CN113082055B

CN113082055B - Regenerated colored hair injection and preparation method thereof

Info

Publication number: CN113082055B
Application number: CN202110400050.9A
Authority: CN
Inventors: 张甜甜; 邢志青; 张平; 王杰; 孙红; 李芬; 楚天云
Original assignee: Jinan Pantheon Biotechnology Co ltd
Current assignee: Jinan Pantheon Biotechnology Co ltd
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2022-09-06
Anticipated expiration: 2041-04-14
Also published as: CN113082055A

Abstract

The invention discloses a regenerative colored hair injection and a preparation method thereof, the method separates epidermal stem cells, melanocytes and dermal fibroblasts from scalp tissues, a cell mass formed by the melanocytes and the dermal fibroblasts and the epidermal stem cells are mixed according to a certain proportion to obtain the regenerative hair injection, and the injection is injected to a hair regeneration part to gradually regenerate the colored hair.

Description

Regenerated colored hair injection and preparation method thereof

Technical Field

The invention relates to a regenerated colored hair injection and a preparation method thereof, belonging to the field of cell engineering.

Background

Alopecia and poliosis are common diseases affecting men, women and children. Currently, the treatment of alopecia mainly depends on drugs and autologous hair transplantation, but the two methods have some difficulties, such as limited drug effect, incapability of increasing the hair quantity of the scalp, and the like. The formation of hair follicles during embryonic development and postnatal hair circulation is regulated by a series of interactions between epithelial-derived hair follicle stem cells (e.g., hair follicle epithelial stem cells, enlarged epithelial stem cells) and mesenchymal-derived hair follicle stem cells (e.g., dermal papilla cells, hair follicle dermal cells). Methods for maintaining this ability have been investigated, including the use of growth factors and signal molecules. For example, loss of fibroblast growth factor-2, Wnt and bone morphogenic proteins may reduce the hair-inducing ability of DP cells. Another approach is to fabricate 3D tissue prior to transplantation and increase DP cell-specific marker expression by forming aggregates of DP cells in hanging drop or non-cell-adherent culture conditions.

The white hair disease can be divided into 2 types of physiological white hair and pathological white hair, and is fundamentally caused by the disorder of the lineage balance of melanocytes in hair follicles regardless of the occurrence mechanism. During hair coloring, melanin granules secreted by melanocytes have a direct effect; once destroyed, the subsequent other progeny melanocytes will eventually lose function. Western medicine has no specific treatment to the disease at present, and the hair dyeing can induce various diseases, such as contact dermatitis, leukemia, osteoporosis, even canceration and the like. In contrast, the traditional Chinese medicine has obvious advantages for treating the disease, can adopt treatment methods such as oral administration, external washing, acupuncture and massage, medicated diet and the like, is worthy of wide application, but needs to be persisted for a long time and has different effects according to different people.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a regenerated colored hair injection and a preparation method thereof, the method separates epidermal stem cells, melanocytes and dermal fibroblasts from scalp tissues, a cell mass formed by the melanocytes and the dermal fibroblasts and the epidermal stem cells are mixed according to a certain proportion to obtain the regenerated hair injection, and the injection is injected to a hair regeneration part to gradually regenerate the colored hair.

A method for preparing an injection for regenerating colored hair comprises the following steps:

1) obtaining scalp tissues, digesting and separating primary epidermal stem cells, primary melanocytes and primary dermal fibroblasts;

2) subculturing the primary epidermal stem cells obtained in the step 1), collecting epidermal stem cells of P3-P5 generations, and inoculating the epidermal stem cells into a cell culture bottle;

3) melanocytes and dermal fibroblasts were measured in a quantitative ratio of 1: (5-20), adding a multifunctional serum-free cell culture medium for resuspension, inoculating the mixture in a low adsorption culture plate after resuspension, culturing for 2-3 days to form a 3D cell mass of melanocytes and dermal fibroblasts, collecting a 3D cell mass suspension, centrifuging, and retaining a supernatant and the centrifuged 3D cell mass;

4) mixing the epidermal stem cells collected in the step 2) and the 3D cell mass after centrifugation in the step 3) according to the quantitative ratio of (2:1) - (1:5), and using the supernatant obtained in the step 3) to resuspend the mixed cells;

5) and (3) cleaning the mixed cells obtained in the step 4) by using DMEM (DMEM) without double antibodies, centrifuging, then discarding the supernatant, and re-suspending the supernatant collected in the step 3) for precipitation, thus obtaining the regenerated colored hair injection.

Further, 8-12 mL of the epidermal cell culture medium is added into the cell culture bottle in the step 2), and the epidermal stem cells of the P3-P5 generation are inoculated into the cell culture bottle according to the bottle of 120-150 ten thousand/T75, wherein the epidermal cell culture medium is prepared according to the patent No. 201610473310.4.

Further, the multifunctional serum-free cell culture medium described in step 3) is a culture medium prepared using the method of patent No. 201610473348.1.

Further, the centrifugation in step 5) is performed at 600-.

Further, the method for separating the epidermal stem cells, the melanocytes and the dermal fibroblasts in the step 1) comprises the following steps:

s1, obtaining scalp tissue, sterilizing, placing in a mixed solution of dissociation enzyme and pancreatin, digesting at 37 ℃ for 10-120min, and neutralizing enzyme reaction by using a DMEM medium containing 10% FBS to form a neutralization solution A;

s2, blowing the neutralization solution A, and filtering the neutralization solution A through a 70-micron filter screen to obtain a filtrate A and a filter residue A;

s3 centrifuging the filtrate A at 600rpm for 4-5min to retain precipitate A;

s4, adding a collagenase I solution with the final concentration of 0.25-5.0 mg/mL into the filter residue A, digesting for 10-120min at 37 ℃, repeatedly beating for 2-60 times by using a DMEM medium containing 10% FBS, and carrying out a neutralization enzyme reaction to form a neutralization solution B;

s5 filtering the neutralization solution B through a 100-micron filter screen, reserving the filtrate B, and centrifuging the filtrate B for 4-5min at the speed of 600-1000rpm to obtain a precipitate B;

s6 the sediment A obtained in the step S3 and the sediment B obtained in the step S5 are respectively counted after being re-suspended by using DMEM culture medium, the cells obtained from the sediment A are divided into 2 parts, one part is cultured by using human skin epidermal cell culture medium, and the other part is cultured by using melanocyte culture medium; culturing the cells obtained from the pellet B using a multifunctional serum-free cell culture medium; the epidermal stem cells, the melanocytes and the dermal fibroblasts can be separated.

Further, the scalp tissue in step S1 is occipital scalp tissue including hair follicles and hair papilla portions.

Further, the sterilization method in the step S1 is: rinsing scalp tissue with DPBS for 2 times, soaking in 75% alcohol for 5min, and soaking in PBS containing streptomycin double antibody for 5min for 2 times.

Further, in the mixed solution of the dissociation enzyme and the pancreatin in the step S1, the final concentration of the dissociation enzyme is 0.5 to 5.0mg/mL, and the final concentration of the pancreatin is 0.05 to 0.5%.

Further, the collagenase i solution and the DMEM medium containing 10% FBS are used in a volume ratio of 1:1 in step S4.

Further, the multifunctional serum-free cell culture medium described in step S6 is a culture medium prepared by the method described in patent No. 201610473348.1

Further, the human epidermal cell culture medium described in step S6 is a medium prepared using the method in patent No. 201610473310.4.

Further, the Melanocyte Medium in step S6 is a TPA-free Medium, preferably one of (1) Medium 254CF available from Gibco, Melanocyte Medium M2 available from Promocell, and Melanocyte Medium-2(MelM-2) available from Sciencell.

Further, the application of the regenerated colored hair injection prepared by the preparation method in promoting the growth of colored hair.

Has the advantages that:

the technical method is simple, and the hair can be regenerated, and the regenerated hair is colored, and the color can be controlled by adjusting different cell proportions.

Drawings

FIG. 1 shows the condition of melanocytes, epidermal cells and dermal cells.

FIG. 23D photograph showing the process of cell mass formation.

FIG. 3 shows the growth of colored regrown hair in mice.

Figure 4 comparison graph of human regenerated colored hair.

Detailed Description

In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.

Example 1

Materials and procedures

1. Scalp tissue extraction and disinfection

(1) Obtaining a tissue sample: tissue origin: the scalp was discarded and the appearance of the scalp was normal. Organization requirements: when taking scalp, ensuring that the taken tissue contains hair follicle and hair papilla part;

(2) disinfection treatment of scalp group: the subcutaneous tissue was removed (removed as clean as possible) with a scalpel or scissors, washed 1 time in 70% ethanol, 5min, and the tissue was washed 5 min/2 times in PBS petri dishes containing 2 × double antibody.

2. Obtaining primary dermal epidermal stem cells and melanocytes

Placing the sterilized skin tissue in a water bath at 37 ℃ in a solution containing 3.5mg/mL of dissociation enzyme and 0.25% of pancreatin for digestion until a large number of cells fall off, wherein the digestion time is 30min, neutralizing the enzyme reaction by using a DMEM medium containing 10% FBS, repeatedly blowing and beating for more than 20 times to obtain a single cell suspension, filtering by using a 70-micron filter screen, centrifuging the filtered suspension for 5 minutes at 600rpm, cleaning once by using 10mL of 10% FBS DMEM to obtain primary skin epidermal stem cells and melanocyte suspension, counting, placing the cell suspension in a centrifuge, centrifuging for 5min at 600rpm, and reserving the cell suspension to obtain primary skin epidermal stem cells and melanocyte mixed sediment. Separating the obtained primary skin epidermal stem cell and melanocyte mixed precipitate into two parts, one part is cultured with human skin epidermal cell culture medium (patent No. 201610473310.4); and culturing the other part by using a melanocyte culture medium to obtain primary skin epidermal stem cells and melanocytes.

3. Obtaining dermal fibroblasts

After filtering the above using a 70 micron filter, the undigested tissue mass on the filter was transferred to a new centrifuge tube and digested with collagenase I at 3.5mg/mL in a water bath at 37 ℃ for 30 min. Then, 150uL of 10mg/mL DNase was added to continue the digestion for 5 min. The tissue was shaken off several times during incubation to disperse the clumped tissue. The cells were neutralized with the same volume of DMEM medium containing 10% FBS (containing double antibody), and gently beaten 20 times. Centrifuge at 1000rpm for 5 min. Resuspending the cell pellet with 10mL DMEM containing double antibody to obtain dermal fibroblast suspension, counting, centrifuging the cell suspension in a centrifuge at 1000rpm for 5min, and retaining the pellet to obtain dermal fibroblast, and culturing the obtained cell with multifunctional serum-free cell culture medium (patent No. 201610473348.1).

4. Obtaining regenerated colored hair injection

(1) When the confluence degree of the cells reaches about 80%, the cells are subjected to passage according to the ratio of 1: 2-1: 5.

(2) Culturing and subculturing the epidermal stem cells, the melanocytes and the dermal fibroblasts obtained in the steps, and selecting the dermal fibroblasts, the melanocytes and the epidermal stem cells of the generations P3-P5 to be respectively inoculated according to the following methods:

the epidermal stem cells were inoculated according to the 120-150 ten thousand/T75 flask;

according to 10 ⁷ Dermal fibroblasts and 10 ⁶ Mixing the melanocytes, adding a multifunctional serum-free cell culture medium for resuspension, inoculating the mixture into a 6-hole low-adsorption culture plate, culturing for 2 days, wherein the final volume of the culture medium in each hole is 3-5 mL, so that dermal fibroblasts and the melanocytes form a 3D cell mass.

(3) Collecting the suspension forming the 3D cell mass, centrifuging at 1000rpm/min for 5min, retaining the supernatant and the centrifuged cell mass, and transferring the supernatant to a new centrifuge tube for later use.

(4) After the epidermal stem cells are digested, collecting the epidermal stem cells, and mixing the collected epidermal stem cells with the centrifuged cell mass obtained in the step (1: 2, and mixing.

(5) Washing the mixed cells once by using DMEM (DMEM) without double antibodies, centrifuging for 5min at 1000rpm, discarding the supernatant, and re-suspending the cell sediment for regenerating the colored hair of the nude mouse by using 1mL of 3D culture supernatant to obtain regenerated colored hair injection; the cell sediment for regenerating colored hair on the head of a human body is resuspended by using 5mL of 3D culture supernatant, and then the regenerated colored hair injection can be obtained.

5. Results

As shown in FIG. 1, A in FIG. 1 is a passaged melanocyte, which is a spindle-shaped bipolar cell with branches; b in FIG. 1 is human dermal fibroblast cells in the shape of an elongated spindle, like a fibroblast, with an oval nucleus; in FIG. 1, C is an epidermal cell which is typically in the shape of a paving stone, has clear intercellular spaces, can obviously distinguish adjacent regions, has better refractive index, and has clear cytoplasm without impurities.

As shown in FIG. 2, A in FIG. 2 is a state where 3D culture cell suspension was inoculated into a 6-well low adsorption plate, and cells were individually dispersed in the culture solution; b in FIG. 2 is a state that 3D culture cell suspension is inoculated to a 6-well low-adsorption culture plate for 6h, cells begin to agglomerate, the cell agglomerates are small, and the cells are irregular; in fig. 2, C is the state that the 3D culture cell suspension is inoculated to the 6-well low-adsorption culture plate for 24 hours, and the cell mass begins to become larger and takes an irregular shape; in FIG. 2, D is the state that the 3D culture cell suspension is inoculated to the 6-well low-adsorption culture plate for 48h, the cell mass is large, and the cell mass is in a more regular circular shape.

EXAMPLE 2 regeneration of colored Hair in nude mice

1. Preparation of injection

(1) Regenerated colored hair injection a was prepared according to the preparation method in example 1.

(2) Preparation of regenerated colored Hair injection B, injection preparation according to the method in example 1, in step (2) according to 10 ⁷ Dermal fibroblast and 2X 10 ⁶ Mixing the melanocytes, adding a multifunctional serum-free cell culture medium for resuspension, inoculating the mixture into a 6-hole low-adsorption culture plate, culturing for 2 days, wherein the final volume of the culture medium in each hole is 3-5 mL, so that the dermal fibroblasts and the melanocytes form a 3D cell mass.

(3) Preparation of regenerated colored Hair injection C, injection C was prepared according to the method of example 1, and 10 was inoculated in step (2) ⁷ Adding a multifunctional serum-free cell culture medium into each dermal fibroblast, carrying out resuspension, inoculating the dermal fibroblast into a 6-hole low-adsorption culture plate, and culturing for 2 days, wherein the final volume of the culture medium in each hole is 3-5 mL, so that the dermal fibroblast forms a 3D cell mass.

(4) Male nude mice, 9, were selected at 6-8 weeks, divided into 3 groups, 3/group: injection a was used in test group 1, injection B was used in test group 2, and injection C was used in test group 3.

2. Method for using injection

(1) Anaesthetizing the nude mice to be transplanted, and injecting the injection of each test group to the back of the nude mice by microneedle injection or other methods;

(2) coating a small amount of antibacterial ointment around the wound, and bandaging the wound with an elastic bandage;

(3) after 10 days of operation, the gauze was removed and hair formation was continued (3-6 months).

3. Results of the experiment

A in FIG. 3 uses a 3D cell mass formed with dermal fibroblasts only (test)Group 3) the hair regenerated after being mixed and injected with the epidermal stem cells according to a certain proportion has more white hair; b in fig. 3 uses 10 ⁷ Dermal fibroblasts and 10 ⁶ The 3D cell mass formed by the melanocytes (test group 1) and the epidermal stem cells are mixed and injected according to a certain proportion, and a small amount of white hair exists in the regenerated hair; c in FIG. 3 uses 10 ⁷ Dermal fibroblast and 2X 10 ⁶ The 3D cell mass (test group 2) formed by the individual melanocytes was mixed with epidermal stem cells at a certain ratio and injected to regenerate black hair.

Example 3 regeneration of colored Hair

1. Preparation of injection

Injection D and injection E from example 2 were prepared according to the procedure in example 2, injection D containing 2X 10 ⁷ Dermal fibroblast and 4X 10 ⁶ Mixing with melanocytes, and making injection E containing 2 × 10 ⁷ Dermal fibroblasts.

2. Application method

The injection D and the injection E are respectively injected into the hair-missing part by microneedle injection or other methods.

Firstly, determining a part of a patient needing to regenerate colored hair, taking a comfortable body position and taking the part as a center, circularly disinfecting skin from inside to outside, and taking the condition that the disinfection radius covers the area needing to regenerate the colored hair as the standard;

secondly, exposing a regeneration area, and injecting the effective anesthetic into a part with less hair to be regenerated by using a micro needle injection method or other methods;

③ other treatments after injection.

3. Results of the experiment

After 4 months, the gradual regrowth of colored hair was observed (see FIG. 4).

In fig. 4, a, the number of hairs regenerated after the 3D cell mass formed only by the dermal fibroblasts and the epidermal stem cells are mixed according to a certain ratio (injection E) is small, and the hairs are all white hairs; b in FIG. 4 is 2X 10 ⁷ Dermal fibroblasts and 4X 10 ⁶ 3D cell formed by melanocyteThe hair regenerated after the ball and the epidermal stem cells are mixed according to a certain proportion (injection D) is more, and the hair is black.

Claims

1. The preparation method of the injection for regenerating colored hair is characterized by comprising the following steps:

1) obtaining scalp tissues, digesting and separating primary epidermal stem cells, primary melanocytes and primary dermal fibroblasts, and specifically comprising the following steps:

s1: obtaining scalp tissue, sterilizing, placing in mixed solution of dissociation enzyme and pancreatin, digesting at 37 deg.C for 10-120min, and neutralizing enzyme reaction with DMEM medium containing 10% FBS to form neutralized solution A; the final concentration of the dissociation enzyme in the mixed solution of the dissociation enzyme and the pancreatin is 0.5-5.0 mg/mL, and the final concentration of the pancreatin is 0.05% -0.5%; the scalp tissue is occipital scalp tissue containing hair follicles and hair papilla parts;

s2: blowing the neutralization solution A, and filtering the neutralization solution A through a 70-micrometer filter screen to obtain a filtrate A and filter residue A;

s3: centrifuging the filtrate A at 600rpm for 4-5min, and retaining precipitate A;

s4: adding collagenase I solution with the final concentration of 0.25-5.0 mg/mL into the filter residue A, digesting for 10-120min at 37 ℃, repeatedly beating for 2-60 times by using a DMEM (DMEM) culture medium containing 10% FBS, and carrying out a neutralization enzyme reaction to form a neutralization solution B;

s5: filtering the neutralization solution B by a filter screen of 100 mu m, reserving the filtrate B, and centrifuging the filtrate B for 4-5min by using 600-1000rpm to obtain a precipitate B;

s6 the sediment A obtained in the step S3 and the sediment B obtained in the step S5 are respectively counted after being re-suspended by using DMEM culture medium, the cells obtained from the sediment A are divided into 2 parts, one part is cultured by using human skin epidermal cell culture medium, and the other part is cultured by using melanocyte culture medium; culturing the cells obtained from the pellet B using a multifunctional serum-free cell culture medium; the epidermal stem cells, the melanocytes and dermal fibroblasts can be separated;

3) melanocytes and dermal fibroblasts were measured in a quantitative ratio of 1: (5-20), adding a multifunctional serum-free cell culture medium for resuspension, inoculating the cells into a culture plate after resuspension, culturing for 2-3 days to form a 3D cell mass of melanocytes and dermal fibroblasts, collecting a 3D cell mass suspension, centrifuging, and reserving a supernatant and the centrifuged 3D cell mass;

4) mixing the epidermal stem cells collected in the step 2) and the 3D cell masses centrifuged in the step 3) according to the number ratio of (2:1) - (1:5), and re-suspending the mixed cells by using the supernatant obtained in the step 3);

5) and (3) cleaning the mixed cells obtained in the step 4) by using DMEM (DMEM) without double antibody, centrifuging, then discarding the supernatant, and re-suspending the precipitate by using the supernatant collected in the step 3) to obtain the regenerated colored hair injection.

2. The method as set forth in claim 1, wherein 8-12 mL of the epidermal cell culture medium is added into the cell culture flask in step 2), and the epidermal stem cells of the P3-P5 generation are inoculated into the cell culture flask according to the 120-150 ten thousand/T75 flask.

3. The method according to claim 1, wherein the centrifugation in step 5) is performed at 600-1000rpm for 5 min.

4. The method of claim 1, wherein the sterilizing step S1 is performed by: rinsing scalp tissue with DPBS for 2 times, soaking in 75% ethanol for 5min, and soaking in PBS containing streptomycin double antibody for 5min for 2 times.

5. The method according to claim 1, wherein the collagenase i solution and the DMEM medium containing 10% FBS are used in a volume ratio of 1:1 in step S4.

6. The method according to claim 1, wherein the melanocyte medium according to step S6 is a medium containing no TPA component.

7. Use of an injection for regenerating colored hair prepared by the preparation method according to any one of claims 1 to 6 for promoting the growth of colored hair.