CN111920828A

CN111920828A - Lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells, preparation method, hair growth nutrient solution and hair regeneration method

Info

Publication number: CN111920828A
Application number: CN202010647925.0A
Authority: CN
Inventors: 刘诚; 马雅婷; 高锦颐
Original assignee: Xi'an Shiyue Regenerative Medicine Research Center Co ltd
Current assignee: Xi'an Shiyue Regenerative Medicine Research Center Co ltd
Priority date: 2020-07-07
Filing date: 2020-07-07
Publication date: 2020-11-13

Abstract

The invention discloses lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells, a preparation method, a hair growth nutrient solution and a hair regeneration method. The preparation method of the freeze-dried powder comprises the following steps: preparing a freeze-dried powder stock solution by adopting umbilical cord mesenchymal stem cells; uniformly mixing the freeze-drying protective agent and the freeze-drying powder stock solution to obtain an intermediate solution; and freeze-drying the intermediate solution under a preset freeze-drying condition to obtain the freeze-dried powder. The prepared freeze-dried powder and hair growth nutrient solution have obvious effect on at least one of promoting hair follicle restoration and promoting hair regeneration of a human body, and are beneficial to relieving the trend of alopecia and/or improving the hair state.

Description

Lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells, preparation method, hair growth nutrient solution and hair regeneration method

Technical Field

The invention relates to the technical field of biology, in particular to umbilical cord mesenchymal stem cells-based freeze-dried powder for hair regeneration, a preparation method of the freeze-dried powder, a hair growth nutrient solution and a hair regeneration method.

Background

In recent years, as social competition has become more intense and the environment has become worse, the population suffering from alopecia has been increasing and is becoming younger. The current situation of alopecia is aggravated by fast-paced work, high-stress life and overweight learning strength. In the face of such a huge hair loss population, it is one of the technical problems to be urgently solved by those skilled in the art to study a product capable of promoting hair regrowth.

Disclosure of Invention

The invention provides lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells, a preparation method of the lyophilized powder, a hair growth nutrient solution and a hair regeneration method, and aims to prepare and provide lyophilized powder capable of promoting hair regeneration.

In a first aspect of the present invention, the present invention provides a method for preparing a lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells, comprising: preparing a freeze-dried powder stock solution by adopting umbilical cord mesenchymal stem cells; uniformly mixing the freeze-drying protective agent and the freeze-drying powder stock solution to obtain an intermediate solution; and freeze-drying the intermediate solution under a preset freeze-drying condition to obtain the freeze-dried powder.

In the preparation method of the invention, the preparation of the lyophilized powder stock solution by using umbilical cord mesenchymal stem cells comprises the following steps: carrying out primary extraction and subculture on the umbilical cord mesenchymal stem cells to obtain a subculture product; pretreating the subculture product to obtain a first liquid; and uniformly mixing the first liquid and the second liquid to obtain the freeze-dried powder stock solution.

In the preparation method of the invention, the primary extraction and subculture of the umbilical cord mesenchymal stem cells are carried out to obtain a subculture product, which comprises the following steps: carrying out umbilical cord pretreatment on an umbilical cord to obtain an umbilical cord tissue; adding 0.1% collagenase type II into the umbilical cord tissue, and digesting under a preset digestion condition to obtain a first transition body; filtering the first transition body, adding an equal amount of first culture medium into the filtered liquid, centrifuging, and sucking a basic culture medium to obtain a second transition body; adding a second culture medium into the second transition body for heavy suspension, and performing adherent culture under a first preset culture condition to obtain a third transition body; sucking a second culture medium, nonadherent cells and cell fragments in the third transition body, and adding the second culture medium again to culture under a second preset culture condition to obtain a fourth transition body; and carrying out cell subculture on the fourth transition body under a third preset culture condition to obtain a subculture product.

In the preparation method of the invention, the umbilical cord is pretreated to obtain the umbilical cord tissue, which comprises the following steps: cleaning the umbilical cord by using alcohol and normal saline; cutting the cleaned umbilical cord into 1-3cm sections; the inner side of the umbilical vein of the umbilical cord is cut open, and the umbilical vein, the umbilical artery and the umbilical envelope are removed from the umbilical cord to obtain umbilical cord tissue; and/or the preset digestion conditions are as follows: digesting in an incubator at 37 ℃ for 30min, and shaking for 1 time every 15 min; and/or adding a second culture medium into the second transition body for heavy suspension, and performing adherent culture under a first preset culture condition to obtain a third transition body, wherein the third transition body comprises: resuspending the second transition body with a second medium at 5000/cm²Inoculating in a culture dish, and standing at 37 deg.C and 5% CO₂Culturing in the incubator; and after culturing for 8-15h in an adherent manner, sucking off the second culture medium, the nonadherent cells and cell fragments to obtain a third transition body.

In the preparation method of the present invention, the pre-treating the subculture product to obtain a first liquid comprises: adding physiological saline into the subculture product for resuspension to obtain a first intermediate; performing freeze thawing and ultrasonic treatment on the first intermediate to obtain a second intermediate; filtering the second intermediate; centrifuging the filtered second intermediate and collecting the centrifuged liquid to obtain the first liquid.

In the preparation method of the present invention, before the mixing the first liquid and the second liquid, the method further comprises: adding 10-50ug/ml vitamin C, 1-10ug/ml insulin, and 1-10ug/ml human transferrin into physiological saline to obtain the second liquid.

In the preparation method of the invention, the freeze-drying protective agent comprises mannitol, trehalose and dextran-40; and/or, the intermediate liquid comprises: 5 to 15 percent of mannitol, 0.1 to 1 percent of trehalose, 0.5 to 1.5 percent of dextran-40, lyophilized powder stock solution and water.

In a second aspect of the invention, the invention provides a lyophilized powder prepared by the method for preparing the lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells in any one of the above embodiments.

In a third aspect of the invention, the invention provides a hair growth nutrient solution, which comprises freeze-dried powder and normal saline, wherein the weight ratio of the freeze-dried powder to the normal saline is 1: 200 of a carrier; the freeze-dried powder is prepared by adopting the preparation method of the freeze-dried powder for hair regeneration based on umbilical cord mesenchymal stem cells in any embodiment.

In a fourth aspect of the present invention, there is provided a method of regenerating hair, the method comprising: dissolving the freeze-dried powder in physiological saline to obtain a hair growth nutrient solution; wherein the weight ratio of the freeze-dried powder to the physiological saline is 1:100, respectively; the freeze-dried powder is prepared by adopting the preparation method of the freeze-dried powder for hair regeneration based on umbilical cord mesenchymal stem cells in any embodiment; the hair growth nutrient solution is smeared on the scalp of a patient with alopecia and is introduced.

The embodiment of the invention provides umbilical cord mesenchymal stem cells-based freeze-dried powder for hair regeneration, a preparation method of the freeze-dried powder, a hair growth nutrient solution and a hair regeneration method. The lyophilized powder is prepared by adopting umbilical cord mesenchymal stem cells to prepare a lyophilized powder stock solution; the freeze-drying protective agent and the freeze-drying powder stock solution are uniformly mixed and freeze-dried after being uniformly mixed, so that the hair conditioner is prepared, has a remarkable effect on at least one of promoting the hair follicle restoration and hair regeneration of a human body, and is beneficial to relieving the trend of alopecia and/or improving the hair state. In addition, the umbilical cord mesenchymal stem cells have fast proliferation and low immunogenicity, are medical waste and are easy to obtain. In addition, the freeze-dried powder of this application only need with normal saline dissolve can, convenient operation when using.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a schematic flow chart of a method for preparing lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells according to an embodiment of the present invention;

FIG. 2 is a schematic representation of a subculture product provided according to an embodiment of the present invention;

FIG. 3 is a schematic representation of free cells digested during passaging as provided by an embodiment of the present invention;

FIG. 4(a) is a schematic diagram of a patient 1 before using the hair growth tonic prepared in example 4 of the present invention;

FIG. 4(b) is a schematic view of patient 1 after 1 month using the hair growth tonic prepared in example 4 according to the present invention according to the method of use in example 5;

fig. 4(c) is a schematic diagram of patient 1 after two 2 months using the hair growth tonic prepared in example 4 of the present invention according to the method of use in example 5;

FIG. 5(a) is a schematic diagram of patient 2 before using the hair growth tonic prepared in example 4 of the present invention;

FIG. 5(b) is a schematic view of patient 2 after 1 month using the hair growth tonic prepared in example 4 according to the method of use in example 5;

fig. 5(c) is a schematic diagram of patient 2 after two 2 months using the hair growth tonic prepared in example 4 of the present invention according to the method of use in example 5;

FIG. 6(a) is a schematic diagram of a patient 3 before using the hair growth tonic prepared in example 4 of the present invention;

FIG. 6(b) is a schematic view of patient 3 after 1 month using the hair growth tonic prepared in example 4 according to the method of use in example 5;

fig. 6(c) is a schematic diagram of patient 3 after using the hair growth tonic prepared in example 4 of the present invention twice for 2 months according to the method of use in example 5.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

The invention provides umbilical cord mesenchymal stem cells-based freeze-dried powder for hair regeneration, a preparation method of the freeze-dried powder, a hair growth nutrient solution and a hair regeneration method. The lyophilized powder can be dissolved in physiological saline and applied on the surface of scalp to promote hair follicle repair and hair regeneration.

Referring to fig. 1, fig. 1 is a schematic flow chart of a method for preparing a lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells according to an embodiment of the present invention. As shown in fig. 1, the preparation method includes steps S101 to S103.

S101, preparing a freeze-dried powder stock solution by adopting umbilical cord mesenchymal stem cells.

Specifically, umbilical cord mesenchymal stem cells (UC-MSCs) are low-immunogenicity cells, exist in umbilical cord tissues of newborns and have strong immunoregulation function. The umbilical cord mesenchymal stem cells have rich sources and are convenient to collect.

The umbilical cord mesenchymal stem cells can not only be differentiated into tissue cells to repair tissue damage, but also secrete active substances such as cell factors and the like for promoting cell growth and regulating and controlling the surrounding environment to the outside of cells in the processes of growth, proliferation and differentiation, such as Stem Cell Factor (SCF), immunoregulatory factor (HGF, LIF), chemotactic factor, trophic factor for supporting progenitor cells (IL-6, FGF-2, PDGF-AA, PDGF-BB, EGF), angiogenesis factor (VEGF165, FGF-2, PDGF-AA, PDGF-BB, EGF), scar inhibiting factor (HGF, FGF-2), anti-apoptosis factor (VEGF165, FGF-2, HGF), wound healing related factor (IL-6, IL-8, TGF-beta, MCP-1, VEGF, GMCSF, TIMP-1), various types of collagen, various lysozyme and the like. Wherein SCF, VEGF, EGF, TGF-beta, FGF and HGF interact with each other, and can repair damaged hair follicle, promote differentiation of hair follicle stem cells, accelerate division of hair mother cells, and promote hair regeneration from root.

In some embodiments, the preparing of the lyophilized powder stock solution using umbilical cord mesenchymal stem cells comprises: carrying out primary extraction and subculture on the umbilical cord mesenchymal stem cells to obtain a subculture product; pretreating the subculture product to obtain a first liquid; and uniformly mixing the first liquid and the second liquid to obtain the freeze-dried powder stock solution.

Compared with the method for preparing the stock solution by collecting the cell culture supernatant and performing ultrafiltration concentration, the method for preparing the hair growth nutrient solution has the advantages that the stock solution of the freeze-dried powder is obtained by using a cell lysis technology, and uncertain influences on the subsequently prepared freeze-dried powder or hair growth nutrient solution are avoided.

In some embodiments, the primary extraction and subculturing of umbilical cord mesenchymal stem cells to obtain a subculture product comprises: carrying out umbilical cord pretreatment on an umbilical cord to obtain an umbilical cord tissue; adding 0.1% collagenase type II into the umbilical cord tissue, and digesting under a preset digestion condition to obtain a first transition body; filtering the first transition body, adding an equal amount of first culture medium into the filtered liquid, centrifuging, and sucking a basic culture medium to obtain a second transition body; adding a second culture medium into the second transition body for heavy suspension, and performing adherent culture under a first preset culture condition to obtain a third transition body; sucking off a second culture medium, nonadherent cells and cell fragments in the third transition body, and adding the second culture medium again to culture under a second preset culture condition to obtain a fourth transition body; and carrying out cell subculture on the fourth transition body under a third preset culture condition to obtain a subculture product.

The double antibody is mixed solution of streptomycin, and can be directly added into cell culture solution for cell culture. However, the involvement of diabodies in cell culture processes can have an uncertain effect on the final product. Compared with the method for preparing the stock solution by collecting the cell culture supernatant, performing ultrafiltration concentration and performing cell pretreatment, which relates to the double antibody preparation stock solution, the freeze-dried powder stock solution is obtained by using a cell lysis technology in the embodiment of the application, the double antibody is not involved in the process of preparing subculture cells, and uncertain influence on subsequently prepared freeze-dried powder or hair growth nutrient solution is avoided.

Induction factors are added in the early cell culture process, and the cost is high if cell pollution occurs; if no cell contamination occurs, the content of the effective components is unstable, and thus experimental data comparison occurs. In the preparation process of the freeze-dried powder stock solution in the embodiment of the application, the first culture medium for early cell culture does not use induction factors, the cost in the culture process is controllable, and the subculture product obtained by culture and the effective components added again can be qualitatively and quantitatively determined.

In some embodiments, the umbilical cord is subjected to umbilical cord pretreatment to obtain umbilical cord tissue, comprising: cleaning the umbilical cord by using alcohol and normal saline; cutting the cleaned umbilical cord into 1-3cm sections; the umbilical cord is dissected from the inside of umbilical vein of umbilical cord, and umbilical vein, umbilical artery and umbilical cord envelope are removed from umbilical cord to obtain umbilical cord tissue. The embodiment of the application can prevent the mixed cells from being mixed into the umbilical cord tissue.

In some embodiments, the predetermined digestion conditions are: digesting in 37 deg.C incubator for 30min, shaking every 15min for 1 time. Illustratively, the adding 0.1% collagenase type ii into the umbilical cord tissue to digest under the preset digestion conditions to obtain a first transition body, specifically comprising: the umbilical cord tissue was cut with scissors, and then placed in a 50ml centrifuge tube, 0.1% collagenase type II (DMEM/F12 for dilution) was added and dissolved in an incubator at 37 ℃ for 30min, and shaken 1 time every 15min, thereby obtaining a first transition body.

In some embodiments, the first transition body is filtered and an equal amount of the first culture medium is added to the filtered liquid for centrifugation and the basal medium is aspirated to obtain the second transition body. Illustratively, the first transition body in the centrifuge tube was poured onto a 100 mesh steel screen, the filtered liquid was collected, an equal amount of the base first medium was added, and transferred to a 50ml centrifuge tube at 2500rpm for 6 minutes.

Illustratively, the first medium is a basal medium, such as DMEM.

In some embodiments, the adding a second culture medium to the second transition body for resuspension and performing adherent culture under a first preset culture condition to obtain a third transition body comprises: resuspending the second transition body with a second medium at 5000/cm²Inoculating in a culture dish, and standing at 37 deg.C and 5% CO₂Culturing in the incubator, and culturing for 8-15h by adherence to obtain a third transition body. The amount of the second medium can be designed according to the amount of cells in the second transition body, for example, the amount of the second medium is 5-6 ml.

The second medium is different from the first medium. Illustratively, the second medium includes DMEM (dulbecco's modified eagle medium), human serum albumin, human transferrin, and selenious acid.

In some embodiments, the second predetermined culture condition is: and (3) culture environment: 37 ℃ and 5% CO₂(ii) a The culture time is 2-3 days.

In some embodiments, the third predetermined culture condition is: and (3) culture environment: 37 ℃ and 5% CO₂(ii) a The cell content of the subculture product is approximately 1250 ten thousand IU to 1500 ten thousand IU.

In some embodiments, the pre-treating the subculture product to obtain a first liquid comprises: adding physiological saline into the subculture product for resuspension to obtain a first intermediate; performing freeze thawing and ultrasonic treatment on the first intermediate to obtain a second intermediate; filtering the second intermediate; centrifuging the filtered second intermediate and collecting the centrifuged liquid to obtain the first liquid.

After adding physiological saline to 1000 ten thousand IU of cells, the cells were resuspended to obtain a first intermediate. And putting the first intermediate into a 15ml centrifugal tube, then putting the centrifugal tube filled with the first intermediate into a refrigerator with the temperature of-80 ℃ for repeated freeze thawing, and then putting the centrifugal tube under ultrasonic conditions for processing so as to carry out ultrasonic lysis on cells in the centrifugal tube, thus obtaining a second intermediate. The cumulative duration of freeze thawing and sonication was 30 min. Illustratively, each sonication is carried out for a period of 5 to 10 seconds, and sonication is carried out in a plurality of times, such as two, three, or more times.

Adopt the screen cloth of aperture not less than 20um to right the second midbody filters, places the liquid that collects after filtering in the high-speed refrigerated centrifuge, and the centrifugation condition is: 10000rpm, 4 ℃, 15 min. And collecting the centrifuged liquid to obtain a first liquid. The first liquid was stored at-20 ℃ for future use.

In some embodiments, before the mixing the first liquid and the second liquid, the method further includes: adding 10-50ug/ml vitamin C, 1-10ug/ml insulin, and 1-10ug/ml human transferrin into physiological saline to obtain the second liquid.

And after obtaining the freeze-dried powder stock solution, carrying out protein concentration measurement and pH measurement on the freeze-dried powder stock solution. The protein concentration of the prepared freeze-dried powder stock solution is more than or equal to 0.5mg/ml, and the pH value is 6.5-7.5. Illustratively, the protein concentration of the lyophilized powder stock solution is 1.1mg/ml, and the pH value is 7.3.

S102, uniformly mixing the freeze-drying protective agent and the freeze-drying powder stock solution to obtain an intermediate solution.

In some embodiments, the lyoprotectant includes mannitol, trehalose, and dextran-40. In some embodiments, the intermediate liquid comprises: 5 to 15 percent of mannitol, 0.1 to 1 percent of trehalose, 0.5 to 1.5 percent of dextran-40, lyophilized powder stock solution and water.

Illustratively, 5% -15% of mannitol, 0.1% -1% of trehalose and 0.5% -1.5% of dextran-40 are dissolved in ultrapure water until the solution is clear, then freeze-dried powder stock solution is added, and finally the volume is fixed to 1L to obtain uniformly dissolved intermediate solution.

S103, freeze-drying the intermediate solution under a preset freeze-drying condition to obtain the freeze-dried powder.

Compared with the method of collecting cell culture supernatant and performing ultrafiltration concentration to obtain freeze-dried powder, the intermediate solution is freeze-dried in the embodiment of the application, and the operation is simple.

In some embodiments, the lyophilizing the intermediate solution under a preset lyophilization condition to obtain the lyophilized powder includes: pre-freezing the intermediate solution for 4 hours at the temperature of not higher than-60 ℃; and (3) carrying out vacuum freeze-drying on the pre-frozen intermediate solution for 48h at the temperature of not higher than-60 ℃ to obtain the freeze-dried powder.

Illustratively, the intermediate solution is filtered in a 0.22um filter and the filtered liquid is collected in a sterile container. And (3) performing aseptic operation on the liquid in the aseptic container, subpackaging the liquid in 5ml penicillin bottles, and half pressing plugs. Each vial contained 3ml of liquid.

Pre-freezing for 4h in a freeze dryer, vacuum freeze-drying for 48h, wherein the temperature of the pre-freezing and the vacuum freeze-drying is not higher than-60 ℃, and the freeze-dried powder is obtained. The prepared freeze-dried powder is stored in an environment of 20 ℃ below zero for standby.

According to the preparation method of the embodiment, the umbilical cord mesenchymal stem cells are adopted to prepare the lyophilized powder stock solution; and (3) uniformly mixing the freeze-drying protective agent and the freeze-drying powder stock solution, and freeze-drying after uniform mixing to prepare the freeze-drying powder. The prepared freeze-dried powder has obvious effects on at least one of promoting the repair of human hair follicles, preventing alopecia and promoting hair regeneration, is beneficial to relieving the trend of alopecia and/or improving the hair state, and is mainly used for early and medium stages of androgenetic alopecia. In addition, the umbilical cord mesenchymal stem cells have fast proliferation and low immunogenicity, are medical waste and are easy to obtain. In addition, the freeze-dried powder of this application only need with normal saline dissolve can, convenient operation when using.

The embodiment of the invention also provides the lyophilized powder for hair regeneration based on the umbilical cord mesenchymal stem cells, and the lyophilized powder is prepared by adopting the preparation method of the lyophilized powder for hair regeneration based on the umbilical cord mesenchymal stem cells in any embodiment.

The freeze-dried powder of the embodiment of the invention is prepared by adopting umbilical cord mesenchymal stem cells to prepare a freeze-dried powder stock solution; the freeze-drying protective agent and the freeze-drying powder stock solution are uniformly mixed and freeze-dried after being uniformly mixed, so that the hair conditioner is prepared, has obvious effects on at least one of promoting hair follicle repair of a human body, preventing alopecia and promoting hair regeneration, and is beneficial to relieving the trend of alopecia and/or improving the hair state. In addition, the umbilical cord mesenchymal stem cells have fast proliferation and low immunogenicity, are medical waste and are easy to obtain. In addition, the freeze-dried powder of this application only need with normal saline dissolve can, convenient operation when using.

The embodiment of the invention also provides a hair growth nutrient solution which comprises freeze-dried powder and normal saline, wherein the weight ratio of the freeze-dried powder to the normal saline is 1: 100. The freeze-dried powder is prepared by adopting the preparation method of the freeze-dried powder for hair regeneration based on umbilical cord mesenchymal stem cells in any embodiment.

According to the hair growth nutrient solution provided by the embodiment of the invention, the lyophilized powder is prepared into lyophilized powder stock solution by adopting umbilical cord mesenchymal stem cells; the freeze-drying protective agent and the freeze-drying powder stock solution are uniformly mixed and freeze-dried after being uniformly mixed, so that the hair conditioner is prepared, has obvious effects on at least one of promoting hair follicle repair of a human body, preventing alopecia and promoting hair regeneration, and is beneficial to relieving the trend of alopecia and/or improving the hair state. In addition, the umbilical cord mesenchymal stem cells have fast proliferation and low immunogenicity, are medical waste and are easy to obtain. In addition, the freeze-dried powder of this application only need with normal saline dissolve can, convenient operation when using.

Specifically, the prepared freeze-dried powder is dissolved and shaken uniformly by normal saline to obtain the hair growth nutrient solution for later use. According to the hair growth nutrient solution provided by the embodiment of the application, the freeze-dried powder is dissolved in the physiological saline, and the sensitization rate can be reduced when the hair growth nutrient solution is applied to scalp parts of patients.

An embodiment of the present invention further provides a hair regeneration method, including: dissolving the freeze-dried powder in physiological saline to obtain a hair growth nutrient solution; wherein the weight ratio of the freeze-dried powder to the physiological saline is 1: 100. The freeze-dried powder is prepared by adopting the preparation method of the freeze-dried powder for hair regeneration based on the umbilical cord mesenchymal stem cells in any embodiment; the hair growth nutrient solution is smeared on the scalp of a patient with alopecia and is introduced.

In some embodiments, the scalp operation area of the patient is cleaned with saline prior to introduction of the hair growth promoting nutritional solution and then disinfected with iodophors. After the scalp operation area is disinfected, the hair growth nutrient solution is uniformly dripped on the scalp operation area, and the nano crystal or the nano needle is used for conducting the leading-in operation. Illustratively, each patient takes 20-40min to introduce the procedure. According to the hair regeneration method, the nano needles are added for disinfection, or the nano crystals are added for disinfection, and the freeze-dried powder is dissolved by using the physiological saline when the method is used, so that the sensitization rate can be reduced.

In the hair regeneration method, the lyophilized powder is prepared into lyophilized powder stock solution by adopting umbilical cord mesenchymal stem cells; the freeze-drying protective agent and the freeze-drying powder stock solution are uniformly mixed and freeze-dried after being uniformly mixed, so that the hair conditioner is prepared, has obvious effects on at least one of promoting hair follicle repair of a human body, preventing alopecia and promoting hair regeneration, and is beneficial to relieving the trend of alopecia and/or improving the hair state. In addition, the umbilical cord mesenchymal stem cells have fast proliferation and low immunogenicity, are medical waste and are easy to obtain. In addition, the freeze-dried powder of this application only need with normal saline dissolve can, convenient operation when using.

Hereinafter, the present invention will be described in detail with reference to examples thereof. These embodiments are merely examples provided to specifically illustrate the present invention, and it will be understood by those skilled in the art that the scope of the present invention is not limited by these embodiments.

Example 1: preparation of subculture product

After the umbilical cord connecting the infant end and the mother is cut off, the umbilical cord is immediately clamped by an umbilical cord clamp to prevent internal pollution. The umbilical cord with the umbilical cord clamps is filled into a sterile bag and sent to a laboratory. The umbilical cord was removed from the sterile bag in a clean bench, and soaked with 75% alcohol for 2min along with the umbilical cord clamps, wherein the alcohol was contained in 150mm petri dishes. After the pretreatment with alcohol, the umbilical cord and the umbilical cord clamps are quickly transferred into normal saline to be soaked and cleaned for 2 times, and each time is 2 min. After cleaning with physiological saline, the umbilical cord clamps are cut off, the umbilical cord is cut into long sections of 1-3cm, the umbilical cord is cut open from the inner side of the umbilical vein with scissors, the umbilical vein, the umbilical artery and the umbilical envelope are removed with forceps with teeth, and the mixed cells are prevented from being mixed, so that the umbilical cord tissue is obtained.

The umbilical cord tissue is cut with scissors, 1 piece of umbilical cord tissue (for example, a piece of umbilical cord tissue of 2cm length) is put into a 50ml centrifuge tube, 0.1% collagenase type II (diluted solution of DMEM/F12) is added and placed in an incubator at 37 ℃ for digestion for 30min, and the mixture is shaken every 15min for 1 time, so that a first transition body is obtained. After the digestion step is completed, the first transition body in the centrifuge tube is poured into a steel screen mesh of 100 meshes, the filtered liquid is collected, the same amount of first culture medium is added, the first transition body is transferred into a 50ml centrifuge tube, centrifugation is carried out at 2500rpm for 6 minutes, and the basic culture medium is sucked off, so that a second transition body is obtained.

Resuspending the second transition body with 5-6ml of second medium at 5000 pieces/cm²Inoculating in a culture dish, and standing at 37 deg.C and 5% CO₂Culturing in an incubator, and performing adherent culture for 8-15h to obtain a third transition body. The second medium, nonadherent cells and cell debris in the third transition body are aspirated off, and then the second medium is added again and continuously placed at 37 ℃ under 5% CO₂Culturing in an incubator. After further culturing for 2-3 days, the cells overgrow to about 90% to obtain a fourth transition body.

And carrying out cell passage operation on the fourth transition body. Taking a 15cm culture dish as an example, after the cell fusion degree exceeds 90%, carrying out cell digestion collection and counting, wherein the cell content is about 1250 ten thousand IU-1500 ten thousand IU, and obtaining a subculture product.

Referring to FIG. 2, FIG. 2 is a schematic representation of a subculture product according to an embodiment of the present invention. As can be seen from FIG. 2, the degree of cell fusion reached 90% or more, which is considered as a passable standard.

Illustratively, referring to fig. 3, fig. 3 is a schematic diagram of free cells digested during passaging according to an embodiment of the present invention, and it can be seen from the diagram that the state of the cells is that healthy cells are so-called live cells. Under a microscope visual field, the morphology of the healthy cells shows that the cells are uniform, bright, high in transparency and strong in refractivity.

Example 2: preparation of lyophilized powder stock solution

The subculture product from example 1 was pretreated: after adding physiological saline to 1000 ten thousand IU of cells, the cells were resuspended to obtain a first intermediate.

And (3) putting the first intermediate into a 15ml centrifugal tube, putting the centrifugal tube filled with the first intermediate into a refrigerator with the temperature of-80 ℃ for repeated freeze thawing, and after freeze thawing, putting the centrifugal tube under an ultrasonic condition for processing so as to ultrasonically crack cells in the centrifugal tube, thereby obtaining a second intermediate. The cumulative duration of freeze thawing and sonication was 30 min. The ultrasonic cracking is carried out for a plurality of times with the time length of 5-10 seconds each time.

And filtering the second intermediate by using a screen with the aperture not less than 20um, placing the collected liquid after filtration into a high-speed freezing centrifuge, and centrifuging for 15min at 10000rpm and 4 ℃. And collecting the centrifuged liquid to obtain a first liquid. The first liquid was stored at-20 ℃ for future use.

4mg of vitamin C, 0.5mg of insulin, and 0.8mg of human transferrin were added to 50ml of physiological saline to obtain a second liquid. And uniformly mixing the first liquid and the second liquid according to the ratio of 1:1 to obtain the freeze-dried powder stock solution.

And (4) carrying out protein concentration determination and pH determination on the freeze-dried powder stock solution. The protein concentration of the prepared freeze-dried powder stock solution is 1.1mg/ml, and the pH value is 7.3.

Example 3: preparation of lyophilized powder

Weighing 85g of mannitol, 6g of trehalose and 8g of dextran 40, dissolving in ultrapure water until the mixture is clear, adding the freeze-dried powder stock solution prepared in the embodiment 2, and finally fixing the volume to 1L to obtain a uniformly dissolved intermediate solution.

Filtering the intermediate solution with a 0.22 μm filter, and collecting the filtered liquid in a sterile container to obtain a sterile liquid. And (3) performing aseptic operation on the aseptic liquid in the aseptic container, subpackaging the aseptic liquid into 5ml penicillin bottles (the filling amount is 3ml), and half pressing and plugging.

Placing the penicillin bottle filled with the sterile liquid in a freeze dryer, and pre-freezing for 4 hours at the temperature of not higher than-60 ℃. And after pre-freezing is finished, performing vacuum freeze-drying for 48 hours at the temperature of not higher than-60 ℃ to obtain freeze-dried powder. Storing the obtained lyophilized powder at-20 deg.C.

Example 4

The freeze-dried powder prepared in the embodiment 3 is dissolved and shaken evenly by normal saline to obtain the hair growth nutrient solution for standby. Wherein the weight ratio of the freeze-dried powder to the physiological saline is 1: 100.

Example 5

Wiping and disinfecting: firstly, the scalp operation area is cleaned by normal saline, and then the scalp operation area is disinfected by iodophor for the second time.

And (3) importing operation: the hair growth nutrient solution prepared in the example 4 is uniformly dripped on a scalp operation area, the introduction operation is carried out by using a nanocrystalline or a nano needle, the redundant residual liquid can be continuously massaged until the residual liquid is completely absorbed, and the operation is finished after the introduced liquid at the scalp part is completely absorbed.

Example 6

After biological safety identification, 3 patients with androgenetic alopecia in early and middle stages are selected as patients using the hair growth nutrient solution, and the patients are used for thinning, softening or losing hair according to the using method of the hair growth nutrient solution under the condition that the patients agree with the conditions. And regularly photographing to observe the effects of alleviating the hair loss tendency, improving the hair state or growing hair.

Patient 1: male, 50 years old. Patient 2: female, 50 years old. Patient 3: male, 51 years old.

Fig. 4(a), fig. 5(a) and fig. 6(a) are schematic diagrams of patient 1, patient 2 and patient 3 before using the hair growth nutrient solution prepared in example 4 of the present invention. Fig. 4(b), fig. 5(b), and fig. 6(b) are schematic views of patient 1, patient 2, and patient 3, respectively, after using the hair growth tonic prepared in example 4 of the present invention once for 1 month according to the method of use in example 5. Fig. 4(c), fig. 5(c), and fig. 6(c) are schematic views of patient 1, patient 2, and patient 3, respectively, after using the hair growth tonic prepared in example 4 of the present invention for two 2 months according to the method of use in example 5.

Each patient uses the hair growth nutrient solution at a fixed time of each month.

It is known that 3 of 3 cases of androgenetic alopecia have significant efficacy in early and middle stages. After the hair growth nutrient solution provided by the embodiment of the invention is applied to treat a patient suffering from alopecia in combination with the nanocrystal or the nanoneedle for one month, the original hair can be obviously thinned and softened, or the color of the scalp at the alopecia part is changed to black, and villi grow out. After the hair growth nutrient solution is used for two months, the original hair becomes thin and soft, or the hair on the scalp of the alopecia part grows with fluff and is uniformly covered. This shows that the hair growth tonic prepared in example 4 is effective in alleviating the tendency of hair loss and improving the condition of hair.

While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for preparing lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells is characterized by comprising the following steps:

preparing a freeze-dried powder stock solution by adopting umbilical cord mesenchymal stem cells;

uniformly mixing the freeze-drying protective agent and the freeze-drying powder stock solution to obtain an intermediate solution;

and freeze-drying the intermediate solution under a preset freeze-drying condition to obtain the freeze-dried powder.

2. The preparation method of claim 1, wherein the preparation of the lyophilized powder stock solution by using umbilical cord mesenchymal stem cells comprises:

carrying out primary extraction and subculture on the umbilical cord mesenchymal stem cells to obtain a subculture product;

pretreating the subculture product to obtain a first liquid;

and uniformly mixing the first liquid and the second liquid to obtain the freeze-dried powder stock solution.

3. The method for preparing the umbilical cord mesenchymal stem cells according to claim 2, wherein the primary extraction and subculture of the umbilical cord mesenchymal stem cells are performed to obtain a subculture product, and the method comprises the following steps:

carrying out umbilical cord pretreatment on an umbilical cord to obtain an umbilical cord tissue;

adding 0.1% collagenase type II into the umbilical cord tissue, and digesting under a preset digestion condition to obtain a first transition body;

filtering the first transition body, adding an equal amount of first culture medium into the filtered liquid, centrifuging, and sucking a basic culture medium to obtain a second transition body;

adding a second culture medium into the second transition body for heavy suspension, and performing adherent culture under a first preset culture condition to obtain a third transition body;

sucking a second culture medium, nonadherent cells and cell fragments in the third transition body, and adding the second culture medium again to culture under a second preset culture condition to obtain a fourth transition body;

and carrying out cell subculture on the fourth transition body under a third preset culture condition to obtain a subculture product.

4. The method of claim 3, wherein the pretreating the umbilical cord to obtain umbilical cord tissue comprises:

cleaning the umbilical cord by using alcohol and normal saline;

cutting the cleaned umbilical cord into 1-3cm sections;

the inner side of the umbilical vein of the umbilical cord is cut open, and the umbilical vein, the umbilical artery and the umbilical envelope are removed from the umbilical cord to obtain umbilical cord tissue; and/or the presence of a gas in the gas,

the preset digestion conditions are as follows: digesting in an incubator at 37 ℃ for 30min, and shaking for 1 time every 15 min; and/or the presence of a gas in the gas,

adding a second culture medium into the second transition body for heavy suspension, and carrying out adherent culture under a first preset culture condition to obtain a third transition body, wherein the third transition body comprises: resuspending the second transition body with a second medium at 5000/cm²Inoculating in a culture dish, and standing at 37 deg.C and 5% CO₂Culturing in the incubator; and after culturing for 8-15h in an adherent manner, sucking off the second culture medium, the nonadherent cells and cell fragments to obtain a third transition body.

5. The method of claim 2, wherein said pre-treating said subculture product to obtain a first liquid comprises:

adding physiological saline into the subculture product for resuspension to obtain a first intermediate;

performing freeze thawing and ultrasonic treatment on the first intermediate to obtain a second intermediate;

filtering the second intermediate;

centrifuging the filtered second intermediate and collecting the centrifuged liquid to obtain the first liquid.

6. The method of claim 2, wherein prior to blending the first liquid and the second liquid, further comprising:

adding 10-50ug/ml vitamin C, 1-10ug/ml insulin, and 1-10ug/ml human transferrin into physiological saline to obtain the second liquid.

7. The method of claim 1, wherein the lyoprotectant comprises mannitol, trehalose, and dextran-40; and/or, the intermediate liquid comprises: 5 to 15 percent of mannitol, 0.1 to 1 percent of trehalose, 0.5 to 1.5 percent of dextran-40, lyophilized powder stock solution and water.

8. A lyophilized powder prepared by the method for preparing the lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells according to any one of claims 1 to 7.

9. The hair growth nutrient solution is characterized by comprising freeze-dried powder and normal saline, wherein the weight ratio of the freeze-dried powder to the normal saline is 1: 100; the freeze-dried powder is prepared by adopting the method for preparing the freeze-dried powder for hair regeneration based on umbilical cord mesenchymal stem cells according to any one of claims 1 to 7.

10. A method of regenerating hair, comprising:

dissolving the freeze-dried powder in physiological saline to obtain a hair growth nutrient solution; wherein the weight ratio of the freeze-dried powder to the normal saline is 1: 100; the freeze-dried powder is prepared by adopting the method for preparing the freeze-dried powder for hair regeneration based on umbilical cord mesenchymal stem cells according to any one of claims 1 to 7;

the hair growth nutrient solution is smeared on the scalp of a patient with alopecia and is introduced.