AU2020103493A4 - A method for isolating hair follicle stem cells - Google Patents
A method for isolating hair follicle stem cells Download PDFInfo
- Publication number
- AU2020103493A4 AU2020103493A4 AU2020103493A AU2020103493A AU2020103493A4 AU 2020103493 A4 AU2020103493 A4 AU 2020103493A4 AU 2020103493 A AU2020103493 A AU 2020103493A AU 2020103493 A AU2020103493 A AU 2020103493A AU 2020103493 A4 AU2020103493 A4 AU 2020103493A4
- Authority
- AU
- Australia
- Prior art keywords
- hair follicle
- follicle stem
- stem cells
- cells
- stem cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 210000003780 hair follicle Anatomy 0.000 title claims abstract description 111
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims abstract description 22
- 210000004918 root sheath Anatomy 0.000 claims abstract description 13
- 239000006143 cell culture medium Substances 0.000 claims abstract description 9
- 238000000746 purification Methods 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 17
- 239000012528 membrane Substances 0.000 claims description 16
- 239000003550 marker Substances 0.000 claims description 14
- 230000029087 digestion Effects 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- 229920000936 Agarose Polymers 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 239000002033 PVDF binder Substances 0.000 claims description 8
- 238000000684 flow cytometry Methods 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 8
- 238000010839 reverse transcription Methods 0.000 claims description 8
- 239000000523 sample Substances 0.000 claims description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 8
- 238000004113 cell culture Methods 0.000 claims description 7
- 229960005322 streptomycin Drugs 0.000 claims description 7
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 claims description 6
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 102000006495 integrins Human genes 0.000 claims description 6
- 108010044426 integrins Proteins 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 102100037589 OX-2 membrane glycoprotein Human genes 0.000 claims description 5
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims description 5
- 239000006285 cell suspension Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 4
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 4
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims description 4
- 102100029966 HLA class II histocompatibility antigen, DP alpha 1 chain Human genes 0.000 claims description 4
- 102100036242 HLA class II histocompatibility antigen, DQ alpha 2 chain Human genes 0.000 claims description 4
- 108010093061 HLA-DPA1 antigen Proteins 0.000 claims description 4
- 108010086786 HLA-DQA1 antigen Proteins 0.000 claims description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 4
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 4
- 238000009396 hybridization Methods 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 4
- 230000005847 immunogenicity Effects 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 4
- 108020004999 messenger RNA Proteins 0.000 claims description 4
- 230000003287 optical effect Effects 0.000 claims description 4
- 238000000751 protein extraction Methods 0.000 claims description 4
- 238000003753 real-time PCR Methods 0.000 claims description 4
- 238000009877 rendering Methods 0.000 claims description 4
- 239000012723 sample buffer Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 210000004556 brain Anatomy 0.000 claims description 3
- 238000002406 microsurgery Methods 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 abstract description 3
- 201000004384 Alopecia Diseases 0.000 description 6
- 238000012136 culture method Methods 0.000 description 6
- 210000004209 hair Anatomy 0.000 description 6
- 102100040443 Keratin, type I cytoskeletal 15 Human genes 0.000 description 5
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 description 4
- 208000024963 hair loss Diseases 0.000 description 4
- 230000003676 hair loss Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 102100040505 HLA class II histocompatibility antigen, DR alpha chain Human genes 0.000 description 3
- 108010067802 HLA-DR alpha-Chains Proteins 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 210000002337 follicle stem cell Anatomy 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000004761 scalp Anatomy 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 108010066330 Keratin-15 Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 231100000360 alopecia Toxicity 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 108010066302 Keratin-19 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000008730 Nestin Human genes 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- 238000010806 PrimeScriptTM RT Reagent kit Methods 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000009583 hair follicle growth Effects 0.000 description 1
- 230000003661 hair follicle regeneration Effects 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 230000003660 hair regeneration Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000005055 nestin Anatomy 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000001732 sebaceous gland Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Classifications
-
- 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0627—Hair cells
- C12N5/0628—Hair stem cells; Hair progenitors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
- C12N2506/092—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells from hair cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4742—Keratin; Cytokeratin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70539—MHC-molecules, e.g. HLA-molecules
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
- G01N2333/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Developmental Biology & Embryology (AREA)
- Dermatology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a method for culturing hair follicle stem cells, which is
obtained by culture and purification in the hair follicle stem cell culture medium with the intact
outer root sheath of hair follicle. The method has the advantages of short cycle, simple
operation and high feasibility, which is suitable for commercialization.
Description
PATENTS ACT 1990
A method for isolating hair follicle stem cells
The invention is described in the following statement:-
A method for isolating hair follicle stem cells
The invention relates to the field of genetic engineering technology and biological
medicine, in particular to a hair follicle stem cell culture method.
Hair follicle stem cells (HFSCs) are a group of multipotential stem cells which exist
in the bulge of outer root sheath of hair follicle, which can self-renew and proliferate. They
can differentiate into hair follicle, sebaceous gland and epidermis, and play an important
role in hair regeneration. Studies have shown that human hair follicle stem cells express
various cell surface molecular markers such as CD200, keratin 15 (KI5), keratin 19 (K9),
integrin 6, integrin Pl, Nestin, etc. However, they do not express endothelial cell marker
molecule CD31; In addition, CD34, a marker molecule of mouse hair follicle stem cells, is
not expressed either. With these positive and negative marker molecules, researchers can
better isolate and purify hair follicle stem cells, which accelerates the research process of
hair follicle stem cells. Compared with embryonic stem cells and other adult stem cells,
hair follicle stem cells have the advantages of abundant sources, convenient materials, no
harm to the body and no ethical problems. It has been reported in the literature that hair is
grown in nude mice by transplanting hair follicle stem cells, which proves that
transplanting healthy hair follicle stem cells has the potential to repair damaged hair
follicles.
However, hair follicle stem cells may have a complex mechanism in promoting hair
regeneration. Garza et al. found that there was no significant difference in the number of
hair follicle stem cells between hair loss tissues and scalp tissues without hair loss, but hair
follicle stem cells in hair loss scalp tissues could not produce source cells for hair growth,
which indicated that hair follicle stem cells in hair loss scalp tissues might have defects.
The research of Matsumura et al. shows that DNA damage accumulated by hair follicle
stem cells will prevent them from working. Other studies have shown that the proliferation
and differentiation of hair follicle stem cells are affected by their surrounding
microenvironment; The signal from the surrounding hair follicles will affect the activity of
hair follicle stem cells, thus stimulating hair follicle growth. Therefore, the study on the
mechanism of hair follicle stem cells promoting hair follicle regeneration is helpful to
promote its clinical application. In addition, although hair follicle stem cells have definite
cell surface marker molecules, the operation of their isolation and culture is complicated
and the time period is long. In view of this, the present invention studies the culture method
of hair follicle stem cells and explores the feasibility of using allogeneic hair follicle stem
cells to treat pathological alopecia.
The purpose of the invention is to study the culture method of hair follicle stem cells,
and to explore the feasibility of treating pathological alopecia with allogeneic hair follicle
stem cells.
The method for culturing hair follicle stem cells, which is characterized in that it is obtained by culture and purification in the hair follicle stem cell culture medium with the intact outer root sheath of hair follicle.
The culture method is characterized in that it comprises the following steps:
(1) The intact hair follicle is taken from the occipital part of the brain and repeatedly
washed with penicillin-streptomycin PBS solution;
(2) The protuberance of the outer root sheath of the hair follicle is cut by microsurgery
under the anatomical microscope and put into the culture dish. The hair follicle stem cell
culture medium is incubated in the incubator of 5% C02 at 37 °C;
(3) The next day, adding 1.5mL of the same amount of culture medium, observing
under the inverted microscope if the spindle-shaped cells grow out of the bulge of the outer
root sheath of the hair follicle. After 80% fusion, subculturing the cells;
(4) After digesting the hair follicle stem cells, preparing a hair follicle stem cell
suspension of 1.Ox105 cells /mL with the hair follicle stem cell culture solution, and
counting;
(5) After mixing 1.2% agarose and DMEM / F12 medium at the ratio of 1:1, injecting
2ml of the mixed solution into the culture dish of 35cm2 . After cooling and solidifying, it
is put into the incubator for standby;
(6) After mixing 0.7% agarose and hair follicle stem cell culture medium in aseptic
test tube according to the ratio of 1:1, 1 / 10 volume of hair follicle stem cell suspension is
added into the tube, which is fully mixed and injected into the culture dish of medium I,
the addition amount is equal to medium I;
(7) The medium is changed every 2 days for a total of 10 days.
Further, the hair follicle stem cell culture solution contains 10% FBS, 100U/mL
streptomycin double antibody and 2ng/ml bFGF DMEM/F12.
In addition, in the culture medium I, after mixing 1.2% agarose and DMEM/F12
culture medium in the ratio of 1:1, taking 2ml of the mixed solution that is injected into a
cm2 culture dish. After cooling and solidifying, putting it into an incubator for later use.
Furthermore, the digestion solution of the hair follicle stem cell digestion contains 0.1%
pancreatin and 0.008% EDTA phosphate buffer.
Plus, the digestion condition is 37 °C for 3-5 minutes.
The culture method also includes the expression of surface marker molecules of hair
follicle stem cells, and the expression of surface marker molecules and negative marker
molecules of hair follicle stem cells is identified by flow cytometry: the 3rd generation hair
follicle stem cells are collected by digestion with 0.25% trypsin, washed once with PBS,
centrifuged and collected, and resuspended with PBS solution containing 1% BSA
respectively. Adding K15, K19, CD200, integrin 1, CD31, and CD34 fluorescent
antibodies added respectively, and labeling the cells by incubation at room temperature for
min in the dark. After washing with PBS, the expression of the labeled molecules is
detected by flow cytometry.
Further, the immunogenicity identification of hair follicle stem cells includes the
following steps:
1) WB detection of the expression of MHC-II molecules in hair follicle stem cells: collecting hair follicle stem cells, adding total cell protein extraction reagent to extract total protein and quantify; Taking 15kg of protein sample and adding 2xSDS gel sample buffer, which are boiled for 5min. Performing centrifugation at 6000rpm for 3min, then taking the supernatant and load the sample. After electrophoresis, transferring the protein on the gel to the PVDF membrane. After the membrane is transferred, it is sealed at room temperature for 2 hours. Putting the PVDF membrane into the hybridization bag and adding appropriately diluted primary antibodies at 4°C overnight. Adding the HRP-labeled secondary antibody corresponding to the primary antibody species, and carrying out shaking incubation at room temperature for 1 h at room temperature. After rinsing the filter membrane, adding developer for color rendering, and putting it into gel imager for exposure imaging; The optical density value of the target zone is analyzed by the Quantity
One software processing system.
2) Real-time fluorescence quantitative PCR to detect the expression of MHC-II
molecules in hair follicle stem cells: collecting hair follicle stem cells, extracting cell total
mRNA by Trizol method, identifying and quantifying by ultraviolet spectrophotometer; By
reverse transcription reaction kit, carrying out reverse transcription of total RNA to cDNA.
Beneficial effects:
The culture method of the invention is simple to operate and has a short time period.
The hair follicle stem cells cultured by the method are used for allogeneic normal human
hair follicle stem cells combined with drug molecules, so as to create a new idea suitable
for hair follicle repairing microenvironment.
Figure 1 represents the skin HE ofnude mice in each group after injection for 5 weeks.
In order to elaborate the technical scheme and technical purpose of the invention, the
following specific implementation mode further introduces the invention.
The method for culturing hair follicle stem cells, which is characterized in that it is
obtained by culture and purification in the hair follicle stem cell culture medium with the
intact outer root sheath of hair follicle.
The cultivation method according to claim 1, which is characterized in that it
comprises the following steps:
(1) The intact hair follicle is taken from the occipital part of the brain and repeatedly
washed with penicillin-streptomycin PBS solution;
(2) The protuberance of the outer root sheath of the hair follicle is cut by microsurgery
under the anatomical microscope and put into the culture dish. The hair follicle stem cell
culture medium is incubated in the incubator of 5% Co2 at 37 °C;
(3) The next day, adding 1.5mL of the same amount of culture medium, observing
under the inverted microscope if the spindle-shaped cells grow out of the bulge of the outer
root sheath of the hair follicle. After 80% fusion, subculturing the cells;
(4) After digesting the hair follicle stem cells, preparing a hair follicle stem cell suspension of 1.Ox105 cells /mL with the hair follicle stem cell culture solution, and counting;
(5) After mixing 1.2% agarose and DMEM / F12 medium at the ratio of 1:1, injecting
2ml of the mixed solution into the culture dish of 35cm2 . After cooling and solidifying, it
is put into the incubator for standby;
(6) After mixing 0.7% agarose and hair follicle stem cell culture medium in aseptic
test tube according to the ratio of 1:1, 1 / 10 volume of hair follicle stem cell suspension is
added into the tube, which is fully mixed and injected into the culture dish of medium I,
the addition amount is equal to medium I;
(7) The medium is changed every 2 days for a total of 10 days.
Further, the hair follicle stem cell culture solution contains 10% FBS, 100U/mL
streptomycin double antibody and 2ng/ml bFGF DMEM/F12.
In addition, in the culture medium I, after mixing 1.2% agarose and DMEM/F12
culture medium in the ratio of 1:1, taking 2ml of the mixed solution that is injected into a
cm2 culture dish. After cooling and solidifying, putting it into an incubator for later use.
Furthermore, the digestion solution of the hair follicle stem cell digestion contains 0.1%
pancreatin and 0.008% EDTA phosphate buffer.
Plus, the digestion condition is 37 °C for 3-5 minutes.
Further, the culture method also includes the expression of surface marker molecules
of hair follicle stem cells and the expression of surface marker molecules and negative
marker molecules of hair follicle stem cells which are identified by flow cytometry: the 3rd generation hair follicle stem cells are collected by digestion with 0.25% trypsin, washed once with PBS, centrifuged and collected, and resuspended with PBS solution containing
1% BSA respectively. Adding K15, K19, CD200, integrin 1, CD31, and CD34 fluorescent
antibodies added respectively, and labeling the cells by incubation at room temperature for
min in the dark. After washing with PBS, the expression of the labeled molecules is
detected by flow cytometry.
Further, the immunogenicity identification of hair follicle stem cells includes the
following steps:
1) WB detection of the expression of MHC-II molecules in hair follicle stem cells:
collecting hair follicle stem cells, adding total cell protein extraction reagent to extract total
protein and quantify; Taking 15kg of protein sample and adding 2xSDS gel sample buffer,
which are boiled for 5min. Performing centrifugation at 6000rpm for 3min, then taking the
supernatant and load the sample. After electrophoresis, transferring the protein on the gel
to the PVDF membrane. After the membrane is transferred, it is sealed at room temperature
for 2 hours. Putting the PVDF membrane into the hybridization bag and adding
appropriately diluted primary antibodies at 4°C overnight. Adding the HRP-labeled
secondary antibody corresponding to the primary antibody species, and carrying out
shaking incubation at room temperature for 1 h at room temperature. After rinsing the filter
membrane, adding developer for color rendering, and putting it into gel imager for
exposure imaging; The optical density value of the target zone is analyzed by the Quantity
One software processing system.
2) Real-time fluorescence quantitative PCR to detect the expression of MHC-II molecules in hair follicle stem cells: collecting hair follicle stem cells, extracting cell total mRNA by Trizol method, identifying and quantifying by ultraviolet spectrophotometer; By reverse transcription reaction kit, carrying out reverse transcription of total RNA to cDNA.
1. Isolation and culture of hair follicle stem cells
1) Isolation and culture of human hair follicle stem cells: recruiting 10 healthy
volunteers, extracting 8-10 intact hair follicles from the back of each volunteer's occipital
part of the head, and washing them repeatedly for 3 times with PBS solution containing
high concentrations of penicillin and streptomycin. Under the dissecting microscope, using
microsurgical scissors to cut off the bulge of the outer root sheath of the hair follicle and
place it in a 35cm2 petri dish; Adding 1.5ml of hair follicle stem cell culture medium
(containing 10% FBS, 100U/mL penicillin double antibody, 2ng /ml bFGF (DMEM/F12
medium), which are incubated at the incubator of 5% C02 at 37C for culture. The next
day, adding 1.5mL of the same amount of culture medium, observing under the inverted
microscope if the spindle-shaped cells grow out of the bulge of the outer root sheath of the
hair follicle. After 80% fusion, subculturing the cells. Cells subcultured to the third
generation are used for subsequent experiments.
2) The expression of surface marker molecules of hair follicle stem cells, and the
expression of surface marker molecules and negative marker molecules of hair follicle stem
cells which are identified by flow cytometry: the 3rd generation hair follicle stem cells are
collected by digestion with 0.25% trypsin, washed once with PBS, centrifuged and
collected, and resuspended with PBS solution containing 1% BSA respectively. Adding
K15, K19, CD200, integrin 1, CD31, andCD34 fluorescent antibodies added respectively, and labeling the cells by incubation at room temperature for 30 min in the dark. After washing with PBS, the expression of the labeled molecules is detected by flow cytometry.
2. Identification of immunogenicity of hair follicle stem cells
1) WB detection of the expression of MHC-II molecules in hair follicle stem cells:
collecting hair follicle stem cells, adding total cell protein extraction reagent to extract total
protein and quantify; Taking 15kg of protein sample and adding 2xSDS gel sample buffer,
which are boiled for 5min. Performing centrifugation at 6000rpm for 3min, then taking the
supernatant and load the sample. After electrophoresis, transferring the protein on the gel
to the PVDF membrane. After the membrane is transferred, it is sealed at room temperature
for 2 hours. Putting the PVDF membrane into the hybridization bag and adding
appropriately diluted primary antibodies at 4°C overnight. Adding the HRP-labeled
secondary antibody corresponding to the primary antibody species, and carrying out
shaking incubation at room temperature for 1 h at room temperature. After rinsing the filter
membrane, adding developer for color rendering, and putting it into gel imager for
exposure imaging; The optical density value of the target zone is analyzed by the Quantity
One software processing system.
2) Detection of MHC-II molecule expression in hair follicle stem cells by real-time
fluorescence quantitative PCR: collecting hair follicle stem cells, extracting cell total
mRNA by Trizol method, which is identified and quantified by ultraviolet
spectrophotometer. Adopting reverse transcription reaction kit (prime script rt reagent kit
with gDNA eraser, TAKARA) for the reverse transcription of Total RNA into cDNA.
According to the GenBank sequence, HLA-DPA1, HLA-DQA1, HLA-DRA 1 and intrinsic
GAPDH primers are designed and synthesized by adopting the Primer 5.0 software.The
QRT-PCR reaction is carried out according to the specification of the kit (Sybr Premix
Extaqtm II, Takara).The HLA-DPA1, HLA-DQA1, HLA-DRA 1, and internal reference
primer GAPDH are shown in the following table:
Gene name Forward primer reverse primer
HLA-DPA1 CTGGACAAGAAGGAGACCGT TCAATGTGGCAGATGAGGGT
HLA-DQA1 AACGCTACAACTCTACCGCT TCTGTGACTGACTGCCCATT
The basic principles and main features and advantages of the present invention have
been shown and described above. Those skilled in the industry should understand that the
present invention is not limited by the foregoing embodiments. The foregoing
embodiments and descriptions only illustrate the principles of the present invention.
Without departing from the spirit and scope of the present invention, the present invention
may have Various changes and improvements, these changes and improvements fall within
the scope of the claimed invention. The scope of protection claimed by the present
invention is defined by the appended claims and their equivalents.
Claims (9)
1. A method for isolating hair follicle stem cells, which is characterized in that it is
obtained by culture and purification in the hair follicle stem cell culture medium with the
intact outer root sheath of hair follicle.
2. The method according to claim 1, it is characterized in that it comprises the
following steps:
(1) The intact hair follicle is taken from the occipital part of the brain and repeatedly
washed with penicillin-streptomycin PBS solution;
(2) The protuberance of the outer root sheath of the hair follicle is cut by microsurgery
under the anatomical microscope and put into the culture dish. The hair follicle stem cell
culture medium is incubated in the incubator of 5% Co2 at 37 °C;
(3) The next day, adding 1.5mL of the same amount of culture medium, observing
under the inverted microscope if the spindle-shaped cells grow out of the bulge of the outer
root sheath of the hair follicle. After 80% fusion, subculturing the cells;
(4) After digesting the hair follicle stem cells, preparing a hair follicle stem cell
suspension of 1.Ox105 cells /mL with the hair follicle stem cell culture solution, and
counting;
(5) After mixing 1.2% agarose and DMEM / F12 medium at the ratio of 1:1, injecting
2ml of the mixed solution into the culture dish of 35cm2 . After cooling and solidifying, it
is put into the incubator for standby;
(6) After mixing 0.7% agarose and hair follicle stem cell culture medium in aseptic test tube according to the ratio of 1:1, 1 / 10 volume of hair follicle stem cell suspension is added into the tube, which is fully mixed and injected into the culture dish of medium I, the addition amount is equal to medium I;
(7) The medium is changed every 2 days for a total of 10 days.
3. The method according to claim 2, it is characterized in that the hair follicle stem
cell culture solution contains 10% FBS, 100U/mL streptomycin double antibody and
2ng/ml bFGF DMEM/F12.
4. The method according to claim 2, it is characterized in that: as for the culture
medium I, after mixing 1.2% agarose and DMEM/F12 culture medium in the ratio of 1:1,
taking 2ml of the mixed solution that is injected into a 35cm2 culture dish. After cooling
and solidifying, putting it into an incubator for later use.
5. The method according to claim 2, it is characterized in that: the digestion solution
of the hair follicle stem cell digestion contains 0.1% pancreatin and 0.008% EDTA
phosphate buffer.
6. The method according to claim 5, it is characterized in that the digestion condition
is 37 °C for 3-5 minutes.
7. The method according to any one of claims 1-6, which is characterized in that, it
also includes the expression of surface marker molecules of hair follicle stem cells, and the
expression of surface marker molecules and negative marker molecules of hair follicle stem
cells is identified by flow cytometry: the 3rd generation hair follicle stem cells are collected
by digestion with 0.25% trypsin, washed once with PBS, centrifuged and collected, and
resuspended with PBS solution containing 1% BSA respectively. Adding K15, K19,
CD200, integrin 1, CD31, and CD34 fluorescent antibodies added respectively, and
labeling the cells by incubation at room temperature for 30 min in the dark. After washing
with PBS, the expression of the labeled molecules is detected by flow cytometry.
8. The method according to any one of claim 1-claim 6, wherein the immunogenicity
identification of hair follicle stem cells includes the following steps:
1) WB detection of the expression of MHC-II molecules in hair follicle stem cells:
collecting hair follicle stem cells, adding total cell protein extraction reagent to extract total
protein and quantify; Taking 15kg of protein sample and adding 2xSDS gel sample buffer,
which are boiled for 5min. Performing centrifugation at 6000rpm for 3min, then taking the
supernatant and load the sample. After electrophoresis, transferring the protein on the gel
to the PVDF membrane. After the membrane is transferred, it is sealed at room temperature
for 2 hours. Putting the PVDF membrane into the hybridization bag and adding
appropriately diluted primary antibodies at 4°C overnight. Adding the HRP-labeled
secondary antibody corresponding to the primary antibody species, and carrying out
shaking incubation at room temperature for 1 h at room temperature. After rinsing the filter
membrane, adding developer for color rendering, and putting it into gel imager for
exposure imaging; The optical density value of the target zone is analyzed by the Quantity
One software processing system.
2) Real-time fluorescence quantitative PCR to detect the expression of MHC-II
molecules in hair follicle stem cells: collecting hair follicle stem cells, extracting cell total
mRNA by Trizol method, identifying and quantifying by ultraviolet spectrophotometer; By
reverse transcription reaction kit, carrying out reverse transcription of total RNA to cDNA.
9. The method according to claim 8, which is characterized in that the first antibodies
are HLA-DPA1, HLA-DQA1, hla-dral and internal reference GAPDH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020103493A AU2020103493A4 (en) | 2020-11-17 | 2020-11-17 | A method for isolating hair follicle stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020103493A AU2020103493A4 (en) | 2020-11-17 | 2020-11-17 | A method for isolating hair follicle stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020103493A4 true AU2020103493A4 (en) | 2021-01-28 |
Family
ID=74192143
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020103493A Ceased AU2020103493A4 (en) | 2020-11-17 | 2020-11-17 | A method for isolating hair follicle stem cells |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2020103493A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115011545A (en) * | 2022-06-06 | 2022-09-06 | 北京熙朵医疗美容门诊部有限公司 | Culture method of hair follicle stem cells |
-
2020
- 2020-11-17 AU AU2020103493A patent/AU2020103493A4/en not_active Ceased
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115011545A (en) * | 2022-06-06 | 2022-09-06 | 北京熙朵医疗美容门诊部有限公司 | Culture method of hair follicle stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101914490B (en) | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof | |
| Akiyama et al. | Lineage differentiation of mesenchymal stem cells from dental pulp, apical papilla, and periodontal ligament | |
| CN102191218B (en) | Complete medium and human amnion-derived mesenchymal stem cell culture method | |
| CN102344906B (en) | Hair follicle stem cell separation culture method | |
| CN102168065A (en) | Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof | |
| CN106834218A (en) | People's amnioic epithelium stem cell serum-free culture medium and its cultural method | |
| Lee et al. | Principal protocols for the processing of cultured meat | |
| CN105331579A (en) | Separation and culture method and application of human testis mesenchymal stem cells | |
| CN109628404A (en) | The construction method and purposes of Preadipocyte immortalized cell line under pigskin | |
| Perczel-Kovách et al. | STRO-1 positive cell expansion during osteogenic differentiation: A comparative study of three mesenchymal stem cell types of dental origin | |
| AU2020103493A4 (en) | A method for isolating hair follicle stem cells | |
| Nejaddehbashi et al. | Isolating human dermal fibroblasts using serial explant culture | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN112342186B (en) | Culture method of hair follicle stem cells | |
| CN108373990B (en) | Culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and method thereof | |
| CN109628388A (en) | With digestive enzyme compositions from placenta blood vessel separating mesenchymal stem cell | |
| CN105886462A (en) | Composition ADSCs for ADSCs culture and ADSCs culture method | |
| Qian et al. | One-step simple isolation method to obtain both epidermal and dermal stem cells from human skin specimen | |
| CN104818243A (en) | Separation method of placenta-derived fetal stem cells | |
| CN110499279A (en) | A method of induce human urine derived stem cells to hepatocyte differentiation | |
| CN106318979A (en) | Method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells | |
| KR101738715B1 (en) | Method for derivation of pluripotent stem cells using plant stem cells or callus extracts, and pluripotent stem cells produced using the same | |
| Wang et al. | Efficient isolation and high yield of epidermal cells from foreskin biopsies by dynamic trypsinization | |
| CN114563330A (en) | Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 | |
| CN102533641B (en) | External serum-free adult stem cell amplifies the method and nutrient solution thereof cultivated |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) |