight deformation and color change.

[0083] The above examples are illustrative of the present application and are not limiting. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein.

Claims

1. A method for preparing semi-biosynthetic fur, characterized in that, Includes the following steps: S1) Obtaining hair: Obtain a mixture of raw hair from animals, remove impurities from the raw hair mixture, and obtain hair; S2) Preparation of hair bundles: The hair obtained in step S1) is sorted and guard hairs and down hairs are obtained separately. The guard hairs and down hairs are arranged and mixed into bundles according to the distribution ratio of the animal’s own guard hairs and down hairs to obtain hair bundles. S3) Integration and culture of the tissue sheath: According to the distribution of guard hairs per unit area of ​​the animal, the hair bundles obtained in step S2) are evenly arranged on the tissue sheath cells and cultured until the cells and hair bundles are fused together. In step S3), in vitro cultured fibroblasts are used as the source of tissue sheath cells; In step S3), DMEM / F-12 medium supplemented with 8–12% fetal bovine serum and 0.5–1.5% antibiotic-antifungal agent was used as the culture medium for tissue sheath cells loaded with hair tufts. The tissue sheath cells loaded with hair tufts were cultured for 15–25 days in a culture environment at 36.5–37.5°C and a CO2 concentration of 4.5–5.5%, allowing interaction between the tissue sheath cells and the hair tufts, while maintaining a cell density of 0.5 × 10⁻⁶ cells / year. 5 ~1.5×10 5 pcs / cm 2 ; S4) Synthesis of epidermal cells: Stem cells are extracted from the blood of animals and reprogrammed to restore them to a pluripotent state. Induced pluripotent stem cells are used to directionally synthesize various types of keratinocytes and fibroblasts required to form the epidermis and / or dermis of animals. S5) Fur integration: Based on the structure of the animal's own epidermis and / or dermis, the various types of keratinocytes and fibroblasts obtained in step S4) grow sequentially on the tissue sheath bearing hair bundles.

2. The method for preparing semi-biosynthetic fur as described in claim 1, characterized in that: In step S1), during the molting season, the raw hair mixture is collected from the animal by combing. The raw hair mixture is then soaked in an alkaline solution with a pH of 8-10 and a temperature of 35-45°C for 0.5-1.5 hours to remove mixed fat and hair.

3. The method for preparing semi-biosynthetic fur as described in claim 1, characterized in that, Step S4) is as follows: S41) Isolation of peripheral blood mononuclear cells: Isolation of peripheral blood mononuclear cells from the blood of animals; S42) Preliminary proliferation of peripheral blood mononuclear cells: The peripheral blood mononuclear cells obtained in step S41) are cultured to form a monolayer of attached cells and non-attached cells are washed off; S43) Directed culture of peripheral blood mononuclear cells: The monolayer of attached cells obtained in step S42) is seeded onto the culture medium, and then electroporation is performed after adding the reprogramming vector; S44) Expansion and passage of peripheral blood mononuclear cells: A population similar to artificially induced pluripotent stem cells was manually selected, expanded in culture medium, and passaged regularly.

4. The method for preparing semi-biosynthetic fur as described in claim 3, characterized in that: In step S42), StemPro 34 SFM medium is used as the culture medium for peripheral blood mononuclear cells. Peripheral blood mononuclear cells are cultured in a culture environment with a temperature of 36.5-37.5℃ and a CO2 concentration of 4.5-5.5%, and non-attached cells are washed off periodically with phosphate-buffered saline and the culture medium is replaced.

5. The method for preparing semi-biosynthetic fur as described in claim 3, characterized in that: In step S43), the culture medium is StemPro 34 SFM medium, the reprogramming vector is Epi5 Episomal reprogramming vector, and electroporation is performed using P2Primary Cell Kit.

6. The method for preparing semi-biosynthetic fur as described in claim 3, characterized in that: In step S44), the culture medium is mTeSR+ medium, and GCDR is used for subculturing every 4 to 6 days. The amplification and subculturing environment is maintained at a temperature of 36.5 to 37.5°C, a CO2 concentration of 4.5 to 5.5%, and an O2 concentration of 18 to 22%.

7. The method for preparing semi-biosynthetic fur as described in claim 1, characterized in that, It also includes the following step S6): S6) Post-processing of fur: Tanning is performed on the fur.

8. Semi-biosynthetic fur, prepared by the method of any one of claims 1 to 7.

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