CN115404203A

CN115404203A - Preparation method and application of mesenchymal stem cells of hair follicle

Info

Publication number: CN115404203A
Application number: CN202110576664.2A
Authority: CN
Inventors: 胡赓熙
Original assignee: Shanghai Iwu Stem Cell Technology Co ltd
Current assignee: Shanghai Iwu Stem Cell Technology Co ltd
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2022-11-29
Also published as: CN117157389A; WO2022247848A1

Abstract

The invention discloses a preparation method of hair follicle mesenchymal stem cells and the hair follicle mesenchymal stem cells prepared by the method. The invention also discloses the medical application of the mesenchymal stem cells of hair follicles in the medicaments for treating osteoporosis or osteopenia.

Description

Preparation method and application of mesenchymal stem cells of hair follicle

Technical Field

The application relates to a preparation method of hair follicle mesenchymal stem cells and medical application of the hair follicle mesenchymal stem cells.

Background

Mesenchymal Stem Cells (MSCs) are a type of undifferentiated adult Stem Cells that exist in differentiated tissues, have self-renewal and multidirectional differentiation potential, can be induced to differentiate into chondrocytes, osteoblasts, smooth muscle Cells, etc., and are a source of seed Cells ideal for regenerative medicine. The mesenchymal stem cells also have strong paracrine capacity, and various secreted growth factors and cytokines can promote angiogenesis and wound repair. Mesenchymal stem cells having self-renewal and multipotential differentiation ability have been isolated from tissues such as bone marrow, hair follicles, fat, pancreas, periosteum, synovium, skeletal muscle, skin, umbilical cord blood, and the like.

The human hair follicle mesenchymal stem cells are positioned in hair papilla in the middle of hair follicle bulbs, are used as a type of adult stem cells which are derived from hair follicles and have self-renewal and multidirectional differentiation potential, play an important role in hair follicle generation, self-renewal and cycle formation, are used as important stem cell sources, have the advantages of rich sources, convenient acquisition, easy amplification, low immunogenicity, few ethical disputes and the like, are seed cells for vascular tissue engineering, nerve regeneration and hair follicle reconstruction, and have important clinical treatment significance. In order to obtain the mesenchymal stem cells of hair follicles, the selection of separation and culture methods is very important, and different separation and culture methods can cause great differences in the separation success rate, proliferation and differentiation capacity and clinical treatment effect of the cells, so that the problem that the preparation method of the mesenchymal stem cells of hair follicles, which has good separation success rate, proliferation and differentiation capacity and treatment effect, needs to be solved is found.

Osteoporosis, osteoporosis (osteoporotis), is a systemic bone disease in which bone fracture easily occurs due to a decrease in bone density and bone quality caused by various causes, a destruction of bone microarchitecture, and an increase in bone fragility. Osteoporosis is divided into two major categories, primary and secondary. Primary osteoporosis is divided into postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis; secondary osteoporosis is a decrease in the amount of bone tissue due to various systemic or endocrine metabolic diseases. Wherein, the postmenopausal osteoporosis generally occurs in women within 5-10 years after menopause; senile osteoporosis generally refers to osteoporosis of the elderly after age 70; idiopathic osteoporosis occurs mainly in adolescents. A serious consequence of osteoporosis is the occurrence of osteoporotic fractures (fragility fractures), i.e. fractures that can occur when slightly traumatized or during daily activity, with good sites being the spine, hips and forearms. Fracture can lead to significant increases in disability and mortality in osteoporotic patients. Therefore, it is very important to find an effective treatment for osteoporosis.

Disclosure of Invention

One aspect of the present invention is a method for preparing mesenchymal stem cells of hair follicle, comprising:

1) Obtaining hair follicle tissue, preferably from a human;

2) Obtaining mesenchymal stem cells of hair follicle from the hair follicle tissue; and

3) Culturing the obtained mesenchymal stem cells of the hair follicle in a culture medium.

Wherein, the step 2) can adopt an enzymolysis method to separate the hair follicle mesenchymal stem cells from the hair follicle tissues, and the enzymolysis liquid can contain collagenase, neutral protease II (Dispase II) and calcium chloride.

Wherein the medium may comprise amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum, and antibiotics.

Or optionally, the method of the invention may further comprise step 4): and (4) carrying out passage on the cultured mesenchymal stem cells of the hair follicle.

In a preferred embodiment, the medium is ScienCell 7501 mesenchymal stem cell medium.

In a preferred embodiment, the enzymatic hydrolysate may comprise collagenase IV, neutral protease ii (Dispase ii) and calcium chloride.

The invention also provides mesenchymal stem cells of hair follicle obtained by the method.

The invention also comprises the application of the mesenchymal stem cells of hair follicles in preparing medicaments for treating osteoporosis and osteopenia diseases; preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is osteoporosis in the middle aged or postmenopausal.

In a preferred embodiment, the medicament is in a liquid dosage form, preferably an intravenous dosage form.

The present invention also includes a method of treating osteoporosis or osteopenia comprising: comprising the step of administering the mesenchymal stem cells of hair follicle to a patient suffering from osteoporosis or osteopenia. Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is osteoporosis in the middle aged or postmenopausal.

In a preferred embodiment, the mode of administration is intravenous administration, preferably intravenous drip or intravenous injection.

Drawings

FIG. 1 is a graph showing the result of the fold expansion of different generation cells of mesenchymal stem cells of hair follicle prepared in example 1 according to the present invention.

FIG. 2 is a graph showing the results of different generation numbers of mesenchymal stem cells of hair follicle prepared in example 1 according to the present invention.

FIG. 3 shows the results of the different medium culture of mesenchymal stem cells of hair follicle to regulate Th lymphocyte subpopulation in example 2.

Fig. 4 shows the results of the different medium culture of the mesenchymal stem cells of hair follicle to regulate the Treg lymphocyte subpopulation in example 2.

Fig. 5 shows the condition that human mesenchymal stem cells of hair follicle delay the aging bone loss of SAMP8 of the mice with premature aging in example 4. Wherein, the sub-graphs A, B, C, D and E respectively show the bone volume fraction, trabecular thickness, trabecular number, trabecular gap and morphological change of the proximal cancellous bone of the tibia.

Detailed description of the preferred embodiments

In the present specification, the components referred to or preferred components thereof may be combined with each other to form a new technical solution, if not specifically stated.

In the present specification, all embodiments and preferred embodiments mentioned herein may be combined with each other to form a new technical solution, if not specifically stated.

In the present description, all the technical features mentioned herein as well as preferred features may be combined with each other to form new technical solutions, if not specifically mentioned.

The terms "a" and "an" as used herein mean "at least one" if not otherwise specified.

The "ranges" disclosed herein are in the form of lower and upper limits. There may be one or more lower limits, and one or more upper limits, respectively. The given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range.

In the present specification, unless otherwise specified, the numerical value or numerical range including the antecedent "about" encompasses the approximate range equivalent to the numerical value or numerical range, such as the range of ± 10% of the numerical value or end value, and the range of ± 5%, 3%, 2%, 1% or ± 0.5%, as would be understood by a person skilled in the relevant art.

The invention provides a method for preparing mesenchymal stem cells of hair follicles, which comprises the following steps:

1) Obtaining hair follicle tissue, preferably from a human;

In some embodiments, the step 1) of obtaining hair follicle tissue comprises extracting intact hair follicles, i.e., removing skin tissue surrounding the hair follicles, and peeling the intact hair follicle tissue away. For example, the specific steps may include: rinsing the skin tissue wrapped with hair follicle stored in the tissue storage liquid with cleaning solution, peeling off the intact hair follicle tissue with tools such as ophthalmologic forceps and scalpel, and storing in fresh tissue storage liquid. Alternatively, the extraction of the intact follicle can also be obtained directly by the follicle extractor.

In some embodiments, the step 2) of obtaining the mesenchymal stem cells of the hair follicle from the hair follicle tissue comprises an enzymatic hydrolysis method or a tissue adherence method, and the obtained mesenchymal stem cells of the hair follicle are also called primary mesenchymal stem cells of the hair follicle. Wherein, the enzymolysis method is to utilize enzymolysis liquid to carry out enzymolysis on hair follicle tissues to ensure that the hair follicle tissues are loosened and the stem cells are easy to creep out. For example, the specific steps may include adding enzymatic hydrolysate to enzymatically digest the hair follicle tissue, and subjecting the hair follicle tissue to enzymatic digestion under suitable conditions. The tissue adherence method is mainly to make stem cells climb out of hair follicle tissues through adherence culture. For example, the specific steps may include cutting the outer root sheath of the hair follicle with a needle under a microscope, adding the culture medium after the hair follicle tissue is completely attached to the wall, and standing and culturing under appropriate conditions to allow the cells around the hair follicle tissue to climb out.

In some embodiments, the culturing of primary cells in step 3) comprises resuspending the primary mesenchymal stem cells in a culture medium, seeding in a culture vessel, and culturing under suitable conditions.

In some embodiments, the passaging of cells in step 4) comprises adding digestive enzymes to disassemble adherent cells from the walls of the cells, terminating digestion, harvesting the cells, adding culture medium to resuspend, inoculating into a culture vessel, and culturing under suitable conditions.

In some embodiments, in step 3), step 4), the medium comprises amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum, and antibiotics. The medium may be prepared by itself or may be purchased commercially.

In some embodiments, the culture medium is about 5-10%, preferably about 5%, fetal bovine serum by volume.

In some embodiments, the antibiotic is gentamicin, penicillin, streptomycin, or a combination thereof.

In some embodiments, the antibiotic, if gentamicin, is present at a concentration of about 50-100U/mL, preferably about 50U/mL; the concentration of the antibiotic, if penicillin, is about 100-150U/mL, preferably about 100U/mL; or about 0.10 to about 0.15mg/mL, preferably about 0.10mg/mL, if streptomycin is used as the antibiotic.

In some embodiments, in step 3), step 4), the medium is ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories).

In some embodiments, the enzymatic hydrolysate used in the step 2) of the enzymatic hydrolysis method comprises collagenase, neutral protease ii (Dispase ii) and calcium chloride.

In some embodiments, the collagenase may be collagenase i or collagenase iv, preferably collagenase iv.

In some embodiments, the collagenase is present in a concentration of about 0.1 to 5mg/mL, preferably about 1mg/mL.

In some embodiments, the neutral protease (Dispase II) concentration is about 1-4mg/mL, preferably about 2mg/mL.

In some embodiments, the calcium chloride concentration is about 5mM.

In some embodiments, the enzymatic hydrolysis in step 2) is carried out for a time period of about 1 to about 3.5 hours, preferably about 3 hours.

In some embodiments, the enzymatic hydrolysis temperature is about 37 ℃.

In some embodiments, when a tissue stock solution is used in the step 1) of obtaining hair follicle tissue, the tissue stock solution can be a basal Medium containing antibiotics and fetal bovine serum, such as DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F12) supplemented with antibiotics and fetal bovine serum. In some embodiments, the antibiotic is selected from gentamicin, penicillin, streptomycin, or a combination thereof. In some embodiments, the tissue stock solution, if it comprises gentamicin, is at a concentration of about 50-100U/mL, preferably about 50U/mL; the tissue stock has a concentration of about 100 to 150U/mL, preferably about 100U/mL, if the tissue stock comprises penicillin, and about 0.10 to 0.15mg/mL, preferably about 0.10mg/mL, if the tissue stock comprises streptomycin. In some embodiments, the fetal bovine serum is about 5% to about 10%, preferably about 10%, by volume.

In some embodiments, the washing solution used for washing the tissue or cells in the preparation method may be a basal medium or a phosphate buffer, preferably a basal medium containing antibiotics and a phosphate buffer. For example, the antibiotic may be selected from gentamicin, penicillin, streptomycin, or a combination thereof. In some embodiments, the wash solution, if comprising gentamicin, is at a concentration of about 50 to about 100U/mL, preferably about 50U/mL; the washing solution, if comprising penicillin, has a concentration of about 100-150U/mL, preferably about 100U/mL; or the wash solution, if comprising streptomycin, is at a concentration of about 0.10 to about 0.15mg/mL, preferably about 0.10mg/mL.

In some embodiments, the digestive enzyme used for adherent cell digestion may be trypsin or a stabilized trypsin replacement enzyme, preferably a stabilized trypsin replacement enzyme (e.g., trypLE) ^TM Express Enzyme(1×)，nophenol red)。

In some embodiments, at said step3) And 4) can be 5% CO ₂ Primary cell culture and subculture were carried out at 37 ℃.

The invention also provides the mesenchymal stem cells of hair follicle prepared by the method. The mesenchymal stem cells of hair follicles obtained by the invention have strong enhanced cell proliferation capacity and enhanced immunoregulation capacity.

The invention also includes the application of the mesenchymal stem cells of hair follicle in preparing the medicine for treating osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is osteoporosis in the middle aged or postmenopausal.

In some embodiments, the pharmaceutical dosage form is a liquid dosage form, preferably an intravenous dosage form.

The present invention also includes a method of treating osteoporosis or osteopenia disorders: comprising the step of administering the mesenchymal stem cells of hair follicle to a patient suffering from osteoporosis or osteopenia. Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is osteoporosis in the middle aged or postmenopausal.

In some embodiments, the mode of administration is intravenous administration, preferably intravenous drip or intravenous injection.

The present invention will be described in further detail with reference to examples. It should be understood that these examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example 1: preparation of human-derived hair follicle mesenchymal stem cells

Material

1) Preparing an enzymolysis liquid:

accurately weighing 0.1g of collagenase IV, dissolving the collagenase IV in 5mL of DMEM/F12 basal medium, after the collagenase IV is completely dissolved, turning upside down and uniformly mixing, and filtering by using a 0.22-micron filter to obtain 20mg/mL of collagenase IV stock solution; accurately weighing 0.1g of neutral protease II (Dispase II) to dissolve in 50mM of hydroxyethyl piperazine ethanesulfonic acid (HEPES), turning upside down and uniformly mixing, and filtering by a 0.22 mu m filter after the neutral protease II (Dispase II) is completely dissolved to obtain 40mg/mL of Dispase II stock solution; 0.11g of calcium chloride was accurately weighed, dissolved in 2mL of sterile water, mixed by inverting the top and bottom, and filtered through a 0.22 μm filter after the calcium chloride was completely dissolved, to obtain a 500mM calcium chloride stock solution.

0.05mL of 20mg/mL collagenase IV stock Solution, 0.05mL of 40mg/mL Dispase II stock Solution, and 0.01mL of 500mM calcium chloride stock Solution were mixed with 0.89mL of a balanced Salt Solution (Hank's balanced Salt Solution (HBSS) to prepare an enzymatic hydrolysate containing 1mg/mL collagenase IV, 2mg/mL Dispase II, and 5mM calcium chloride, respectively.

2) Preparing a cleaning solution: a washing solution containing 50U/mL of gentamicin (purchased from Huazhong pharmaceutical products Co., ltd.) and 10% fetal bovine serum was prepared in a DMEM/F12 basal medium.

3) Preparing a tissue storage solution: a tissue stock containing 50U/mL gentamicin (from Huazhou pharmaceutical industries, ltd.) and 10% fetal bovine serum was prepared in DMEM/F12 basal medium.

The method comprises the following steps:

1) Extraction of intact hair follicles:

putting the scalp tissue wrapped with the human hair follicle into a culture dish filled with 10mL of cleaning solution, rinsing twice, soaking the rinsed scalp tissue into the tissue storage solution, using elbow ophthalmic forceps, cutting off the skin epidermal tissue with an auxiliary surgical knife, transversely clamping the skin tissue with the elbow ophthalmic forceps by the left hand, and carefully separating the skin tissue wrapped with the hair follicle by the right hand using micro forceps. The dissected follicle will carry a small portion of the skin connective tissue, and microscopic forceps are used under a stereomicroscope to carefully separate the connective tissue around the follicular tissue. The stripped intact hair follicle tissue was placed in a petri dish containing a tissue storage solution.

2) Obtaining primary cells:

after obtaining the complete hair follicle tissue according to the step 1), carefully placing the hair follicle tissue at the bottom of an EP (EP) tube by using a micro-forceps, and sucking the tissue storage liquid carried by the hair follicle tissue as much as possible by using a liquid transfer gun. Adding enzymatic hydrolysate to each hair follicle in an amount of 8 μ L, 37 deg.C, 5% ₂ The incubator was allowed to stand for 3 hours, the bottom of the tube was gently flicked every 1 hour, and gently mixed. After 3 hours of enzymolysis, the outer hair root sheath layer of the hair follicle can be seen under the microscopeComplete enzymolysis, the hair shaft part can not be completely enzymolyzed. Gently blowing and beating for 30 times by using a 100-1000 mu L pipette, completely mixing the enzymolysis liquid, and sucking all the cell suspension obtained by enzymolysis.

3) Primary cell culture:

the cell suspension after enzymolysis was added to the culture flask, and then the ScienCell 7501 mesenchymal stem cell culture medium (purchased from ScienCell Research Laboratories) was added for resuspension. Placing the culture flask at 37 deg.C, 5% CO ₂ Culturing in a cell culture box. The culture was continued on the fifth and ninth days by replacing the fresh medium. When the cell fusion degree reaches 90% or above, the cells can be harvested or passaged.

4) Cell passage:

subculturing the mesenchymal stem cells of hair follicle prepared by the steps with the generation of 5.0 × 10 ³ Individual cell/cm ² The density of the cells was inoculated into a T75 flask containing ScienCell 7501 mesenchymal stem cell culture medium (purchased from ScienCell Research Laboratories), the cell density in the flask was observed to be more than 90%, the medium was discarded, and then the flask was washed twice with a washing solution, and the washing solution was discarded. 3mL of stabilized trypsin replacement enzyme (TrypLE) was added to the cell pellet at the bottom of the flask ^TM Express Enzyme (1 ×), no phenol red), left to stand at room temperature for digestion for 3 minutes. Adding 6mL PBS into a culture bottle by using a 10mL pipette to dilute the enzyme solution, stopping digestion, taking out the supernatant, placing the supernatant into a 15mL centrifuge tube, adding 10mL PBS to rinse the bottom of the bottle for 1 time, adding the rinse solution into the centrifuge tube, 1500r/min, centrifuging for 5 minutes, discarding the supernatant, adding 1mLScien cell 7501 mesenchymal stem cell culture medium for resuspension, detecting the number of cells harvested per generation by using AO/PI (AO (Acridine Orange) Acridine Orange, PI (Propidium Iodide) double-staining apoptosis detection kit (DNA probe double-staining cell nucleus method, purchased from Shanghai Rui Yu Biotech Co., ltd.), and calculating the amplification factor of each generation, wherein the experimental results are shown in figure 1 and figure 2.

And (4) experimental conclusion: the mesenchymal stem cells of hair follicle prepared by the preparation method can be harvested 3 multiplied by 10 after P3 generation ⁸ The total amount of cells of the cells meets the production requirement. P1-P3 generation, cell amplification multiple above 10 times, P4-P7 generation,the amplification factor is more than 5 times, and the strong cell proliferation capacity is shown.

Example 2: comparison of regulation effect of human-derived hair follicle mesenchymal stem cells cultured in different culture media on immune function

Human mesenchymal stem cells were prepared as described in example 1, using human embryonic stem cell complete medium (purchased from science biotechnology limited, seiko), amniotic fluid medium (purchased from bydy biomedicine limited, guangzhou) instead of ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories), respectively.

The regulation effect of human mesenchymal stem cells of hair follicle cultured in different culture media on the immune function is compared according to the following experimental method.

1) Mesenchymal stem cells of hair follicle regulate helper T cell (Th lymphocyte subgroup) function

The experimental method comprises the following steps: the mesenchymal stem cells of hair follicle obtained by different culture medium culture have the cell concentration of 1 multiplied by 10 respectively ⁵ Laying in twelve-well plate, culturing for 24 hr, inoculating 5 × 10 ⁵ Individual Peripheral Blood Mononuclear Cells (PBMC) were co-cultured so that the ratio of the number of mesenchymal stem cells of hair follicle and Peripheral blood mononuclear cells in the well was 1. Separately culturing peripheral blood mononuclear cells (5 × 10 cells) ⁵ One/well as control group. Phytohemagglutinin (PHA) was added to each well of the test group and the control group, and the concentration was 5% by weight ₂ Incubate at 37 ℃ for 67 hours, and add Protein Transport Inhibitor (Protein Transport Inhibitor, purchased from BD GolgiStop) per well ^TM ) And Ionomycin (Ionomycin), and incubation was continued in the incubator for 5 hours. Peripheral blood mononuclear cells of the test group and the control group were collected, and cytokines (CD 4+ and IFN-. Gamma. +) associated with Th1 cells and cytokines (CD 4+ and IL-17 +) associated with Th17 cells were detected by flow cytometry, and the results were shown in Table 1 and FIG. 3.

TABLE 1 results of regulating Th lymphocyte subpopulation by hair follicle mesenchymal stem cells cultured in different culture media

Kind of culture medium	ScienCell	7501 culture medium	Complete medium (Saiyehbio)	Amniotic fluid medium (Baydi organism)
					Th1 inhibitory Rate	-95.8％	-88.76％	-79.97％
Th17 inhibition rate	-84％	-73％	-22％

The experimental conclusion is that: the mesenchymal stem cells of hair follicles can effectively inhibit the proliferation of proinflammatory lymphocyte subpopulations Th1 and Th17, the action effects of the mesenchymal stem cells of hair follicles cultured by different culture media are obviously different, and the suppression effect of the mesenchymal stem cells of hair follicles cultured by the mesenchymal stem cell culture medium ScienCell 7501 is the best.

2) Function of mesenchymal stem cells of hair follicle for regulating Regulatory T cells (Treg)

The experimental method comprises the following steps: the mesenchymal stem cells of hair follicle obtained by different culture medium culture have the cell concentration of 1 multiplied by 10 respectively ⁵ The culture medium is spread in a twelve-well plate per mL/well, and inoculated with 5X 10 seeds per well after 24 hours of adherent culture ⁵ A single nucleus cell of peripheral blood to fill the hair follicle in the holeThe test groups were prepared by co-culturing the plasma stem cells and peripheral blood mononuclear cells at a ratio of 1. Separately culturing peripheral blood mononuclear cells (5 × 10 cells) ⁵ One/well as a control group. Put into 5% of CO ₂ And cultured in an incubator at 37 ℃ for 72 hours. Peripheral blood mononuclear cells of the test group and the control group were collected, the cells were stained according to the operation method of the Treg detection kit (purchased from Biolegend), and Treg cell-associated cytokines (CD 4+, CD25+, and FoxP3 +) were detected by a flow cytometer, and the results are shown in table 2 and fig. 4.

Table 2 results of regulating Treg lymphocyte subpopulation by mesenchymal stem cells cultured in different culture media

Kind of culture medium	ScienCell	7501 culture medium	Complete medium (Saiyehbio)	Amniotic fluid medium (Baydi organism)
					Treg promotion rate	191.78％	94.98％	75.34％

And (4) experimental conclusion: the mesenchymal stem cells of the hair follicle can promote the proliferation function of the Treg lymphocyte subpopulation, the action effects of the mesenchymal stem cells of the hair follicle cultured by different culture media have obvious difference, and the promotion effect of the mesenchymal stem cells of the hair follicle cultured by the ScienCell 7501 mesenchymal stem cell culture medium is the best.

Example 3: influence of enzymolysis liquid formula and enzymolysis time on separation of human hair follicle mesenchymal stem cells

Human-derived mesenchymal stem cells were prepared as described in example 1, except that the hair follicle tissue was enzymatically hydrolyzed using the enzymatic hydrolysates of the following formula 1, formula 2 and formula 3, respectively, instead of the enzymatic hydrolysate in example 1.

Formula 1 of the enzymolysis liquid: accurately weighing 0.1g of collagenase I, dissolving the collagenase I in 5mL of DMEM/F12 basal medium, reversing the collagenase I and mixing the collagenase I evenly after the collagenase I is completely dissolved, filtering the mixture by a 0.22-micron filter to obtain 20mg/mL of collagenase I stock Solution, and adding 95mL of Balanced Salt Solution (Hank's Balanced Salt Solution (HBSS) to dilute the Solution to obtain 1mg/mL of collagenase I enzymatic hydrolysate.

Formula 2 of the enzymatic hydrolysate: accurately weighing 0.1g of collagenase IV, dissolving the collagenase IV in 5mL of DMEM/F12 basic culture medium, after the collagenase IV is completely dissolved, turning upside down and uniformly mixing, filtering by using a 0.22 mu m filter to obtain 20mg/mL collagenase IV stock Solution, and adding 95mL Balanced Salt Solution (Hank's Balanced Salt Solution (HBSS) for dilution to obtain 1mg/mL collagenase IV enzymolysis Solution.

Formula 3 of the enzymolysis liquid: an enzymatic hydrolysate was prepared as described in example 1, except that collagenase IV was replaced with collagenase I, thereby obtaining an enzymatic hydrolysate containing collagenase I of 1mg/mL, dispase II of 2mg/mL and calcium chloride of 5mM.

The enzymolysis effect of the hair follicle tissue is observed under an optical microscope after enzymolysis for 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours and 3.5 hours.

The results under the microscope show that the inner hair root sheath and the outer hair root sheath can be separated from the hair shaft after 1 hour of enzymolysis, the inner hair root sheath and the outer hair root sheath can be obviously separated from the hair shaft after 3 hours of enzymolysis, and the inner hair root sheath and the outer hair root sheath can be dispersed into single cells after the enzymolysis through blowing. The longer the time, the more sufficient the enzymolysis is, but the microscopic examination of the enzymolysis for 3.5 hours is not much different from the 3 hours. The mesenchymal stem cells of hair follicle prepared by enzymolysis for 3 hours are taken to be cultured in a ScienCell 7501 mesenchymal stem cell culture medium, the growth state is observed under a microscope, and the number of the finally harvested cells is counted, and the result is shown in Table 3.

TABLE 3 results of the number of mesenchymal stem cells of hair follicle finally harvested from hair follicle tissue by enzymolysis of different enzymolysis solutions

Formula of enzymolysis liquid	Days for culturing primary mesenchymal stem cells	Cell number harvested
			Example 1 enzymatic hydrolysate formulation	12 days	175000
Formula 1 of enzymolysis liquid	12 days	55700
			Formula 2 of enzymolysis liquid	12 days	98600
Formula 3 of enzymolysis liquid	12 days	91500

And (4) conclusion: the effect of the combination of collagenase and neutral protease II (Dispase II) is obviously better than that of the single use of collagenase; and the effect of the combination of collagenase IV and neutral protease II (Dispase II) is better than that of the combination of collagenase I and neutral protease II (Dispase II). In contrast, the enzymatic hydrolysate formulation of example 1 proliferated rapidly after enzymolysis, and the number of cells harvested at the same culture time was the greatest.

Example 4: human-derived hair follicle mesenchymal stem cell preparation for treating primary osteoporosis and osteopenia

The inventor selects 20-week-old presenility mouse SAMP8 as a research object and injects a low dose (1 multiplied by 10) through tail vein ⁵ One/one) and high dose (4X 10) ⁵ One/one) of human-derived hair follicle mesenchymal stem cells (prepared in example 1) are injected for 3 times, and the interval between two injections is 1 week, so that the effect of the human-derived hair follicle mesenchymal stem cells on delaying the age-increasing bone loss of SAMP8 of the premature aging mouse is researched. SAMP8 mice are a common model of rapid aging animals, and are used for the study of age-related primary osteoporosis and osteopenia due to the spontaneous development of osteopenia or even osteoporosis at an early stage.

Specifically, the test was performed in 5 groups: 1. normal control group, SAMR1 mice (12), with the same genetic background as the precocious aging mouse, SAMP8, but without the precocious aging phenotype; 2. blank group, presenile mice SAMP8 (12), without any treatment; 3. the solvent group, early-aging mice SAMP8 (12), received the same dose of physiological saline (cell resuspension solvent) tail vein injection, injected 3 times in total, with 1 week interval between each injection; 4. hair follicle mesenchymal stem cell low dose group, presenile mouse SAMP8 (12) received human derived hair follicle mesenchymal stem cells (1X 10) ⁵ One or only) tail vein injection, 3 times of injection are carried out, and the interval between every two injections is 1 week; 5. high dose group of mesenchymal Stem cells of Hair follicle, presenile mouse SAMP8 (12) received mesenchymal Stem cells of human origin (4X 10) ⁵ One/one) tail vein injection, 3 times in total, with 1 week interval between each injection.

And (3) killing the mice 2 months after the last 1 cell injection, separating the tibia, detecting the improvement effect of the exogenous stem cells on the proximal cancellous bone aging bone loss of the tibia of the SAMP8 mice by means of a Micro-computer tomography (Micro-CT) technology, and specifically detecting the improvement condition of the bone body integral number and other bone microstructure parameters (such as the thickness, the number and the gap of the bone trabeculae) and 3D images thereof, wherein the statistical result and the 3D images are shown in an attached figure 5.

As can be seen from fig. 5, the high/low dose of mesenchymal stem cells of hair follicle can improve the integral number of the bone body of the proximal cancellous bone of mouse tibia (fig. 5A) and other bone microstructural parameters, including trabecular thickness (fig. 5B), trabecular number (fig. 5C) and trabecular gap (fig. 5D), to different degrees; as shown in fig. 5E, the 3D image shows the same trend that the bone loss phenomenon of the mice with premature aging can be effectively improved by injecting high/low dose of mesenchymal stem cells of hair follicle. The result of SAMP8 of a premature-aging mouse shows that the human-derived hair follicle mesenchymal stem cells prepared by the invention can effectively delay the age-increasing bone loss.

Although specific examples have been described herein, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. It is, therefore, intended that the appended claims cover all such modifications that are within the scope of this present invention.

Claims

1. A method for preparing mesenchymal stem cells of hair follicle, the preparation method comprising:

1) Obtaining hair follicle tissue, preferably from a human;

3) Culturing the obtained mesenchymal stem cells of the hair follicle in a culture medium;

wherein, the step 2) adopts an enzymolysis method to separate the hair follicle tissue to obtain the hair follicle mesenchymal stem cells, and the used enzymolysis solution contains collagenase, neutral protease II (Dispase II) and calcium chloride; or/and

the culture medium comprises amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.

2. The method of claim 1, further comprising step 4): and (4) carrying out passage on the cultured mesenchymal stem cells of the hair follicle.

3. The method according to claim 1, wherein the fetal bovine serum is 5-10% by volume, preferably 5%;

or/and the antibiotic is gentamicin, penicillin, streptomycin or a combination thereof;

preferably, the concentration of the gentamicin is 50-100U/mL, preferably 50U/mL; the concentration of the penicillin is 100-150U/mL, preferably 100U/mL; or/and the streptomycin concentration is 0.10-0.15mg/mL, preferably 0.10mg/mL.

4. The method according to claim 1, wherein the medium is ScienCell 7501 mesenchymal stem cell medium.

5. The method of claim 1, further having one or more of the following features:

the collagenase is collagenase I or collagenase IV, preferably collagenase IV;

the collagenase concentration is 0.1-5mg/mL, preferably 1mg/mL;

the concentration of the neutral protease II (Dispase II) is 1-4mg/mL, preferably 2mg/mL;

the concentration of the calcium chloride is 5mM;

the enzymolysis time of the enzymolysis method is 1-3.5 hours, preferably 3 hours;

the enzymolysis temperature is 37 ℃.

6. The method of claim 1, wherein in step 1), the hair follicle tissue is contained in a tissue stock solution, the tissue stock solution being a basal medium containing antibiotics and fetal bovine serum;

preferably, the volume percentage of the fetal bovine serum is 5% -10%, preferably 10%;

preferably, the antibiotic is selected from gentamicin, penicillin, streptomycin, or a combination thereof;

more preferably, the concentration of gentamicin is 50-100U/mL, preferably 50U/mL; the concentration of the penicillin is 100-150U/mL, preferably 100U/mL; or/and the streptomycin concentration is 0.10-0.15mg/mL, preferably 0.10mg/mL.

7. The method according to claim 2, wherein in step 4) comprises digestion with a digestive enzyme, which is trypsin or a stabilized trypsin replacement enzyme, preferably a stabilized trypsin replacement enzyme.

8. Mesenchymal stem cells of hair follicle obtained by the method of any one of claims 1 to 7.

9. Use of mesenchymal stem cells of hair follicles according to claim 8 in the preparation of a medicament for the treatment of osteoporosis or osteopenia;

preferably, the medicament is in a liquid dosage form, preferably an intravenous administration dosage form;

preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is osteoporosis in the middle aged or postmenopausal.

10. A method of treating osteoporosis or osteopenia; characterized by comprising the step of administering the mesenchymal stem cells of hair follicle according to claim 8 to a patient suffering from osteoporosis or osteopenia;

preferably, the mode of administration is intravenous administration, preferably intravenous drip or intravenous injection;