CN110699316A

CN110699316A - A method for preparing cell suspension for promoting hair regeneration and preventing alopecia

Info

Publication number: CN110699316A
Application number: CN201911034606.6A
Authority: CN
Inventors: 张平; 邢志青; 吴训伟; 张甜甜; 王杰
Original assignee: JINAN PANSHENG BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: JINAN PANSHENG BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2020-01-17

Abstract

The invention discloses a preparation method of cell suspension for promoting hair regeneration and preventing alopecia, which utilizes scalp tissues to separate scalp epidermal cells and dermal papilla cells, and prepares the cells after the expansion culture into cell suspension to prepare injection for treating androgenetic alopecia, wherein the injection is derived from patients without immunological rejection reaction, has good cell activity and strong regeneration capacity, and has high efficiency for treating the androgenetic alopecia; and the injection can be prepared only by taking a small amount of scalp tissues, so that the transplanting efficiency is higher.

Description

A method for preparing cell suspension for promoting hair regeneration and preventing alopecia

Technical Field

The invention relates to a preparation method of a cell suspension for promoting hair regeneration and preventing alopecia, belonging to the field of cell culture.

Background

Alopecia is a common disease in clinical dermatology, and the incidence rate is gradually increased and the trend of youthfulness is present, which troubles the life of modern people, so that the alopecia is concerned more and more widely. The normal hair loss is the hair in the catagen and telogen phases, and the normal amount of hair can be maintained because the hair entering the catagen phase and the hair newly entering the anagen phase are constantly in dynamic balance. Abnormal or excessive hair loss is an abnormal condition of alopecia, and the reasons are many. Hereditary, pathological and bad life habits cause alopecia.

Androgenetic alopecia (also known as male pattern alopecia, MA) is the most common clinical progressive and characteristic alopecia of the head. The hair loss occurs gradually in a predictable mode along with the increase of age, the hair loss is mainly at the top of the head, the hair loss area begins from the two sides of the forehead, the hair loss area gradually expands upwards from the top, the hair is gradually thinned and thin, and finally most or all of the hair at the top of the head falls off, but the hair at the back of the pillow and above the two sides of the head are dependent on the horseshoe-shaped appearance. The pathological feature of androgenetic alopecia is that the ratio of anagen phase follicles to telogen phase follicles is reduced from the normal 12:1 to 5: 1.

Hair follicles have a periodic growth characteristic throughout the life of mammals, undergoing periodic changes of anagen, catagen and telogen phases, and the growth cycle of hair follicles is an intrinsic characteristic of biological individuals. Through the research on the growth of the hair follicle, the following results are found: in the regulation of hair follicle growth, the central link is the epithelial-mesenchymal interaction. The hair papilla, the major mesenchymal component of the hair follicle, is composed of a population of very distinct fibroblasts that produce a distinct extracellular matrix (ECM) required for the growth and development of the hair follicle, and alterations in the hair papilla ECM are closely related to the growth cycle of the hair follicle. It can therefore be speculated that the hair papilla constitutes the microenvironment for hair follicle growth, and the hair papilla cells send or receive signals through a paracrine or autocrine mechanism, thereby regulating the development and periodic growth of the hair follicle. The effects of hair papilla cells on hair follicle development and periodic growth regulation depend on a continuous and tight junction (through the native ECM) with hair matrix cells.

Currently, autologous free hair transplantation has become the most common surgical method for the clinical treatment of hair defects, especially androgenetic alopecia. The hair transplantation uses the pillow part as a hair supply area, and has limited sources, so that the problems of insufficient supply area, scar supply area, unstable transplantation survival rate and the like often exist for a large-area bald person, and the operation effect is obviously influenced.

Disclosure of Invention

The present invention overcomes the above-mentioned deficiencies of the prior art and provides a method for preparing a cell suspension that promotes hair regrowth and prevents hair loss. The method uses scalp tissues to separate scalp epidermal cells and dermal papilla cells, and prepares the cells into cell suspension after the scalp tissues are amplified and cultured to prepare the injection for treating androgenetic alopecia, and the transplantation efficiency can reach more than 20 times by only taking a small amount of scalp tissues.

A method for preparing a cell suspension for promoting hair regrowth and preventing hair loss, comprising the steps of:

(1) disinfecting a collected fresh scalp tissue sample, then treating the scalp tissue sample by using a PBS buffer solution, draining the treated tissue to remove redundant liquid, weighing, and chopping the tissue after weighing to obtain chopped tissue;

(2) adding the minced tissue obtained in the step (1) into an enzyme digestive juice, uniformly mixing, performing shake digestion at 37 ℃ for 55-60min, adding a trypsin solution with the concentration of 0.25%, continuing digestion for 25-30min, adding a DNA enzyme solution with the concentration of 10g/L after digestion is finished, and continuing digestion for 5min to obtain a tissue digestive juice;

(3) mixing and neutralizing the tissue digestive juice obtained in the step (2) with a DMEM medium containing 10% fetal calf serum, repeatedly sucking, filtering, centrifuging, removing supernatant, and dividing cells into two parts, namely a cell cluster A and a cell cluster B;

(4) resuspending the cell pellet a obtained in step (3) using DMEM medium containing 10% fetal bovine serum supplemented with 10mmol/L Rock protein kinase inhibitor, transferring the resuspended cell culture fluid (including cells and medium) into a cell culture flask, culturing for 3d, then changing to CnT07 medium, and then changing the fluid 1 time every 2 d; observing the cells under a microscope, and carrying out passage when the cells grow to reach 80-90% coverage rate in a culture vessel; the cells were cultured as follows 1: 2-4, and carrying out passage for 2-4 generations to obtain human scalp epidermal cells;

(5) taking the cell mass B obtained in the step (3), suspending the cells by using a DMEM (DMEM) culture medium containing 10% fetal calf serum, and inoculating the suspended cells into a cell culture bottle; observing the cells under a microscope, and carrying out passage when the cells grow to reach the coverage rate of 80-90% of a cell culture bottle; according to the following steps: 3-6 passages, and 2-6 passages to obtain human scalp dermal papilla cells;

6) and (3) mixing the culture solution of the human scalp epidermal cells obtained in the step (4) and the culture solution of the human scalp dermal papilla cells obtained in the step (5) according to the volume ratio of 1:2-5 to obtain a cell suspension for promoting hair regeneration and preventing alopecia.

Further, the PBS buffer in the above step (1) contains 200 units/ml of penicillin and 200. mu.g/ml of streptomycin.

Further, the enzyme digestion solution in the step (2) contains 2.5g/L of collagenase type I and 2.5g/L of dissociation enzyme; 20ml of the enzyme digest was used per gram of tissue.

Further, the 0.25% trypsin solution in step (2) above was added at a rate of 5ml per gram of tissue, so that the final concentration of trypsin in the digestion solution was 0.05%.

Further, the DNase solution in the step (2) is added according to the proportion of 100 mu L per gram of the tissue.

Further, the tissue digest and the DMEM medium are mixed in a volume ratio of 1:1 in the step (3).

Further, the cell culture flask described in the above step (4) was previously subjected to rinsing treatment with a coating substrate purchased from Gibco, Inc. under the trade name of R011K.

The application also protects a cell suspension prepared by the preparation method.

The application also protects the use of the above cell suspension in promoting hair regrowth and preventing hair loss.

The application also protects the application of the cell suspension in preparing injection for treating androgenetic alopecia.

Has the advantages that:

(1) the transplanting efficiency can be more than 20 times (namely 2 cm) only by taking a small amount of scalp tissue²The tissue can be injected by 40-100cm²Scalp area) without the need for large scale harvesting of scalp tissue.

(2) The mixed cell suspension prepared by the invention is derived from patients without immunological rejection, has good cell activity and strong regeneration capacity, so that the injection has high effective rate for treating androgen-derived alopecia, and avoids the problems of 'dismantling east wall and supplementing west wall' and poor treatment effect of the traditional hair follicle transplantation.

Drawings

FIG. 1 cell suspension preparation.

Fig. 2A is a diagram of hair follicles formed at an injection site, and B is a diagram of follicles in hair cysts and hair papillae.

Fig. 3C is a graph showing the results of paraffin sectioning after transplantation, and D is a graph showing the regeneration of hair follicles.

Fig. 4 shows the regenerated hair under-the-mirror image (7 days after injection).

Fig. 5 comparative graph of regrown hair (3 months after injection).

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 preparation of cell suspension for promoting Hair regrowth and preventing alopecia

Materials and methods

(1) Treatment of scalp tissue: shearing off the fat and connective tissue in the hair outside the scalp and the subcutaneous tissue, disinfecting the collected fresh scalp tissue sample for 3min by using alcohol, and then treating the fresh scalp tissue sample for two times by using PBS containing 2 times of double antibody, wherein each time lasts for 3min to achieve the aim of disinfection. The treated tissue was drained of excess fluid, weighed and recorded and then the tissue was plated out on 100mm cell culture dishes. The tissue was cut into a uniform minced meat shape with a scalpel.

(2) Enzyme digestion: the minced tissue was added to an enzyme digest (20 ml of enzyme digest containing 2.5g/L collagenase type I and 2.5g/L resolvase) and mixed well. Digestion was carried out in a 37 ℃ water bath at 80 Xg for 60min with shaking. Then, 0.25% trypsin solution (5 ml of 1g tissue) was added to make the final concentration 0.05% for further digestion for 30min, and finally 10g/L DNase (100. mu.L of 1g tissue) was added for digestion for 5 min.

(3) Collecting cells: after the tissue is digested, the same volume of DMEM medium containing 10% fetal calf serum is added to neutralize the digestive juice, and the sucking and beating are repeated. Filtration was performed using a 100 μm cell filter. Centrifuge at 1000 Xg for 5min, discard the supernatant. The cells were divided in two halves, one for the culture of hair follicle stem cells and one for the culture of dermal papilla cells.

(4) Culturing scalp epidermal cells: a portion of the collected cells was taken, resuspended in DMEM medium containing 10% fetal bovine serum containing 10mmol/L Rock protein kinase inhibitor, inoculated into a cell culture flask previously treated with a coating matrix (the number of cells per T75 cell culture flask was about 300 to 500 ten thousand), cultured for 3 days, and then changed to CnT07 medium 1 time per 2 days. Cells are observed under a microscope, and passage or subsequent study is carried out when the cells grow full. Changing the culture solution every 2 days; when the cell growth reaches 80-90% coverage rate in a culture vessel, carrying out passage; the cells were cultured as follows 1: passage at the ratio of 2-4, passage for 2-4 generations.

(5) Culturing dermal papilla cells: an aliquot of the collected cells was taken, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded into cell culture flasks (approximately 300 to 500 ten thousand cells per T75 cell culture flask). Cells are observed under a microscope, and passage or subsequent study is carried out when the cells grow full. When the cell growth reaches 80-90% coverage rate in a culture vessel, carrying out passage; according to the following steps: 3-6 passages, and 2-6 passages; the culture medium was changed every 5 days after the passage of dermal cells.

(6) Preparation of suspensions of scalp epidermal cells and dermal papilla cells: the cultured qualified human scalp epidermal cells and dermal papilla cells are prepared into suspension according to a certain proportion.

Second, result analysis

As shown in FIG. 1, A in the figure is the prepared scalp epidermal cells, and B in the figure is the dermal papilla cells obtained by culture. Example 2 experiment of the Effect of injecting cell suspension into the dorsal part of immunized mice to regenerate hair

Materials and methods

(1) Scalp epidermal cell and dermal papilla cell suspension injection

The cultured qualified human scalp epidermal cells and dermal papilla cells are in a certain ratio (in this embodiment, the number of scalp epidermal cells is 1 × 10)⁶P3 for each generation; the number of dermal papilla cells was 2X 10⁶P4) mixed with 75 μ l of F12 medium to prepare a cell suspension. Four needles were injected into the back of immunodeficient mice.

(2) Analysis of Hair regrowth

The regeneration of hair follicles in mice was examined 12 weeks after injection using a dissecting light microscope, and regenerated skin samples were excised and collected, frozen 10 μm sections were made, and stained with hematoxylin-eosin: fixed by immersion in 10% formalin, washed with 1xPBS, stained with hematoxylin-violet for three minutes, washed with distilled water, and then stained with eosin for 30 seconds. Then washing and dehydrating with 70%, 90% and 100% ethanol respectively. After dehydration, the sections were rinsed with xylene, mounted, and the tissue structure of the regenerated skin was observed with an optical microscope.

The immunofluorescence staining method comprises the following steps: frozen sections were fixed with 4% paraformaldehyde for 10 min, washed with PBS, and incubated with blocking buffer for 1 hour at room temperature. Primary antibody was incubated overnight at 4 ℃. The next day, sections were washed in PBS solution and incubated for 1 hour with secondary antibody. The expression of each protein was analyzed by confocal microscopy.

Secondly, hair regrowth results

The hair follicle formation is shown in FIG. 2. The hair follicle was formed at the injection site (fig. 2A), and the bulb of the formed hair follicle was black, presumably melanin. The hair cyst has a mature follicle with a dark black hair shaft (white arrow in fig. 2B) and a clear hair papilla (oval indicated by black arrow in the middle-lower part in fig. 2B). In FIG. 3, C is the paraffin section result after transplantation (the regenerated region has normal epidermis and dermis structures, Der represents the dermis, Epi represents the epidermis), and D is the hair follicle regeneration (black arrow represents the hair papilla).

Example 3 clinical trial of injection of cell suspension from autologous scalp tissue

First, injection of epidermal cell and dermal cell suspension

20 androgenetic alopecia patients with disease course within 2 years of age of 30-50 years and other male with healthy physical indexes are selected, and informed consent is given and signed before use. 2X 2cm of scalp tissue was removed from the patient's occiput with a scalpel, and human scalp epidermal cells and dermal papilla cells were cultured according to the above experimental method. The cultured qualified human scalp hair follicle cells and dermal papilla cells are in a certain ratio (in this embodiment, the number of scalp epidermal cells is 2 × 10)⁶P3 for each generation; the number of dermal papilla cells was 5X 10⁶P4) mixed with 75 μ l of F12 medium to prepare a cell suspension. The patient's skin was routinely disinfected and injected subcutaneously at multiple points along the median sagittal line in a conventional hair growth zone (5cm by 8cm) and observed for 3 months after treatment. The judgment standard of the curative effect is as follows: 1) and (3) curing: the newly grown terminal hair of the skin at the lesion part is more than 20/cm²；

2) The method has the following advantages: the number of new hairs in affected part is less than 20/cm²；

3) And (4) invalidation: no change after treatment.

Secondly, hair regrowth results

The formation of the hair follicle 2 weeks after injection is shown in figure 4. The fine hair is new hair, and the thick hair is small hair at the original injection position. The number of regenerated hairs is large.

After 3 months, the total effective rate can reach 95%, wherein the cure number is 12, the effective number is 7, and the ineffective number is 1. In fig. 5, the left image is before treatment and the right image is after treatment.

Claims

1. A method for preparing a cell suspension for promoting hair regrowth and preventing hair loss, comprising the steps of:

(2) adding the minced tissue obtained in the step (1) into an enzyme digestive juice containing collagenase and dissociation enzyme, uniformly mixing, carrying out shake digestion at 37 ℃ for 55-60min, then adding a trypsin solution with the concentration of 0.25%, continuing digestion for 25-30min, adding a DNA enzyme solution with the concentration of 10g/L after digestion is finished, and continuing digestion for 5min to obtain a tissue digestive juice;

(3) mixing the tissue digestive juice obtained in the step (2) with a DMEM medium containing 10% fetal calf serum, repeatedly sucking, filtering, centrifuging, removing supernatant, and dividing cells into two parts, namely a cell cluster A and a cell cluster B;

(4) resuspending the cell pellet a obtained in step (3) using DMEM medium containing 10% fetal bovine serum supplemented with 10mmol/L Rock protein kinase inhibitor, transferring the resuspended cell culture fluid into a cell culture flask, culturing for 3d, then changing to CnT07 medium, and changing the fluid 1 time every 2d thereafter; when the cell growth reaches 80-90% coverage rate of the cell culture bottle, carrying out passage; the cells were cultured as follows 1: 2-4, and carrying out passage for 2-4 generations to obtain a culture solution of human scalp epidermal cells;

(5) taking the cell mass B obtained in the step (3), suspending the cells by using a DMEM (DMEM) culture medium containing 10% fetal calf serum, and inoculating the suspended cells into a cell culture bottle; when the cell growth reaches 80-90% coverage rate of the cell culture bottle, carrying out passage; according to the following steps: 3-6 passages, and passing for 2-6 passages to obtain human scalp dermal papilla cell culture solution;

(6) and (3) mixing the culture solution of the human scalp epidermal cells obtained in the step (4) and the culture solution of the human scalp dermal papilla cells obtained in the step (5) according to the volume ratio of 1:2-5 to obtain a cell suspension for promoting hair regeneration and preventing alopecia.

2. The method of claim 1, wherein the PBS buffer in step (1) contains 200 units/ml penicillin and 200. mu.g/ml streptomycin.

3. The method according to claim 1, wherein the enzyme digest of step (2) contains 2.5g/L collagenase type I and 2.5g/L dissociation enzyme; 20ml of the above enzymatic digest was used per gram of tissue.

4. The method of claim 1, wherein the 0.25% trypsin solution of step (2) is added at a rate of 5ml per gram of tissue to provide a final concentration of 0.05% trypsin in the digestive fluid.

5. The method according to claim 1, wherein the DNase solution in the step (2) is added at a rate of 100. mu.L per gram of the tissue.

6. The method according to claim 1, wherein the tissue digest and the DMEM medium are mixed in a volume ratio of 1:1 in step (3).

7. The method according to claim 1, wherein the cell culture flask in the step (4) is previously rinsed with a coating substrate purchased from Gibco, Inc. under the trade name of R011K.

8. A cell suspension prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the cell suspension of claim 8 for promoting hair regrowth and preventing hair loss.

10. Use of the cell suspension of claim 8 for the preparation of an injection for the treatment of androgenetic alopecia.