CN116875537B - Method for constructing hair follicle organoids - Google Patents
Method for constructing hair follicle organoids Download PDFInfo
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- CN116875537B CN116875537B CN202311132945.4A CN202311132945A CN116875537B CN 116875537 B CN116875537 B CN 116875537B CN 202311132945 A CN202311132945 A CN 202311132945A CN 116875537 B CN116875537 B CN 116875537B
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Classifications
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- 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
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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- 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
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- C12N2502/092—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells hair cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention relates to a method for constructing a hair follicle organoid, which utilizes a human occipital hair follicle to obtain hair follicle stem cells and hair follicle fibroblasts, prepares the hair follicle stem cells and the hair follicle fibroblasts into single cell suspension, reversely cultures the single cell suspension, and then adds organoid culture solution to form the hair follicle organoid. According to the physiological mechanism of hair regeneration, hair follicle stem cells and hair follicle fibroblasts are utilized to directionally differentiate hair follicle organoids, the differentiated hair follicle organoids have cell characteristics and structures similar to those of normal primary hair follicle structures of human beings, and can be frozen and revived.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for constructing a hair follicle organoid.
Background
The number of people suffering from alopecia in China is about 2 hundred million, the trend of younger people is shown, the young and middle-aged people become the main force of 'the large army of alopecia', and the incidence of alopecia is increased by more than 10 times than 20 years ago. Alopecia causes remarkable damage to appearance, often brings a certain psychological burden to patients, and has strong treatment demands. However, methods for treating alopecia are very limited, and until now, only minoxidil and finasteride have been approved for treatment by the FDA, but the therapeutic effect of the drug is not satisfactory, the treatment onset time is long, and symptoms are repeated after drug withdrawal. Therefore, development of a novel drug for inducing hair follicle regeneration is accompanied by a great market demand, which depends on a preclinical research model capable of performing safety and efficacy tests, and a common mouse model has substantial differences from human biology, so that clinical transformation of research results has a certain difficulty, and therefore development of a skin model which is close to a hair follicle structure in a normal human skin physiological environment is needed, and the development of a therapeutic effect test research and a regenerative medicine research which are widely put into a drug development process are needed.
Organoids refer to stem cells having multipotential differentiation potential that divide and differentiate under in vitro three-dimensional culture conditions to form organ-like biological structures, have self-renewal and self-assembly capabilities, reproduce the function of the corresponding organ, and provide a highly similar physiological system. The organoids as novel disease models have obvious advantages, compared with two-dimensional culture, the organoids not only can be subjected to long-term subculture, but also have stable phenotype and genetic characteristics, and can be subjected to genome sequencing and expression profile analysis; compared with an animal model, the method has the characteristics of high operability, high timeliness and economy and low ethical risk. The organoid technology has been used by scholars at home and abroad to culture various organoid models for research of tissue and tumor development, drug efficacy research, personalized therapy research and regenerative medicine research. Therefore, organoid technology has great potential in the field of cultured skin models, becoming the best choice for differentiation of hair follicle structures.
In the last decade, research on the use of organoid technology to culture skin and follicular organoid models has resulted in a certain accumulation. Researchers digest and culture the epidermis and dermis of a newborn mouse into single cells respectively, or directly mix and transplant, or integrate into a spherical cell mass and transplant to the surface of a nude mouse after culture, and can see the hair regeneration on the surface of the nude mouse skin, however, the research is stopped at the murine stage. In 2020, lee et al differentiated organoids containing epidermis, dermis and hair follicle structures after 3 months of culture by induction of human embryonic stem cells and human induced pluripotent stem cells, and transplanted them to nude mice with visible hair of about 5 mm. The human embryonic stem cells and the human induced pluripotent stem cells respectively have ethical problems and canceration probability, and the current differentiation method has long period and low efficiency, and the hair follicle structure transplanted to the surface of the nude mice is smaller. In the process of human hair regeneration regulation, hair follicle stem cells play a vital role, have high self-renewal and differentiation capacity, and are continuously split and differentiated into hair after normal activation, so that the hair follicle stem cells are hopeful to become optimal seed cells for differentiation of hair follicle organoids.
Chinese patent CN201911171516.1 discloses a method for culturing early micro-hair follicle in vitro and its application. The culture method comprises the following steps: mixing dermal progenitor cells and epidermal stem cells, performing suspension culture by using a culture medium to form dermal-epidermal aggregates, and collecting the cultured hair follicle-like structure, namely the cultured hair follicle-like micro-organ. The cultured cells of the patent are scalp dermal group cells and foreskin epidermal stem cells, the sources of the cells are complex, and the technology has no universality if the transformation of the autologous hair follicle regeneration application is considered.
Chinese patent CN202110982838.5 discloses a method for constructing androgenetic alopecia cell model, comprising the following steps: collecting hair follicle bulb part of forehead part of androgenetic alopecia patient, separating hair papilla of hair follicle bulb part, and culturing; taking a carina region of the occipital part of an androgenic alopecia patient, separating outer root sheath cells of the carina region and culturing; 3D culturing the hair papilla cells obtained after the culture treatment to enable the hair papilla cells to gather to form hair papilla cell balls; the hair papilla cell pellet is inoculated in the upper chamber of a transwell culture dish, the outer root sheath cell is inoculated in the lower chamber of a pre-coated transwell culture dish, and dihydrotestosterone is added to obtain the cell model of androgenetic alopecia. The patent inoculates hair papilla cell ball and outer root sheath cell respectively in upper chamber and lower chamber of transwell culture dish, and culture system is comparatively complicated, and complex operation.
Disclosure of Invention
The invention aims to provide a method for constructing a hair follicle organoid, which utilizes hair follicle stem cells and hair follicle fibroblasts to directionally differentiate the hair follicle organoid according to a physiological mechanism of hair regeneration, and the differentiated hair follicle organoid has cell characteristics and structures similar to those of a normal primary hair follicle structure of a human body, can be frozen and revived, and has short time consumption and high differentiation efficiency, thereby laying a foundation for future application in the aspects of medicine curative effect prediction, hair follicle regulation research, regenerative medicine research and the like.
The aim of the invention can be achieved by the following technical scheme:
a method for constructing hair follicle organoids comprises obtaining hair follicle stem cells and hair follicle fibroblasts from human occipital hair follicle, preparing single cell suspension from Hair Follicle Stem Cells (HFSC) and Hair Follicle Fibroblasts (HFFB), culturing in inverted manner, and adding organoid culture solution to form hair follicle organoids.
Further, the follicular organoids have hair follicle-like structures resembling hair shafts and hair bulbs, rather than simple 3D aggregates.
Further, the specific steps of the method for constructing the hair follicle organoids are as follows:
s1, taking hair follicles of the occipital part of a patient, separating Hair Follicle Stem Cells (HFSCs) and Hair Follicle Fibroblasts (HFFBs), and culturing;
s2, preparing Hair Follicle Stem Cells (HFSCs) obtained after the culture treatment in the step S1 into HFSC single cell suspension, inoculating the HFSC single cell suspension into a 96-well plate for inverted culture, and adding a organoid culture solution to form HFSCO (hair follicle organoids constructed by HFSC differentiation);
s3, preparing the Hair Follicle Fibroblasts (HFFB) obtained after the culture treatment in the step S1 into HFFB single cell suspension, inoculating the HFFB single cell suspension into a 96-well plate for inverted culture, and adding the organoid culture solution to form HFSCFBO (HFFB differentiation-constructed hair follicle organoid).
The specific steps in step S1 are as follows:
s1-1, taking hair follicles of the occipital part of a patient and flushing;
s1-2, transferring the washed hair follicle into disperse enzyme, and stopping after incubation for a certain time;
s1-3, separating the hair follicle after incubation into hair follicle fragments, adding digestive enzyme for digestion for a certain time, and stopping;
s1-4, adding the digested hair follicle fragments into HFSC or HFFB culture solution for resuspension to prepare HFSC cell suspension or HFFB cell suspension, and inoculating the HFSC cell suspension or HFFB cell suspension into a culture dish for static culture;
step S1-5, when the cells grow to more than 80% of the area of the culture dish, single HFSC or HFFB suspension cells are obtained by digestion with digestive enzymes, and transferred to a new culture dish for passage in a ratio of 1:3.
In step S1-1, the hair follicle is washed 3 times with Du' S phosphate buffered saline (DPBS).
In step S1-2, the washed hair follicle is transferred to a dispersion enzyme diluted with HFSC or HFFB culture medium, and incubated in an incubator, and after incubation, an equal volume of HFSC or HFFB culture medium is added to stop digestion.
The above further, the Dispase II is a Dispase II concentration of 2U/ml.
The incubator is further incubated at 37 deg.c for 2-4 hr in 95% air and 5% CO 2 。
The HFSC broth was further prepared from 100ml KM-d medium (scientific # 2111), 10-100ul N-2 supplement (100X), 10-100ul B-27 supplement (50X), 1-200ng/ml FGF-2, 1-100ng/ml EGF and 1-100 μg/ml Normocin.
Further, fibroblast Medium culture solution (scientific # 2301) was used as the HFFB culture solution.
In the step S1-3, the hair follicle after incubation is separated into hair follicle fragments of 1-2mm, the supernatant is removed by centrifugation, 1ml of TrypLE ™ Express Enzyme digestive Enzyme is added, the hair follicle fragments are blown away, the hair follicle fragments are fully contacted with the digestive Enzyme, the hair follicle fragments are placed in an incubator for digestion for 8-12 minutes, and 4ml of culture solution is added to stop digestion; adding 1ml of TrypLE ™ Express Enzyme again, placing in an incubator for digestion for 8-12 minutes, and adding 4ml of culture solution to stop digestion; the supernatant was centrifuged off to obtain digested hair follicle fragments.
The above steps S1-4, inoculating HFSC cell suspension into culture dish coated in advance, inoculating HFFB cell suspension into common culture dish, spreading cell suspension on bottom of dish by cross method, and standing at 37deg.C under 5% CO 2 Culturing in an incubator under the condition.
The specific method for the culture dish coated in advance comprises the following steps: 10cm dishes were coated with 1-20. Mu.g/ml type IV collagen at 37℃4 hours in advance.
In step S1-5, when the cells grow over 80% of the area of the dish, single HFSC or HFFB suspension cells are obtained by digestion with TrypLE ™ Express Enzyme, the digestion is stopped by adding 4 volumes of DPBS, the supernatant is centrifuged off, the HFSC culture solution or HFFB culture solution is added to resuspend the cells, and the cells are transferred to a new dish in a ratio of 1:3, and the HFSC or HFFB is inoculated.
The specific steps in step S2 are as follows:
HFSC with substitution number P1-P3 is digested by TrypLE ™ Express Enzyme when the HFSC enters logarithmic phase growth, filtered to prepare HFSC single cell suspension, resuspended by organoid culture fluid, inoculated into 96-well plates, centrifuged and cultured upside down.
The incubator is further incubated at 37 deg.c for 2-4 hr in 95% air and 5% CO 2 。
The above further, the organoid culture solution is an organoid culture solution containing 1% matrigel.
The specific steps in step S3 are as follows:
HFFB with substitution number P1-P3 is digested by TrypLE ™ Express Enzyme when the HFFB enters logarithmic phase growth, filtered to prepare HFFB single cell suspension, resuspended by organoid culture fluid, inoculated into 96-well plates, centrifuged and cultured upside down.
The incubator is further incubated at 37 deg.c for 2-4 hr in 95% air and 5% CO 2 。
The above further, the organoid culture solution is an organoid culture solution containing 1% matrigel.
Compared with the prior art, the invention has the following advantages:
1. according to the physiological mechanism of hair regeneration, the invention uses hair follicle stem cells and hair follicle fibroblasts to directionally differentiate hair follicle organoids, the differentiated hair follicle organoids have similar cell characteristics and structures as those of normal primary hair follicle structures of human beings, and can be frozen and revived, and the method has short time consumption and high differentiation efficiency, and lays a foundation for future application in the aspects of medicine curative effect prediction, hair follicle regulation research, regenerative medicine research and the like.
2. The invention is to use the hair follicle stem cells from human follicular bulge sites as seed cells for induction differentiation for the first time, and to combine the hair follicle fibroblasts in parallel to successfully construct a hair follicle organoid model with a hair follicle structure, and the multi-layer differentiated structure and the high expression of hair follicle-related keratin are observed through H & E staining and immunofluorescence staining.
3. The invention has larger induction differentiation amplification multiple, and about hundreds of hair follicle organoids can be induced by 1 hair follicle unit.
4. Compared with the Chinese patent CN201911171516.1, the invention adopts the hair follicle stem cells of the scalp as the main body, has the unique characteristic of tissue regeneration and has regeneration medical application in skin, bones, cardiovascular and nerve and other tissue organs, and the invention successfully induces and differentiates hair follicle organoids by using the hair follicle stem cells and fibroblasts as seed cells through a method of improving differentiation culture solution, and has hair follicle-like structures similar to hair shafts and hair bulbs, but not simple 3D aggregates.
5. Compared with the Chinese patent CN202110982838.5, the invention has completely different culture conditions, carries out suspension culture on hair follicle stem cells in a low-adhesion 96-well plate, can differentiate into a hair follicle-like structure with hair shafts and hair bulbs in 5-7 days of culture, has simpler and more convenient model construction and large model quantity, and screens a precise medical research model for potential medicines.
Drawings
FIG. 1 is a schematic diagram showing the morphology of hair follicle stem cells extracted from occipital hair follicles at P0, P1, P2 and P3;
FIG. 2 is a schematic diagram of RT-qPCR detection results of different batches of HFSCs, wherein EKC is a control group of cells and represents P <0.01;
FIG. 3 is a schematic representation of immunofluorescent staining expression of HFSC and EKC (bar=20 μm);
FIG. 4 is a schematic representation of the morphology of fibroblasts extracted from occipital hair follicles at P0, P1, P2 and P3;
FIG. 5 is a schematic diagram of RT-qPCR detection results of HFFB in different batches, wherein DFB is a control cell;
fig. 6 is a schematic representation of immunofluorescent staining expression of HFFB and DFB (bar=20 μm);
FIG. 7 is a plot of the morphology of HFSCO follicular organoids under an inverted microscope at various days of culture (fold: 100X);
FIG. 8 is a plot of the morphology of HFSCFBO follicular organoids under an inverted microscope at various days of culture (fold: 40X);
FIG. 9 is a schematic representation of H & E staining of the bulb and tail ends of HFSCO and HFSCBO follicular organoids with normal hair follicles in a similar anagen phase, HFSCO follicular organoids from HFSC differentiation, and HFSCBO follicular organoids from HFSCSC and HFFB co-differentiation (fold: 200X);
FIG. 10 is a schematic diagram showing the fluorescent expression profiles of KRT15, CD200, LHX2, P-cadherin, ki-67, of HFSCO and HFSCBO in which the upper behavioural paraffin section tissue of each antibody staining was immunofluorescent stained, the lower behavioural whole tissue permeabilized, red Alexa Fluor 647 fluorescence, green Alexa Fluor 488 fluorescence, yellow Alexa Fluor 555 fluorescence, blue Hoechst fluorescence (bar=20μm) for HFSCO and HFSCBO on day 7 of expression culture of stem cells and proliferating cell related markers;
FIG. 11 is a schematic diagram showing the fluorescent expression of ASMA, SOX2, KRT5, VIMENTIN, KRT6, KRT16, KRT81, TRP1 and PMEL of HFSCO and HFSCBO showing the expression of hair follicle-characteristic structure-related markers in HFSCO and HFSCBO on day 7 of culture, wherein red is Alexa Fluor 647 fluorescence, green is Alexa Fluor 488 fluorescence, yellow is Alexa Fluor 555 fluorescence, and blue is Hoechst fluorescence representing nuclei (bar=20 μm).
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
A method for constructing hair follicle organoids comprises obtaining hair follicle stem cells and hair follicle fibroblasts from human occipital hair follicle, preparing single cell suspension from Hair Follicle Stem Cells (HFSC) and Hair Follicle Fibroblasts (HFFB), culturing in inverted manner, and adding organoid culture solution to form hair follicle organoids.
Further, the specific steps of the method for constructing the hair follicle organoids are as follows:
s1, taking hair follicles of the occipital part of a patient, separating Hair Follicle Stem Cells (HFSCs) and Hair Follicle Fibroblasts (HFFBs), and culturing;
s2, preparing Hair Follicle Stem Cells (HFSCs) obtained after the culture treatment in the step S1 into HFSC single cell suspension, inoculating the HFSC single cell suspension into a 96-well plate for inverted culture, and adding a organoid culture solution to form HFSCO (hair follicle organoids constructed by HFSC differentiation);
s3, preparing the Hair Follicle Fibroblasts (HFFB) obtained after the culture treatment in the step S1 into HFFB single cell suspension, inoculating the HFFB single cell suspension into a 96-well plate for inverted culture, and adding the organoid culture solution to form HFSCFBO (HFFB differentiation-constructed hair follicle organoid).
The specific steps in step S1 are as follows:
s1-1, taking hair follicles of the occipital part of a patient and flushing;
s1-2, transferring the washed hair follicle into disperse enzyme, and stopping after incubation for a certain time;
s1-3, separating the hair follicle after incubation into hair follicle fragments, adding digestive enzyme for digestion for a certain time, and stopping;
s1-4, adding the digested hair follicle fragments into HFSC or HFFB culture solution for resuspension to prepare HFSC cell suspension or HFFB cell suspension, and inoculating the HFSC cell suspension or HFFB cell suspension into a culture dish for static culture;
step S1-5, when the cells grow to more than 80% of the area of the culture dish, single HFSC or HFFB suspension cells are obtained by digestion with digestive enzymes, and transferred to a new culture dish for passage in a ratio of 1:3.
In step S1-1, the hair follicle is washed 3 times with Du' S phosphate buffered saline (DPBS).
In step S1-2, the washed hair follicle is transferred to a dispersion enzyme diluted with HFSC or HFFB culture medium, and incubated in an incubator, and after incubation, an equal volume of HFSC or HFFB culture medium is added to stop digestion.
The above further, the Dispase II is a Dispase II concentration of 2U/ml.
The incubator is further incubated at 37 deg.c for 2-4 hr in 95% air and 5% CO 2 。
The HFSC broth was further prepared from 100ml KM-d medium (scientific # 2111), 10-100ul N-2 supplement (100X), 10-100ul B-27 supplement (50X), 1-200ng/ml FGF-2, 1-100ng/ml EGF and 1-100 μg/ml Normocin.
Further, fibroblast Medium culture solution (scientific # 2301) was used as the HFFB culture solution.
In the step S1-3, the hair follicle after incubation is separated into hair follicle fragments of 1-2mm, the supernatant is removed by centrifugation, 1ml of TrypLE ™ Express Enzyme digestive Enzyme is added, the hair follicle fragments are blown away, the hair follicle fragments are fully contacted with the digestive Enzyme, the hair follicle fragments are placed in an incubator for digestion for 8-12 minutes, and 4ml of culture solution is added to stop digestion; adding 1ml of TrypLE ™ Express Enzyme again, placing in an incubator for digestion for 8-12 minutes, and adding 4ml of culture solution to stop digestion; the supernatant was centrifuged off to obtain digested hair follicle fragments.
The above steps S1-4, inoculating HFSC cell suspension into culture dish coated in advance, inoculating HFFB cell suspension into common culture dish, spreading cell suspension on bottom of dish by cross method, and standing at 37deg.C under 5% CO 2 Culturing in an incubator under the condition.
The specific method for the culture dish coated in advance comprises the following steps: 10cm dishes were coated with 1-20. Mu.g/ml type IV collagen at 37℃4 hours in advance.
In step S1-5, when the cells grow over 80% of the area of the dish, single HFSC or HFFB suspension cells are obtained by digestion with TrypLE ™ Express Enzyme, the digestion is stopped by adding 4 volumes of DPBS, the supernatant is centrifuged off, the HFSC culture solution or HFFB culture solution is added to resuspend the cells, and the cells are transferred to a new dish in a ratio of 1:3, and the HFSC or HFFB is inoculated.
The specific steps in step S2 are as follows:
HFSC with substitution number P1-P3 is digested by TrypLE ™ Express Enzyme when the HFSC enters logarithmic phase growth, filtered to prepare HFSC single cell suspension, resuspended by organoid culture fluid, inoculated into 96-well plates, centrifuged and cultured upside down.
The incubator is further incubated at 37 deg.c for 2-4 hr in 95% air and 5% CO 2 。
The above further, the organoid culture solution is an organoid culture solution containing 1% matrigel.
The specific steps in step S3 are as follows:
HFFB with substitution number P1-P3 is digested by TrypLE ™ Express Enzyme when the HFFB enters logarithmic phase growth, filtered to prepare HFFB single cell suspension, resuspended by organoid culture fluid, inoculated into 96-well plates, centrifuged and cultured upside down.
The incubator is further incubated at 37 deg.c for 2-4 hr in 95% air and 5% CO 2 。
The above further, the organoid culture solution is an organoid culture solution containing 1% matrigel.
The above embodiments are described in more detail below with reference to specific examples.
Examples
A method for constructing hair follicle organoids comprises the steps of preparing culture solution, separating, culturing and passaging human hair follicle stem cells, differentiating and culturing hair follicle organoids, freezing and recovering cells and organoids and the like.
1. Preparation of culture solution
(1) Hair Follicle Organoid Differentiation Medium (HFODM): based on 49% Advanced DMED/F12 and 49% Neurobasal Medium, 1X GlutaMax supplement,0.5 Xvitamin A free B27 supplement,0.5X N2 supplement,0.1mM 2-Mercap toehanol,10-100ng/ml IGF-1, 10-100uM CHIR99021 are added
(2) HFODM1% M: on the basis of HFODM, adding 0.1-10% Corning Matrigel Growth Factor Reduced Basement Membrane Matrix.
(3) HFSC culture solution: 100ml KM-d medium (scientific # 2111), 10-100ul N-2 support (100X), 10-100ul B-27 support (50X), 1-200ng/ml FGF-2, 1-100ng/ml EGF and 1-100 μg/ml Normocin;
(3) HFFB culture solution: fibroblast Medium culture (Sciencell # 2301) was used. HFSC is cultured by coating 1-20 μg/ml type IV collagen at 37 ℃ in advance of 4 hours in a 10cm dish.
2. Isolated culture, passaging, cryopreservation and resuscitation of human primary HFSC and HFFB
(1) Hair follicle preparation: the isolated occipital hair follicle needs to be transported in a low-temperature and moist environment, and in order to keep the activity of hair follicle cells, HFSC and HFFB need to be isolated and cultured in a biosafety cabinet as soon as possible.
(2) Isolation culture of human primary HFSC and HFFB: hair follicles were washed 3 times with DDPBS in 6cm low adhesion dishes, gently transferred with ophthalmic forceps to 2U/ml dispese II diluted with HFSC culture solution or HFFB culture solution, and placed in 37 ℃ 5% co 2 Incubate in incubator under conditions for 2-4 hours. Digestion is stopped by adding an equal volume of culture medium, the follicle is separated into 1-2mm small segments using surgical blades or ophthalmic scissors, transferred to a 15ml centrifuge tube, and centrifuged for 5 minutes in a centrifuge at 500 g. The supernatant was discarded, 1ml of TrypLE ™ Express Enzyme digestive Enzyme was added, and the hair follicle fragments were blown off and allowed to come into full contact with the digestive Enzyme and placed in an incubator for digestion for 10 minutes. Digestion was stopped by adding 4ml of culture medium, allowed to stand for a short time, and the supernatant was transferred to a new centrifuge tube after the hair follicle had settled to the bottom of the tube. 1ml of TrypLE ™ Express Enzyme was added again and placed in an incubator for digestion for 10 minutes. Digestion was stopped by adding 4ml of culture medium, allowed to stand for a moment, the above supernatants were integrated, and centrifuged in a centrifuge for 5 minutes at 500 and g. The supernatant was discarded, HFSC or HFFB broth was added for resuspension, the suspension was filtered through a 100 mesh screen into a new centrifuge tube, the hair follicle tissue was gently ground with the syringe plunger face to flush more cells down, and the cell suspension was centrifuged in a centrifuge for 5 minutes at 500 g. The supernatant was discarded, the cell pellet was resuspended with HFSC or HFFB medium, the HFSC cell suspension was inoculated into a dish coated in advance, the HFFB cell suspension was inoculated into a normal 10cm dish, and the cell suspension was plated on the bottom of the dish in a "cross" manner.Placed at 37 ℃ with 5% CO 2 Culturing in an incubator under the condition. No replacement of the broth was performed for the first two days after inoculation, followed by replacement of the broth every 2 days. The cells were observed with an inverted microscope and photographed for recording.
(3) Passaging of human primary HFSC and HFFB: after 2 weeks, the cell clones can be passaged. When the cells are expanded to a density of about 80%, the next passage can be performed. Passage of HFSC also required seeding in type IV collagen-coated dishes. Single suspension cells were obtained using TrypLE ™ Express Enzyme digestion, digestion was stopped by adding 4 volumes of DPBS and the cell suspension was transferred to a centrifuge tube and centrifuged at 500 g for 5 minutes. The supernatant was discarded, the cells were resuspended in culture medium, and the cells were uniformly blown with a gun head, and inoculated with HFSC or HFFB at a ratio of 1:3. When the cell expansion number is large, the cells can be transferred to a T175 culture flask for culture. Cells of different algebra were recorded by observation with an inverted microscope and photographed.
3. Differentiation culture of hair follicle organoids
First, a follicular organoid is constructed using HFSC differentiation, and the follicular organoid constructed by this system is hereinafter referred to as HFSCO (Hair Follicle Stem Cells derived Organoids); secondly, the invention adds HFFB differentiation to construct the hair follicle organoid based on the system, and the hair follicle organoid constructed by the system is called HFSCFBO (Hair Follicle Stem Cells and Hair Follicle Fibroblasts derived Organoids) in the following.
(1) Differentiation of HFSC into follicular organoids
1) Construction of HFSCO: HFSCs were selected with algebra P1-P3 and digested with TrypLE ™ Express Enzyme as they enter log phase growth and the cell pellet was filtered through a filter screen to prepare a single cell suspension. Subsequently, living cells were counted using a cell counting plate, and data were recorded for calculation of fold expansion. The cells were then number-regulated by resuspension with organoid culture medium containing 1% matrigel and blow-homogenized, and HFSC was inoculated into low-adhesion round-bottomed 96-well plates using a multichannel pipette at a concentration of 2000 HFSC per well in a volume of 100 μl. The 96-well plate was centrifuged at 110-300g for 5-15 minutes at room temperature, after which 3D spherical aggregates were observed by an inverted microscope. Is placed in37°C、5%CO 2 Incubation in incubator.
2) Differentiation and maturation of HFSCO: on day 2, 100. Mu.l of organoid culture medium was added to the wells. Transfer it to 37℃and 5% CO 2 The rotation speed is 30 rpm on the horizontal shaking table in the incubator. Subsequently, the culture medium was replaced every 2 days. Morphological changes of HFSCO were observed and recorded by an inverted microscope.
(2) HFFB is involved in differentiation of HFSC into follicular organoids
1) Construction of HFSCBO: like HFSC, HFFB of the same patient source algebra P1-P3 was selected and digested with TrypLE ™ Express Enzyme to prepare a single cell suspension when it entered log phase growth. The organoid culture broth containing 1% matrigel was resuspended and blown uniformly. After HFSCO had formed 3D spherical aggregates, a 100. Mu.l volume of HFFB cell suspension in an amount proportional to HFSC was gently added, and the two cells were assembled by centrifugation (110 g at room temperature, 6 min centrifugation) and non-centrifugation (natural standing) respectively according to the above conditions to form 3D spherical aggregates of HFSCBO. Placed at 37 ℃ with 5% CO 2 Incubation in incubator.
2) Differentiation maturation of HFSCFBO: similarly, the culture medium was replenished on day 2 and 96-well plates were transferred to 37℃with 5% CO 2 The culture medium was replaced every 2 days on a horizontal shaker in the incubator. By day 7, HFSCFBO aggregates were washed off with pre-chilled DPBS solution, washing off residual matrigel. It was transferred to a low adhesion 24 well plate and the total amount of culture medium was 500 μl, and culture was continued on a horizontal shaker in a 37 ℃ 5% co2 incubator with medium changes every 2 days. Morphological changes of HFSCFBO were observed and recorded by inverted microscopy.
4. Cryopreservation and resuscitation of hair follicle organoids
(1) Cryopreserved cells: the differentiated hair follicle organoids can be stored using specialized organoid cryopreservation solutions, or can be stored using DMSO-containing serum.
(2) Resuscitates cells: the primary cultured human hair follicle stem cells can be further cultured using DK-SFM, or can be cultured using Keratinocyte Medium-defined (KM-d). Stable passaging of human hair follicle stem cells typically does not require a coated culture dish, but coating the culture dish according to the previously mentioned methods of the invention increases the survival rate of the cells after passaging. Human hair follicle stem cells are generally stable for 3-5 passages. After the hair follicle organoids are revived, the culture is performed by using HFODM with the concentration of 1% M, and after 5 days, the culture medium is replaced by HFODM. The culture process still requires the use of low adhesion plates and shaker tables.
EXAMPLE 2 isolation, expansion and identification of human follicular stem cells
The method can obtain a large amount of high-quality hair follicle stem cells by utilizing human occipital hair follicle tissues. The morphology of hair follicle stem cells extracted from occipital hair follicles under an inverted microscope is shown in the figure 1, wherein the morphology of the hair follicle stem cells extracted from occipital hair follicles is shown in the figure 1, the cells initially form cell colonies, can grow in an adherent manner, are in a polygonal cell morphology, can stably proliferate, passaged, frozen and revived, and the morphologies of different algebraic cells are not obviously different.
HFSC related genes are identified by RT-qPCR from different batches and different algebra in logarithmic growth phase, and cells in a control group are human primitive representative skin keratinocytes (epidermal keratinocytes, EKC). As shown in FIG. 2, the expression of HFSC-related genes was detected in HFSCs, and the expression levels of the dry related genes KRT15, LHX2, SOX9, LGR5 and ITGA6 were higher than those of EKC, which indicates that the primary HFSCs extracted by the present invention have stem cell characteristics and can be used for the induction differentiation of hair follicle organoids.
The immunofluorescent staining expression patterns of HFSC and EKC are shown in FIG. 3, and the markers are specific markers (bar=20μm) related to hair follicle stem cells, and HFSC highly expresses HFSC related proteins such as KRT15, KRT19, CD200, ITGA6, CD71, WIF1, FST, p63 and the like. These proteins differ in the expression site of the cell, and the cell membrane protein CD200 is observed in HFSC cell membranes or plasma membrane vesicles; integrin ITGA6 can be observed on HFSC cell membranes or in the cytoskeleton; transferrin receptor CD71 can also be observed on HFSC cell membranes; two keratins KRT15 and KRT19, wnt signaling pathway regulator WIF1, BMP signaling pathway regulator FST can be observed in HFSC cytoplasm; transcription factor p63 can be observed in HFSC nuclei.
EXAMPLE 3 isolation, expansion and identification of human follicular fibroblasts
The method can obtain a large amount of high-quality hair follicle fibroblasts from human occipital hair follicle tissues. The morphological diagrams of the fibroblasts extracted from occipital hair follicles under an inverted microscope are shown in fig. 4, wherein the cells initially form cell monoclonals, can grow in an adherent manner and stretch into a spindle-shaped cell morphology, have elongated and strip-shaped cytoblast, are rich in cytoplasm and accord with the morphology of activated fibroblasts. And can stably proliferate, passaged, frozen and revived, and the morphology of different algebraic cells is not obviously different.
HFFB in different batches and different algebra in the logarithmic growth phase is taken, the identification of the fibroblast related genes is carried out by RT-qPCR, and the cells in the control group are human primary dermal fibroblasts (dermal fibroblasts, DFB), so that no obvious statistical difference is found. As shown in FIG. 5, the expression of the fiber-forming related genes including COL1A1, FN1, FBN1, SOX2, ALPL, NOG, PLCG and LEF/TCF was detected in HFFB. Wherein, the gene expression level of the specific related genes SOX2, ALPL, NOG, PLCG2 and LEF/TCF of the hair papilla fibroblast is higher than that of the DFB, which indicates that the primary HFFB extracted by the invention has the characteristic of hair follicle-derived fibroblast and can be used for inducing differentiation of hair follicle organoids.
In order to clearly extract the expression of the primary HFSC related protein markers and the positions of the primary HFSC related protein markers in cells, the primary HFSC related protein markers are subjected to immunofluorescent staining, and the markers are specific markers (bar=20μm) related to hair follicle stem cells, as shown in fig. 6, and HFFB high-expresses VIMENTIN, ASMA, S A4 and COL1A1 fibroblast related proteins. These proteins are predominantly located in the cytoplasm at the site of cell expression, where ASMA is an actin, which is associated with the conversion of fibroblasts to smooth muscle cells and is often used as a cellular marker for hair papilla fibroblasts.
Example 4 construction of human follicular organoids Using obtained human follicular Stem cells and fibroblasts
The hair follicle stem cells obtained by the method can be used for constructing HFSCO (Hair Follicle Stem Cells derived Organoids); the hair follicle organoids constructed by adding HFFB on the basis of this system are called HFSCFBO (Hair Follicle Stem Cells and Hair Follicle Fibroblasts derived Organoids).
As can be seen in fig. 7, HFSCO self-assembles to form a bud-like structure protruding from the sphere, and HFSC differentiate into follicular organoids in a vesicle-hair bulb-like bipolarized structure. With the increase of the culture time, the structure continues to proliferate and expand gradually to form a vesicle-hair bulb-like dual-polarization structure, namely, a vesicle with a transparent head end and a hair bulb-like structure with a substantial tail end. Through observation of the differentiation of different batches of HFSCs into hair follicle organoids, organoid structures tended to stabilize on day 14 of culture, with no further increase seen on day 30 of structure compared to it.
As shown in FIG. 8, HFSCFBO was observed to germinate organoid spheres under an inverted microscope about 5-7 days of culture, showing a rod-like structure extending from the sphere and a hair-like structure at the end. With the increase of the culture time, the hair bulb-like structure at the tail end is continuously increased.
As shown in fig. 9, HFSCO can see concentric layers of cells arranged in layers, similar to the outer most layers of the hair follicle cross section, the center layer being similar to the hair shaft and the overall structure being thinner, compared to normal hair follicles. HFSCFBO is similar in size to normal hair follicles. The bulb can observe a spherical outer layer formed by HFFB, which approximates to a hair papilla structure; under the wrapping of HFFB, the HFSC is eccentrically differentiated and extends out of the rod-shaped structure; the rod-like structure at its tail end is "C" -shaped basophilic stained, similar to the cortex of the hair shaft in a normal hair follicle.
As shown in FIG. 10, positive expression of stem cells and proliferation-related markers, including hair follicle stem cell-related marker Keratin15 (KRT 15, keratin 15), CD200, epidermal stem cell-related marker LIM homeobox protein 2 (LHX 2, LIM homeobox protein 2), P-cadherin (P-cadherin), and hair matrix proliferation-related marker Ki-67, was detected in both HFSCO and HFSCBO on day 7 of culture. Importantly, marker expression in HFSCFBO is structural in that the hair follicle stem cell related markers KRT15 and CD200 are both cell markers specific for the carina region, while LHX2 and P-cadherein positive epidermal stem cells are also located in the carina region, and the location of their expression in HFSCFBO can reflect the structure of hair follicles.
Meanwhile, the identification of relevant markers of hair follicle characteristic structures was performed on HFSCO and HFSCFBO on day 7 of culture, respectively. As shown in fig. 11, in HFSCFBO, expression of related markers of hair follicle characteristic structures including papillary related marker SRY box transcription factor 2 (SOX 2, SRY-box transcription factor 2), pilus related marker alpha smooth muscle actin (ASMA, alpha smooth muscle Actin), dermal sheath related marker VIMENTIN (VIMENTIN), basal stratum corneum keratinocyte related marker Keratin 5 (KRT 5, haratin 5), medullary related marker Keratin6 (KRT 6, haratin 6), keratin16 (KRT 16, haratin 16), cortical related marker Keratin 81 (KRT 81, haratin 81), melanocyte related marker tyrosinase related protein 1 (TRP 1, tyrosinase related protein 1), melanocyte precursor protein (PMEL, pre-melanosome protein) could be detected; and the expression of the related markers has the characteristic structural characteristic distribution situation of hair follicles, for example, the hair papilla related marker SOX2 is detected in a hemispherical form in a bud structure protruding from a sphere, and the dermis sheath related marker VIMENTIN is detected in a packaged form in the sphere; in contrast, in HFSCO, the expression of the dermal sheath related marker VIMENTIN and the melanin related marker TRP1 was not detected.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A method for constructing a hair follicle organoid, comprising the specific steps of:
s1, taking hair follicles of the occipital part of a patient, separating hair follicle stem cells and hair follicle fibroblasts, and culturing;
s2, preparing the hair follicle stem cells obtained after the culture treatment in the step S1 into HFSC single cell suspension, inoculating the HFSC single cell suspension into a 96-well plate for suspension culture, and adding an organoid culture solution to form HFSCO;
s3, preparing the hair follicle fibroblasts obtained after the culture treatment in the step S1 into HFFB single cell suspension, adding the HFFB single cell suspension into the HFSCO obtained in the step S2, performing suspension culture, and adding the organoid culture solution to form HFSCBO;
the organoid culture solution is an organoid culture solution containing 1% matrigel.
2. The method of constructing a hair follicle organoid according to claim 1, wherein the specific steps in step S1 are as follows:
s1-1, taking hair follicles of the occipital part of a patient and flushing;
s1-2, transferring the washed hair follicle into disperse enzyme, and stopping after incubation for a certain time;
s1-3, separating the hair follicle after incubation into hair follicle fragments, adding digestive enzyme for digestion for a certain time, and stopping;
s1-4, adding digested hair follicle fragments into HFSC or HFFB culture solution for resuspension to prepare HFSC cell suspension or HFFB cell suspension, inoculating into a culture dish, and standing for culture, wherein the HFSC culture solution is prepared from 100ml KM-d culture medium, 10-100ul N-2 support, 10-100ul B-27 support, 1-200ng/ml FGF-2, 1-100ng/ml EGF and 1-100 mu g/ml Normocin;
the HFFB culture solution adopts Fibroblast Medium culture solution;
step S1-5, when the cells grow to more than 80% of the area of the culture dish, single HFSC or HFFB suspension cells are obtained by digestion with digestive enzymes, and transferred to a new culture dish for passage in a ratio of 1:3.
3. The method of constructing a hair follicle organoid according to claim 2, wherein in step S1-1, the hair follicle is washed 3 times with a dolphin phosphate buffer solution;
in step S1-2, the washed hair follicle is transferred to a dispersion enzyme diluted by HFSC culture solution or HFFB culture solution, placed in an incubator for incubation, and after incubation is completed, an equal volume of HFSC culture solution or HFFB culture solution is added to stop digestion.
4. A method of constructing a follicular organoid according to claim 3, wherein the Dispase is Dispase II at a concentration of 2U/ml;
the incubation temperature of the incubator is 37 ℃, the incubation time is 2-4 hours, and the incubation environment is 95% of air and 5% of CO 2 。
5. The method for constructing a hair follicle organoid according to claim 2, wherein in step S1-3, the hair follicle after incubation is separated into 1-2mm hair follicle fragments, the supernatant is centrifuged off, 1ml of TrypLE ™ Express Enzyme digestive Enzyme is added, the hair follicle fragments are blown off and allowed to fully contact with the digestive Enzyme, and the hair follicle fragments are placed in an incubator to digest for 8-12 minutes, and 4ml of culture solution is added to stop the digestion; adding 1ml of TrypLE ™ Express Enzyme again, placing in an incubator for digestion for 8-12 minutes, and adding 4ml of culture solution to stop digestion; the supernatant was centrifuged off to obtain digested hair follicle fragments.
6. The method of constructing a hair follicle organoid according to claim 2, wherein in steps S1-4, the HFSC cell suspension is inoculated into a culture dish coated in advance, the HFFB cell suspension is inoculated into a common culture dish, the cell suspension is spread on the bottom of the dish by a "cross" method, and the dish is placed at 37 ℃ with 5% co 2 Culturing in an incubator under the condition;
the specific method of the culture dish coated in advance comprises the following steps: 10cm dishes were coated with 1-20. Mu.g/ml type IV collagen at 37℃4 hours in advance.
7. A method of constructing a hair follicle organoid according to claim 2, wherein in steps S1-5, when the cells grow over 80% of the area of the culture dish, single HFSC or HFFB suspension cells are obtained by digestion with TrypLE ™ Express Enzyme, digestion is stopped by adding 4 volumes of DPBS, the supernatant is centrifuged off, and the cells are resuspended in HFSC or HFFB culture solution and transferred to a new culture dish in a ratio of 1:3, and inoculated with HFSC or HFFB.
8. The method of constructing a hair follicle organoid according to claim 1, wherein the specific steps in step S2 are as follows:
HFSC with substitution number of P1-P3 is digested by TrypLE ™ Express Enzyme when the HFSC enters logarithmic phase growth, filtered to prepare HFSC single cell suspension, resuspended by organoid culture fluid, inoculated into 96-well plates, centrifuged and cultured upside down;
the culture temperature is 37 ℃, the culture time is 5-7 days, and the culture environment is 95% of air and 5% of CO 2 。
9. The method of constructing a hair follicle organoid according to claim 1, wherein the specific steps in step S3 are as follows:
and (2) taking HFFB with the number of P1-P3, digesting the HFFB with TrypLE ™ Express Enzyme when the HFFB enters the logarithmic phase growth, filtering the HFFB to prepare HFFB single cell suspension, re-suspending the HFFB single cell suspension by using the organoid culture solution, adding the HFFB single cell suspension into the HFSCO obtained in the step S2, and performing natural static culture, thus forming the HFSCBO after adding the organoid culture solution.
10. The method of claim 9, wherein the step of constructing a follicular organoid comprises,
the culture temperature is 37 ℃, the culture time is 5-7 days, and the culture environment is 95% of air and 5% of CO 2 ，
The organoid culture solution is an organoid culture solution containing 1% matrigel.
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