WO2022247848A1 - Preparation method for and application of hair follicle mesenchymal stem cell - Google Patents

Abstract

Disclosed are a preparation method for a hair follicle mesenchymal stem cell and a hair follicle mesenchymal stem cell prepared thereby. Disclosed is a pharmaceutical application of the hair follicle mesenchymal stem cell in a drug for treating osteoporosis or diseases related to bone mass decrease.

Description

Preparation method and application of hair follicle mesenchymal stem cells technical field

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

Background technique

Mesenchymal Stem Cells (MSCs) are a type of undifferentiated adult stem cells that exist in differentiated tissues. They have the potential of self-renewal and multidirectional differentiation, and can be induced to differentiate into chondrocytes, osteoblasts, smooth muscle cells, etc. It is an ideal seed cell source for regenerative medicine. Mesenchymal stem cells also have a strong paracrine ability, secreting a variety of growth factors and cytokines that can promote angiogenesis and wound repair. Mesenchymal stem cells with self-renewal and multidirectional differentiation capabilities have been isolated from bone marrow, hair follicle, fat, pancreas, periosteum, synovium, skeletal muscle, skin, umbilical cord blood and other tissues.

Human hair follicle mesenchymal stem cells are located in the hair papilla in the middle of the hair follicle. As a type of adult stem cells derived from hair follicles, they have self-renewal and multidirectional differentiation potential. It plays an important role in the formation of the cycle. As an important source of stem cells, it also has the advantages of abundant sources, convenient acquisition, easy expansion, low immunogenicity, and less ethical controversy. It is the seed cell for vascular tissue engineering, nerve regeneration, and hair follicle reconstruction. important clinical implications. In order to obtain hair follicle-derived mesenchymal stem cells whose quality and quantity meet the needs of clinical treatment, the selection of isolation and culture methods is very important. Different isolation and culture methods will lead to differences in the success rate of cell isolation, proliferation and differentiation capabilities, and clinical treatment effects. Therefore, it is a problem that must be solved to find a preparation method of hair follicle mesenchymal stem cells with better separation success rate, proliferation and differentiation ability and therapeutic effect.

Osteoporosis, namely osteoporosis (osteoporosis), is a systemic bone disease in which bone density and bone quality decrease and bone microstructure is destroyed due to various reasons, resulting in increased bone fragility and prone to fracture. Osteoporosis is divided into primary and secondary two categories. Primary osteoporosis is divided into three types: postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis; secondary osteoporosis is due to Reduced bone mass caused by various systemic or endocrine and metabolic diseases. Among them, postmenopausal osteoporosis generally occurs in women within 5 to 10 years after menopause; senile osteoporosis generally refers to the osteoporosis that occurs in the elderly after the age of 70; and idiopathic osteoporosis mainly occurs in adolescents. The serious consequence of osteoporosis is the occurrence of osteoporotic fractures (fragility fractures), that is, fractures that can occur during minor trauma or daily activities, and the most common sites are the spine, hip and forearm. Fractures can lead to significantly increased morbidity and mortality in patients with osteoporosis. Therefore, it is extremely important to find an effective method for treating osteoporosis.

Contents of the invention

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

1) obtaining hair follicle tissue, preferably from human hair follicle tissue;

2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue; and

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

Wherein, step 2) can adopt enzymatic hydrolysis method to obtain hair follicle mesenchymal stem cells by separating from hair follicle tissue, and the enzymatic hydrolysis solution used can contain collagenase, dispase II (Dispase II) and calcium chloride.

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

Alternatively, the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.

In one of the preferred embodiments, the culture medium is ScienCell 7501 mesenchymal stem cell culture medium.

In one of the preferred embodiments, the enzymolysis solution may contain collagenase IV, dispase II (Dispase II) and calcium chloride.

The content of the present invention also includes the hair follicle mesenchymal stem cells obtained by the method of the present invention.

The content of the present invention also includes the application of the hair follicle mesenchymal stem cells in the preparation of drugs for the treatment of osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the Described osteoporosis is middle-aged and elderly osteoporosis or postmenopausal osteoporosis.

In one of the preferred embodiments, the drug is in a liquid dosage form, preferably an intravenous administration dosage form.

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

In one of the preferred embodiments, the administration method is intravenous administration, preferably intravenous drip or intravenous injection.

Description of drawings

FIG. 1 shows the result graph of the amplification factor of different generations of hair follicle mesenchymal stem cells prepared by the present invention in Example 1.

FIG. 2 shows the results of the total amount of hair follicle mesenchymal stem cells prepared by the present invention in Example 1 at different generations.

FIG. 3 shows the results of regulation of Th lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2. FIG.

FIG. 4 shows the results of regulation of Treg lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2. FIG.

Figure 5 shows the situation of human-derived hair follicle mesenchymal stem cells delaying the age-related bone loss of premature aging mice SAMP8 in Example 4. Among them, sub-figures A, B, C, D, and E respectively show the changes in bone volume fraction, trabecular thickness, number of trabecular bone, trabecular bone gap, and shape of cancellous bone in the proximal tibia.

specific implementation plan

In this specification, unless otherwise specified, the various components involved or their preferred components can be combined with each other to form a new technical solution.

In this specification, unless otherwise specified, all the embodiments and preferred embodiments mentioned herein can be combined with each other to form new technical solutions.

In this specification, if there is no special description, all the technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

The term "a" used in this specification means "at least one" unless otherwise specified.

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

In this specification, if there is no special description, the numerical value or numerical range, regardless of whether it is preceded by the word "about", covers the approximate range that can be understood by those skilled in the relevant art to be equivalent to the numerical value or numerical range, for example, the numerical value Or the range of ±10% of the end value, or the range of ±5%, ±3%, ±2%, ±1% or ±0.5%.

The invention provides a method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:

1) obtaining hair follicle tissue, preferably from human hair follicle tissue;

2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue; and

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

Alternatively, the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.

In some embodiments, step 1) obtaining the hair follicle tissue includes extracting the complete hair follicle, that is, removing the skin tissue surrounding the hair follicle, and peeling off the complete hair follicle tissue. For example, the specific steps may include: rinsing the skin tissue surrounding the hair follicle stored in the tissue storage solution with a cleaning solution, using ophthalmic forceps, a scalpel and other tools to peel off the complete hair follicle tissue, and storing it in a fresh tissue storage solution. middle. Alternatively, the extraction of intact hair follicles can also be obtained directly through a hair follicle extraction machine.

In some embodiments, step 2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue includes enzymatic hydrolysis or tissue attachment method, and the obtained hair follicle mesenchymal stem cells are also called primary hair follicle mesenchymal stem cells. Among them, the enzymatic hydrolysis method is to use enzymatic hydrolysis solution to enzymatically hydrolyze the hair follicle tissue to make it loose and facilitate stem cells to climb out. For example, the specific steps may include adding an enzymatic hydrolysis solution to enzymatically hydrolyze the hair follicle tissue, and placing it under suitable conditions for enzymatic hydrolysis. The tissue-adherence method mainly allows stem cells to climb out of the hair follicle tissue through adherent culture. For example, specific steps may include using a needle under a stereomicroscope to make an incision at the outer hair root sheath of the hair follicle, adding medium after the hair follicle tissue is completely adhered to the wall, and culturing statically under suitable conditions to wait for the cells around the hair follicle tissue to crawl out.

In some embodiments, the cultivation of the primary cells in step 3) includes resuspending the primary hair follicle mesenchymal stem cells in a culture medium, inoculating them in a culture vessel, and culturing them under suitable conditions.

In some embodiments, the cell subculture in step 4) includes adding digestive enzymes to digest the adherent cells from the wall of the vessel, after terminating the digestion, harvesting the cells, adding medium to resuspend, inoculating in a culture container, and placing them under suitable conditions nourish.

In some embodiments, in step 3) and step 4), the medium contains amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics. The culture medium can be prepared by oneself or purchased through commercial channels.

In some embodiments, the volume percentage of fetal calf serum in the culture medium is about 5-10%, preferably about 5% or about 10%.

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

In some embodiments, if the antibiotic is gentamicin, its concentration is about 50-100U/mL, preferably about 50U/mL; if the antibiotic is penicillin, its concentration is about 100-150U/mL, preferably about 100 U/mL; or if the antibiotic is streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL.

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

In some embodiments, the enzymatic hydrolysis solution used in step 2) includes collagenase, dispase II and calcium chloride.

In some embodiments, the collagenase may be collagenase I or collagenase IV, preferably collagenase IV.

In some embodiments, the concentration of collagenase is about 0.1-5 mg/mL, preferably about 1 mg/mL.

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

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

In some embodiments, when the enzymatic hydrolysis method is used in the step 2), the enzymatic hydrolysis time is about 1-3.5 hours, preferably about 3 hours.

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

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

In some embodiments, the cleaning solution used for cleaning tissues or cells in the preparation method may be basal medium or phosphate buffer, preferably basal medium and phosphate buffer containing antibiotics. For example, the antibiotic can be selected from gentamicin, penicillin, streptomycin or combinations thereof. In some embodiments, if the cleaning solution contains gentamicin, its concentration is about 50-100 U/mL, preferably about 50 U/mL; if the cleaning solution contains penicillin, its concentration is about 100-150 U/mL , preferably about 100 U/mL; or if the cleaning solution contains streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL.

In some embodiments, the digestive enzyme used for digestion of adherent cells may be trypsin or a stable trypsin replacement enzyme, preferably a stable trypsin replacement enzyme (eg TrypLE ^™ Express Enzyme (1×), no phenol red).

In some embodiments, in the step 2), the primary cells can be obtained by enzymatic hydrolysis at 5% CO ₂ at 37°C; more preferably, the primary cells can be obtained by enzymatic hydrolysis in a constant temperature oscillating metal bath at 37°C. Generation cells, and set the rotation speed to 600rpm.

In some embodiments, in the steps 3) and 4), primary cell culture and subculture can be carried out under the conditions of 5% CO ₂ and 37°C.

The invention also provides hair follicle mesenchymal stem cells prepared by the method of the invention. The hair follicle mesenchymal stem cells obtained in the invention have enhanced cell proliferation ability and enhanced immune regulation ability.

The present invention also includes the application of the hair follicle mesenchymal stem cells in the preparation of medicines for treating osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the bone Osteoporosis is middle-aged and elderly osteoporosis or postmenopausal osteoporosis.

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

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

In some embodiments, the administration method is intravenous administration, preferably intravenous infusion or intravenous injection.

The order of magnitude of the dosage can be, for example, 10 ⁶ -10 ⁹ cells/time. The administered dose may be a fixed dose (also known as a flat dose) or a dose based on the patient's weight. The frequency of administration and the period of administration are not particularly limited, for example, once every 10 days to 3 years, for a total of 1 to 10 administrations. In a preferred embodiment, the administration is administered every three months for a total of two administrations.

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

Example 1: Preparation of human hair follicle mesenchymal stem cells

Material

1) Preparation of enzymolysis solution:

Accurately weigh 0.1g of collagenase IV and dissolve it in 5mL of DMEM/F12 basal medium. After the collagenase IV is completely dissolved, mix it upside down and filter it through a 0.22μm filter to obtain a 20mg/mL collagenase IV stock solution; accurately weigh Dissolve 0.1g of dispase Ⅱ (Dispase Ⅱ) in 50mM hydroxyethylpiperazineethanesulfonic acid (HEPES). Filter to obtain 40mg/mL Dispase II storage solution; accurately weigh 0.11g of calcium chloride, dissolve it in 2mL of sterile water, mix it upside down, and after the calcium chloride is completely dissolved, filter through a 0.22μm filter to obtain 500mM chlorine Calcium storage solution.

Take 0.05 mL of the prepared 20 mg/mL collagenase Ⅳ stock solution, 0.05 mL of 40 mg/mL Dispase Ⅱ stock solution, and 0.01 mL of 500 mM calcium chloride stock solution and mix them with 0.89 mL of Hank's Balanced Salt Solution (HBSS). Homogenize, and thus prepare an enzymatic hydrolysis solution containing 1 mg/mL collagenase Ⅳ, 2 mg/mL Dispase Ⅱ and 5 mM calcium chloride.

2) Prepare cleaning solution: prepare a cleaning solution containing 50 U/mL gentamicin (purchased from Huazhong Pharmaceutical Co., Ltd.) and 10% fetal bovine serum in DMEM/F12 basal medium.

3) Prepare tissue storage solution: prepare tissue storage solution containing 50 U/mL gentamicin (purchased from Huazhong Pharmaceutical Co., Ltd.) and 10% fetal bovine serum in DMEM/F12 basal medium.

method:

1) Extraction of complete hair follicles:

Put the scalp tissue wrapped with human hair follicles into a petri dish containing 10mL of cleaning solution, rinse twice, soak the rinsed scalp tissue in the tissue storage solution, use elbow ophthalmic tweezers, and assist a scalpel to cut off For the skin epidermis, the left hand clamps the skin tissue laterally with ophthalmic tweezers, and the right hand uses micro tweezers to carefully separate the skin tissue wrapped around the hair follicles. The stripped hair follicles will carry a small portion of skin connective tissue, and the connective tissue around the hair follicle tissue is carefully separated using microscopic forceps under a stereo microscope. Place the stripped complete hair follicle tissue in a petri dish containing tissue storage solution.

2) Primary cell acquisition:

After obtaining the complete hair follicle tissue according to step 1), carefully place the hair follicle tissue at the bottom of the EP tube with microscopic tweezers, and use a pipette gun to suck out the tissue storage solution carried by the hair follicle tissue as much as possible. Add 8 μL of enzymatic hydrolysis solution to each hair follicle, and let it stand in a 5% CO ₂ incubator at 37°C for 3 hours, carefully flick the bottom of the tube every 1 hour, and mix gently. After 3 hours of enzymatic hydrolysis, it can be seen under the microscope that the outer hair root sheath of the hair follicle has been completely enzymatically hydrolyzed, and the hair shaft cannot be completely enzymatically hydrolyzed. Use a 100-1000μL pipette gun to gently pipette 30 times to mix the enzymolysis solution completely, draw up all the cell suspension obtained by enzymolysis, and then pass it through a 100μm cell sieve.

3) Primary cell culture:

Add the cell suspension after enzymatic hydrolysis and sieving to the culture flask, and then add ScienCell 7501 mesenchymal stem cell culture medium (purchased from ScienCell Research Laboratories) to resuspend. Place the culture flask in a 37°C, 5% _CO2 cell incubator for culture. On the fifth day and the ninth day, fresh medium was replaced to continue culturing. When the cell confluency reaches 90% or above, it can be harvested or passaged.

4) Cell passage:

The hair follicle mesenchymal stem cells prepared through the above steps were subcultured, and each passage was inoculated at a density of 5.0× ¹⁰³ cells/ ^cm2 into a culture medium containing ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories). In the T75 culture flask, observe that the cell density in the culture flask reaches more than 90%, discard the medium, wash twice with the cleaning solution, and discard the cleaning solution. Add 3 mL of stable trypsin replacement enzyme (TrypLE ^TM Express Enzyme (1×), no phenol red) to the cell pellet at the bottom of the culture flask, and place it at room temperature for 3 minutes for digestion. Use a 10mL pipette to add 6mL of PBS to the culture bottle to dilute the enzyme solution, take out the supernatant and place it in a 15mL centrifuge tube, then add 10mL of PBS to rinse the bottom of the bottle once, add the same rinse solution to the centrifuge tube, 1500r /min, centrifuge for 5 minutes, discard the supernatant, add 1mL of ScienCell 7501 mesenchymal stem cell medium to resuspend, and use AO/PI (AO (Acridine Orange) acridine orange, PI (Propidium Iodide) propidium iodide) double staining Apoptosis detection kit (DNA probe double-stained nucleus method, purchased from Shanghai Ruiyu Biotechnology Co., Ltd.) detects the number of cells harvested at each generation, and calculates the amplification factor of each generation. The experimental results are shown in Figures 1 and 2.

Experimental conclusion: the hair follicle mesenchymal stem cells prepared by the preparation method of the present invention can harvest a total of 3×10 ⁸ cells after passage P3, which meets the production requirements. In the P1-P3 generation, the cell amplification factor is more than 10 times, and in the P4-P7 generation, the cell expansion factor is more than 5 times, showing a strong cell proliferation ability.

Example 2: Comparison of the regulatory effects of human hair follicle mesenchymal stem cells cultured in different media on immune function

According to Example 1, human embryonic stem cell complete medium (purchased from Saiye Biotechnology Co., Ltd.) and amniotic fluid medium (purchased from Guangzhou Baidi Biomedicine Co., Ltd.) were used to replace ScienCell 7501 mesenchymal stem cell medium ( Purchased from ScienCell Research Laboratories) to prepare human hair follicle mesenchymal stem cells.

According to the following experimental method, the regulation effect of human hair follicle mesenchymal stem cells cultured in different media on immune function was compared.

1) Hair follicle mesenchymal stem cells regulate helper T cells (Th lymphocyte subsets)

Experimental method: Hair follicle mesenchymal stem cells obtained from different culture media were spread in twelve-well plates at a cell concentration of 1×10 ⁵ /mL/well, and after 24 hours of adherent culture, 5×10 ⁵ cells were inoculated in each well. Peripheral blood mononuclear cells (PBMC) were co-cultured so that the ratio of hair follicle mesenchymal stem cells and peripheral blood mononuclear cells in the wells was 1:5, and they were used as the test group. Peripheral blood mononuclear cells were cultured separately, and the number of inoculated cells was 5×10 ⁵ per well, which was used as a control group. Each well of the test group and the control group was added with phytohemagglutinin (Phytohemagglutinin, PHA), placed in 5% CO ₂ , incubated at 37°C for 67 hours, and then added a protein transport inhibitor (Protein Transport Inhibitor, purchased from BD GolgiStop ^™ ) and ionomycin (Ionomycin), continue to cultivate in the incubator for 5 hours. The peripheral blood mononuclear cells of the test group and the control group were collected, and the cytokines related to Th1 cells (CD4+ and IFN-γ+) and the cytokines related to Th17 cells (CD4+ and IL-17+) were detected by flow cytometry. ), the results are shown in Table 1 and Figure 3.

Table 1 Results of hair follicle mesenchymal stem cells cultured in different media regulating Th lymphocyte subsets

Medium type		ScienCell 7501 Medium	Complete Medium (Saiye Biological)	Amniotic fluid culture medium (Baidi Bio)
Th1 inhibition rate	-95.8%	-88.76%	-79.97%
Th17 inhibition rate	-84%	-73%	-twenty two%

Experimental conclusion: hair follicle mesenchymal stem cells can effectively inhibit the proliferation of pro-inflammatory lymphocyte subsets Th1 and Th17, and the effects of hair follicle mesenchymal stem cells cultured in different media are significantly different, among which ScienCell 7501 mesenchymal stem cell medium The cultured hair follicle mesenchymal stem cells had the best inhibitory effect.

2) Hair follicle mesenchymal stem cells regulate the role of regulatory T cells (Regulatory cell, Treg for short)

Experimental method: Hair follicle mesenchymal stem cells obtained from different culture media were spread in twelve-well plates at a cell concentration of 1×10 ⁵ /mL/well, and after 24 hours of adherent culture, 5×10 ⁵ cells were inoculated in each well. Peripheral blood mononuclear cells, so that the ratio of hair follicle mesenchymal stem cells and peripheral blood mononuclear cells in the wells is 1:5, were co-cultured as the test group. Peripheral blood mononuclear cells were cultured separately, and the number of inoculated cells was 5×10 ⁵ per well, which was used as a control group. Place them in a 5% CO ₂ , 37°C incubator for 72 hours. The 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 the Treg cell-related cytokines (CD4+, CD25+ and FoxP3+) were detected by flow cytometry , the results are shown in Table 2 and Figure 4.

Table 2 Results of hair follicle mesenchymal stem cells cultured in different media regulating Treg lymphocyte subsets

Medium type

ScienCell 7501 Medium

Complete Medium (Saiye Biological)

Amniotic fluid culture medium (Baidi Bio)

Treg promotion rate

191.78%

94.98%

75.34%

Experimental conclusion: hair follicle mesenchymal stem cells can promote the proliferation of Treg lymphocyte subsets, and the effects of hair follicle mesenchymal stem cells cultured in different media are significantly different, among which hair follicle mesenchymal stem cells cultured in ScienCell 7501 mesenchymal stem cell The promotion effect of mesenchymal stem cells is the best.

Example 3: Effect of Enzymolysis Solution Formula and Enzymolysis Time on Isolation of Human Hair Follicle Mesenchymal Stem Cells

Human-derived hair follicle mesenchymal stem cells were prepared as described in Example 1, except that the enzymatic hydrolysis solutions of the following formula 1, formula 2 and formula 3 were used instead of the enzymatic hydrolysis solution in Example 1 to enzymolyze the hair follicle tissue.

Enzyme solution formula 1: Accurately weigh 0.1 g of collagenase I and dissolve it in 5 mL of DMEM/F12 basal medium. After the collagenase I is completely dissolved, mix it upside down and filter it through a 0.22 μm filter to obtain 20 mg/mL collagenase Add 95 mL of Hank's Balanced Salt Solution (HBSS) to the Ⅰ storage solution and dilute it to obtain a 1 mg/mL collagenase Ⅰ hydrolysis solution.

Enzyme solution formula 2: Accurately weigh 0.1g of collagenase IV and dissolve in 5ml of DMEM/F12 basal medium. After the collagenase IV is completely dissolved, mix upside down and filter through a 0.22μm filter to obtain 20mg/mL collagen Add 95 mL of Hank's Balanced Salt Solution (HBSS) to the enzyme IV stock solution to dilute it to obtain a 1 mg/mL collagenase IV hydrolyzate.

Enzymolysis Solution Formula 3: Prepare the enzymolysis solution as described in Example 1, except that collagenase IV is replaced with collagenase I, thus obtaining a solution containing 1 mg/mL collagenase I, 2 mg/mL Dispase II and 5mM calcium chloride enzymatic solution.

After enzymatic hydrolysis for 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, and 3.5 hours, the effect of enzymatic hydrolysis on hair follicle tissue was observed under an optical microscope.

The results under the microscope showed that the inner hair root sheath and the outer hair root sheath were separated from the hair shaft after 1 hour of enzymatic hydrolysis. and outer root sheath can be dispersed into single cells after blowing. The longer the time, the more sufficient the enzymatic hydrolysis, but there is little difference between 3.5 hours of enzymatic hydrolysis and 3 hours of microscopic examination. The hair follicle mesenchymal stem cells prepared by enzymatic hydrolysis for 3 hours were cultured in ScienCell 7501 mesenchymal stem cell medium. The growth status was observed under a microscope and the number of finally harvested cells was counted. The results are shown in Table 3.

Table 3 The results of the number of hair follicle mesenchymal stem cells harvested by enzymolysis of hair follicle tissue with different enzymatic solutions

Enzyme solution formula	Culture days of primary hair follicle mesenchymal stem cells	Harvested cell number
Embodiment 1 enzymolysis solution formula	12 days	175000
Enzyme solution formula 1	12 days	55700

Enzyme solution formula 2	12 days	98600
Enzyme solution formula 3	12 days	91500

Conclusion: The effect of the combination of collagenase and dispase Ⅱ (Dispase Ⅱ) is significantly better than that of collagenase alone; and the effect of the combination of collagenase Ⅳ and dispase Ⅱ (Dispase Ⅱ) is better than that of collagenase Ⅰ and dispase Ⅱ (Dispase II) combination. Relatively speaking, the enzymolysis solution formula of Example 1 proliferated rapidly after enzymolysis, and the number of cells harvested at the same culture time was the largest.

Example 4: Treatment of Primary Osteoporosis and Osteopenia with Human Hair Follicle Mesenchymal Stem Cell Preparation

The inventors selected 20-week-old progeria mice SAMP8 as the research object, and injected low-dose (1×10 ⁵ cells/mouse) and high-dose (4×10 ⁵ cells/mouse) human hair follicle-derived mesenchymal stem cells ( Example 1), a total of 3 injections, with an interval of 1 week between the two injections, in order to explore the effect of human hair follicle mesenchymal stem cells on delaying the age-related bone loss of premature aging mice SAMP8. SAMP8 mice are a commonly used animal model of rapid aging. They are used in the study of age-related primary osteoporosis and osteoporosis because they can spontaneously develop osteopenia and even osteoporosis in the early stage. Specifically, it was divided into 5 groups for the test: 1. Normal control group, SAMR1 mice (12), which have the same genetic background as the progeria mouse SAMP8, but do not have the progeria phenotype; 2. Blank group, progeria mice SAMP8 (12 rats), without any treatment; 3. Vehicle group, progeria mice SAMP8 (12 rats), received the same dose of normal saline (cell resuspension solvent) tail vein injection, a total of 3 injections, every two injections The interval was 1 week; 4. In the low-dose hair follicle mesenchymal stem cell group, premature aging mice SAMP8 (12) received human hair follicle mesenchymal stem cell (1× ¹⁰⁵ /mouse) tail vein injection, a total of 3 injections , 1 week between each two injections; 5. Hair follicle mesenchymal stem cell high-dose group, progeria mice SAMP8 (12) received human hair follicle mesenchymal stem cells (4×10 ⁵ /only) tail vein injection , a total of 3 injections, with an interval of 1 week between each two injections.

The mice were sacrificed 2 months after the last injection of cells, and the tibiae were isolated, and the effect of exogenous stem cells on the aging bone loss of proximal tibial cancellous bone in SAMP8 mice was detected by micro-computed tomography (Micro-CT) , specifically detected the improvement of bone volume fraction and other bone microstructural parameters (such as bone trabecular thickness, number and gap) and their 3D images. The statistical results and 3D images are shown in Figure 5.

It can be seen from accompanying drawing 5 that high/low doses of hair follicle mesenchymal stem cells can improve mouse proximal tibial cancellous bone volume fraction (accompanying drawing 5A) and other bone microstructural parameters, including bone trabecular thickness ( Accompanying drawing 5B), number of bone trabeculae (accompanying drawing 5C) and bone trabecular space (accompanying drawing 5D); It can be seen from accompanying drawing 5E, 3D image also shows the same trend, namely injection of high/low dose of interstitial hair follicle Mesenchymal stem cells can effectively improve bone loss in progeria mice. The results in premature aging mice SAMP8 show that the human hair follicle mesenchymal stem cells prepared by the present invention can effectively delay age-related bone loss.

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

Claims (10)

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

   1) obtaining hair follicle tissue, preferably from human hair follicle tissue;

   2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue; and

   3) culturing the obtained hair follicle mesenchymal stem cells in the culture medium;

   Wherein, step 2) adopts enzymatic hydrolysis method to obtain hair follicle mesenchymal stem cells from hair follicle tissue, and the enzymatic hydrolysis solution used comprises collagenase, dispase II (Dispase II) and calcium chloride; or/and

   The medium contains amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.
2. The method according to claim 1, further comprising step 4): passage of the cultured hair follicle mesenchymal stem cells.
3. The method according to claim 1, wherein the fetal bovine serum volume percentage is 5-10%, preferably 5% or 10%;

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

   Preferably, the gentamicin concentration is 50-100U/mL, preferably 50U/mL; the penicillin concentration is 100-150U/mL, preferably 100U/mL; or/and, the streptomycin concentration is 0.10 - 0.15 mg/mL, preferably 0.10 mg/mL.
4. The method according to claim 1, wherein the culture medium is ScienCell 7501 mesenchymal stem cell culture medium.
5. The method of claim 1, further comprising one or more of the following features:

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

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

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

   The calcium chloride concentration is 5mM;

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

   The enzymatic hydrolysis temperature is 37°C.
6. The method according to claim 1, wherein in said step 1), the hair follicle tissue is contained in a tissue storage solution, and the tissue storage solution is a basal medium containing antibiotics and fetal bovine serum;

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

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

   More preferably, the gentamicin concentration is 50-100U/mL, preferably 50U/mL; the penicillin concentration is 100-150U/mL, preferably 100U/mL; or/and, the streptomycin concentration is 0.10-0.15 mg/mL, preferably 0.10 mg/mL.
7. The method according to claim 2, characterized in that, in the step 4), it includes digesting with a digestive enzyme, and the digestive enzyme is trypsin or a stable trypsin replacement enzyme, preferably a stable trypsin replacement enzyme.
8. The hair follicle mesenchymal stem cells obtained by the method according to any one of claims 1-7.
9. The application of hair follicle mesenchymal stem cells described in claim 8 in the preparation of medicines for the treatment of osteoporosis or osteopenia;

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

   Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
10. A method for treating osteoporosis or osteopenia; characterized in that, comprising the step of administering hair follicle mesenchymal stem cells according to claim 8 to patients suffering from osteoporosis or osteopenia;

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

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

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