WO2022247848A1 - Procédé de préparation et application d'une cellule souche mésenchymateuse de follicule pileux - Google Patents

Procédé de préparation et application d'une cellule souche mésenchymateuse de follicule pileux Download PDF

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WO2022247848A1
WO2022247848A1 PCT/CN2022/094871 CN2022094871W WO2022247848A1 WO 2022247848 A1 WO2022247848 A1 WO 2022247848A1 CN 2022094871 W CN2022094871 W CN 2022094871W WO 2022247848 A1 WO2022247848 A1 WO 2022247848A1
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hair follicle
mesenchymal stem
osteoporosis
stem cells
tissue
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PCT/CN2022/094871
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胡赓熙
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上海我武干细胞科技有限公司
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Publication of WO2022247848A1 publication Critical patent/WO2022247848A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0666—Mesenchymal stem cells from hair follicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the application relates to a preparation method of hair follicle mesenchymal stem cells and a medical application of hair follicle mesenchymal stem cells.
  • MSCs Mesenchymal Stem Cells
  • Mesenchymal Stem Cells 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.
  • Osteoporosis namely osteoporosis (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.
  • 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.
  • One of the contents of the present invention is a method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:
  • 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.
  • the culture medium may contain amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.
  • the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.
  • the culture medium is ScienCell 7501 mesenchymal stem cell culture medium.
  • 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.
  • 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.
  • the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
  • the administration method is intravenous administration, preferably intravenous drip or intravenous injection.
  • 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. 4 shows the results of regulation of Treg lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2.
  • 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.
  • 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.
  • 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.
  • the numerical value or numerical range 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:
  • the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.
  • 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.
  • 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.
  • the extraction of intact hair follicles can also be obtained directly through a hair follicle extraction machine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the medium in step 3) and step 4), 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.
  • the volume percentage of fetal calf serum in the culture medium is about 5-10%, preferably about 5% or about 10%.
  • the antibiotic is gentamicin, penicillin, streptomycin or a combination thereof.
  • 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.
  • the medium is ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories).
  • the enzymatic hydrolysis solution used in step 2) includes collagenase, dispase II and calcium chloride.
  • the collagenase may be collagenase I or collagenase IV, preferably collagenase IV.
  • the concentration of collagenase is about 0.1-5 mg/mL, preferably about 1 mg/mL.
  • the concentration of the neutral protease (Dispase II) is about 1-4 mg/mL, preferably about 2 mg/mL.
  • the calcium chloride concentration is about 5 mM.
  • the enzymatic hydrolysis time is about 1-3.5 hours, preferably about 3 hours.
  • the enzymatic hydrolysis temperature is about 37°C.
  • the tissue storage solution when used in the process of obtaining hair follicle tissue in step 1), 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).
  • the antibiotic is selected from gentamicin, penicillin, streptomycin or combinations thereof.
  • 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%.
  • 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.
  • the antibiotic can be selected from gentamicin, penicillin, streptomycin or combinations thereof.
  • 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.
  • 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 TM Express Enzyme (1 ⁇ ), no phenol red).
  • a stable trypsin replacement enzyme eg TrypLE TM Express Enzyme (1 ⁇ ), no phenol red.
  • the primary cells in the step 2), 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.
  • 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.
  • 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.
  • the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
  • 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.
  • Example 1 Preparation of human hair follicle mesenchymal stem cells
  • 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.
  • 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
  • the left hand clamps the skin tissue laterally with ophthalmic tweezers
  • 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.
  • step 1) 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 ⁇ L 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.
  • 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).
  • a culture medium containing ScienCell 7501 mesenchymal stem cell medium purchased from ScienCell Research Laboratories.
  • 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.
  • 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.
  • the cell amplification factor is more than 10 times
  • 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
  • human embryonic stem cell complete medium purchased from Saiye Biotechnology Co., Ltd.
  • amniotic fluid medium purchased from Guangzhou Baidi Biomedicine Co., Ltd.
  • ScienCell 7501 mesenchymal stem cell medium Purchased from ScienCell Research Laboratories
  • PBMC Peripheral blood mononuclear cells
  • Peripheral blood mononuclear cells 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.
  • 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.
  • Hair follicle mesenchymal stem cells regulate the role of regulatory T cells (Regulatory cell, Treg for short)
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • HBSS Hank's Balanced Salt 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 0.22 ⁇ m 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.
  • HBSS Hank's Balanced Salt Solution
  • 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.
  • 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
  • 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.
  • 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.
  • Micro-CT micro-computed tomography

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Abstract

L'invention concerne un procédé de préparation d'une cellule souche mésenchymateuse de follicule pileux et une cellule souche mésenchymateuse de follicule pileux ainsi préparée. L'invention concerne une application pharmaceutique de la cellule souche mésenchymateuse de follicule pileux dans un médicament pour le traitement de l'ostéoporose ou de maladies liées à la diminution de la masse osseuse.
PCT/CN2022/094871 2021-05-26 2022-05-25 Procédé de préparation et application d'une cellule souche mésenchymateuse de follicule pileux WO2022247848A1 (fr)

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