AU2020103546A4 - A hair follicle microarray co-isolation system and its application in the treatment of pathological hair loss - Google Patents
A hair follicle microarray co-isolation system and its application in the treatment of pathological hair loss Download PDFInfo
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Abstract
A hair follicle microarray co-isolation system and its application in the treatment
of pathological hair loss, which includes a suspension of hair follicle stem cells and a
damaged hair follicle to be repaired. The invention provides a new idea of adopting
allogeneic normal human hair follicle stem cells in combination with drug molecules to
create a microenvironment suitable for hair follicle repair, and offers a hair follicle
microarray co-isolation system for in vitro 3D co-culture of hair follicle stem cells and
damaged hair follicles by using soft agar.
-1/2
HFSC(low HFSC(medium HFSC(high
Control 09% NaCl dose) do3e) dose)
U
-A
0v
Figure 1.
The skin HE of nude mice in each group after injection for 5 weeks
Description
-1/2
HFSC(low HFSC(medium HFSC(high
Control 09% NaCl dose) do3e) dose)
0v
Figure 1.
The skin HE of nude mice in each group after injection for 5 weeks
PATENTS ACT 1990
A hair follicle microarray co-isolation system and its application in the treatment of
pathological hair loss
The invention is described in the following statement:-
The invention relates to the field of genetic engineering technology and
biopharmaceuticals, in particular to a hair follicle microarray co-culture system and its
application in treating pathological alopecia.
Hair follicle stem cells (HFSCs) are a group of self-renewing and proliferating
multipotential stem cells present in the bulge of the outer root sheath of hair follicles, which
can differentiate into hair follicles, sebaceous glands, and epidermis and play an important
role in hair regeneration. Studies have shown that human hair follicle stem cells express a
variety of cell surface molecular markers such as CD200, keratin 15 (K15), keratin 19
(K19), integrin a6, integrin Pl, Nestin, etc., except for the endothelial cell marker CD31;
In addition, they also do not express CD34, a marker molecule for mouse hair follicle stem
cells. With these positive and negative marker molecules, researchers can better isolate and
purify hair follicle stem cells, which accelerates the process of hair follicle stem cell
research. Compared to embryonic stem cells and other adult stem cells, hair follicle stem
cells have the advantages of being abundant, easy to obtain, harmless to the organism, and
free from ethical problems. It has been reported in the literature that hair growth in nude
mice is achieved by transplantation of hair follicle stem cells, demonstrating that transplantation of 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 acting. 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. Therefore, if allogeneic hair follicle stem cells can be used as
donors, it will greatly expand the application scope of hair follicle stem cells. In view of
this, the present invention studies the related mechanism of hair follicle stem cells
promoting hair regeneration, and explores the feasibility of using allogeneic hair follicle
stem cells to treat pathological alopecia.
The purpose of the invention is to study the related mechanism of hair follicle stem
cells to promote hair regeneration, and to explore the feasibility of adopting hair follicle
stem cells to treat pathological alopecia.
The hair follicle microarray co-culture system comprises a hair follicle stem cell
suspension and damaged hair follicles to be repaired.
Further, the culture method of the hair follicle stem cell suspension comprises the
following steps:
(1) Taking healthy hair follicles and washing them repeatedly with penicillin
streptomycin PBS solution;
(2) Under the dissecting microscope, cutting off the bulge of the outer root sheath of
the hair follicle by microsurgery, which is placed in a petri dish; Adding hair follicle stem
cell culture medium, which is incubated in the incubator of 5%CO2 at 37°C;
(3) Supplementing the same amount of medium 1.5mL the next day, observing under
an inverted microscope whether there is spindle-shaped cells grow in the outer root sheath
of the hair follicle or not, and carrying out subculturing after the cells have reached 80%
fusion;
(4) 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 culture medium according to the mass
ratio of 1:1, taking 2ml of the mixed solution which is injected into a 35cm 2 culture dish, and putting it into an incubator for later use after cooling and solidification;
(6) After mixing 0.7% agarose and hair follicle stem cell culture medium in the ratio
of 1:1 in a sterile test tube, adding 1/10 volume of hair follicle stem cell suspension to the
tube, which are mixed well. Then injecting medium I into the petri dish, the amount added
is equal to medium I;
(7) Replacing the culture medium once every 2 days for a total of 10 days.
Further, the hair follicle stem cell culture medium contains 10% FBS, 100U/mL
penicillin double antibody, 2ng/ml bFGF DMEM/F12.
Furthermore, the culture medium I is mixed with 1.2% agarose and DMEM/F12
culture medium according to the ratio of 1:1, then 2ml of the mixed solution is injected into
a 35cm 2 culture dish, which is put into an incubator for later use after being cooled and
solidified.
Further, the digestion solution of the hair follicle stem cell digestion contains a
phosphate buffer solution containing 0.1% pancreatin and 0.008% EDTA by mass.
Additional, the healthy hair follicle is taken from the occipital part of the head.
In addition, the digestion condition is 37 °C for 3-5 minutes.
Further, the damaged hair follicles to be repaired are repeatedly washed with
penicillin-streptomycin PBS solution, and the follicles are inserted into the upper agar of
the solidified hair follicle stem cell suspension. Then they are put into an incubator for 3D
co-culture to form a hair follicle microarray co-culture system.
The application of a hair follicle microarray co-culture system in drugs for treating pathological alopecia is characterized in that the hair follicle microarray co-culture system according to any one of claims 1-6 is prepared into drugs for treating pathological alopecia, damaged hair follicles in hair loss areas are implanted into the drugs for repair culture, and repaired hair follicles are implanted into hair loss areas.
Furthermore, implanting successfully repaired hair follicles in the hair loss area, and
offering an oral drug.
Further, the oral drug is thymosin.
Figure 1 shows the skin HE of nude mice in each group after injection for 5 weeks.
Figure 2 represents the hair growth status after subcutaneous injection of HFSC.
In order to elaborate the technical scheme and technical purpose of the invention, the
following specific implementation mode further introduces the invention.
1. Animal experiments
1.1 Establishment of androgen-induced alopecia rat model: selecting male SD rats
aged 6 weeks, which at treated with depilatory cream to remove their back hair, then
gavaging them 1 mL of (1.5 mg) methyltestosterone (60 tablets of methyltestosterone, 0.5
mg for each pill, ground into fine powder, which is mixed with 20 m L of distilled water
and stirred evenly) and 0.2 mL of lard once a day for 60 days continuously to establish androgen alopecia rat model.
1.2 Animal grouping and hair follicle stem cell transplantation: the successfully
modeled SD rats are randomly divided into control group, hair follicle stem cell group, and
hair follicle stem cell+drug group, with six rats in each group. After digestion, hair follicle
stem cells are collected and washed twice with sterile saline, and then resuspended to
1.0x105 cells/mL cell suspension. In the hair follicle stem cell group, the cell suspension
is injected subcutaneously at multiple points, 100 L at each point, for a total of 500 [L;
Hair follicle stem cells are injected every 2 days for a total of 3 injections. The control
group is injected with the same volume of saline in the same manner. In addition to hair
follicular stem cell transplantation, the hair follicular stem cell+drug group is given
intraperitoneal injection of appropriate amount of drug twice a week.
Control Experimen Experiment Experiment Experimenta Experimental Experimental group 6 group tal group 1 al group 2 al group 3 1 group 4 group 5 Physiological Saline Saline Saline Culture Culture Culture supernatant saline +HFSC(1x +HFSC(2x +HFSC(4x supernatant supernatant +HFSC(1xl06)+HU 106) 106) 106) +HFSC(1x106) VSMC(1x106)
1.3 Observing the hair growth of the depilated part of rats every week and making
records. After 8 weeks, the rats are killed and the skin tissue of the depilated part of the
back is made into paraffin tissue specimens.
Experim Experim Experim Experim Experim Experime Control Crol ental ental ental ental ental ntal group group group 1 group 2 group 3 group 4 group 5 6
Ow 0 0 0 0 0 0 0
1w 0 0 0 0 0 0 0
2w 0 0 2 2 0 1 2 3w 0 2 3 2 3 2 3
4w 0 2 3 3 2 3 3 w 0 2 3 3 3 3 3
1.4 HE staining of hair follicles: The paraffin blocks of skin tissue are cut into paraffin
sections with the thickness of 5[m, followed by xylene dewaxing and gradient alcohol
hydration. After washing with tap water for 3 times, soaking and staining the tissues with
Harris hematoxylin staining solution for 3-5min, then washing them with tap water;
Differentiating the material above with 1% hydrochloric acid alcohol for 3-5s, and washing
it with tap water; Soaking the tissue in 0.2% ammonia solution for 1 minute, then it turns
blue, and washing the tissues with tap water; Soaking the tissues with eosin staining
solution for 30 s; Dehydrating with 95% alcohol and absolute ethanol, and sealing with
xylene transparent and neutral gum; Observing the samples and taking pictures under the
microscope, as shown in Figure 1. Comparing the pathological differences between the
experimental groups and the control group.
1.5 Immunofluorescence staining of hair follicles: paraffin sections of skin tissue were
dewaxed with xylene and hydrated with gradient alcohol, washed with distilled water for
3-5 times, treated with 10% goat serum blocking solution containing 0.25% Triton-100 for
minutes, and incubated at 4°C overnight with appropriately diluted primary antibodies
(K19, CD200 and integrin P1). The next day, after washing with PBS for three times, the
fluorescent secondary antibody corresponding to the species of the primary antibody was
added to react at 37°C for 30min; Cleaning with PBS, re-staining with DAPI, sealing,
observing under fluorescence microscope and taking pictures. Comparing the difference of
hair follicle stem cells between the experimental group and the control group.
2. Isolation and culture of hair follicle stem cells
(1) Isolation and culture of human hair follicle stem cells: 10 healthy volunteers are recruited. 8-10 intact hair follicles are extracted from the posterior occipital region of the brain of each volunteer and washed three times with PBS solution containing high concentrations of penicillin and streptomycin. Under the dissecting microscope, hair follicles are cut off from the bulge of the outer root sheath with microsurgical scissors and placed in a petri dish of 35cm 2 ; Adding 1.5ml of hair follicle medium (DMEM/F12 medium containing 10% FBS, 100U/mL penicillin and 2ng/ml bFGF), which are incubated in a 5%
C02 incubator at 37°C. On the next day, adding 1.5mL of medium and the cells are
observed under the inverted microscope to see if there are any shuttle-shaped cells grow
out of the hair follicle outer root sheath projections. Cells subcultured to the third
generation is for the subsequent experiments.
2) Identification of the expression of surface marker molecules and negative marker
molecules of hair follicle stem cells by flow cytometry: the 3rd generation hair follicle stem
cells are digested with 0.25% trypsin, washed once with PBS, centrifuged and collected,
and resuspended with PBS solution containing 1% BSA respectively. Adding the
fluorescent antibodies of K15, K19, CD200, integrin 1, CD31 and CD34 respectively, and
the cells are labeled by incubation at room temperature for 30 min in the dark. After
washing with PBS, the expression of these labeled molecules is detected by flow cytometry.
2. Identification of immunogenicity of hair follicle stem cells
1) Detecting the expression of MHC-II molecules in hair follicle stem cells by WB:
collecting hair follicle stem cells, adding a cell total protein extraction reagent to extract
and quantify the total protein. Adding 15kg protein sample into 2xSDS gel loading buffer,
which are boiled for 5min, centrifuged at 6000rpm for 3min. Taking the supernatant which is transferred to the protein on gel to PVDF membrane after electrophoresis. Sealing the membrane at room temperature for 2 h after transferring, putting PVDF membrane into the hybridization bag, and adding appropriately diluted primary antibodies (HLA-DPA1,
HLA-DQA1, HLA-DRA1 and internal reference GAPDH) 4 respectively. Adding HRP
labeled secondary antibody corresponding to the primary antibody species, which are
incubate at room temperature with shaking for 1 h. Rinsing the filter membrane, adding
developer for color rendering, and putting it into a gel imager for exposure imaging. The
optical density value of the target zone is analyzed by 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, identifying and quantifying by ultraviolet spectrophotometer.
Total RNA is reverse transcribed into cDNA by adopting reverse transcription reaction kit
(prime script rt reagent kit with gDNA eraser, TAKARA). According to Genebank
sequence, HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH primers
are designed by Primer 5.0 software and synthesized by our company. The qRT-PCR
reaction is performed according to the instructions of the kit (SYBR Premix ExTaqTM II,
TAKARA). HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH
primers are shown in the following table:
Table 1. Primer sequences of HLA-dpal, HLA-DQA1, HLA-DRA1 and internal
reference GAPDH
Gene name Forward primer Reverse primer
HLA-DPA1 CTGGACAAGAAGGAGACCGT TCAATGTGGCAGATGAGGGT
HLA-DQA1 AACGCTACAACTCTACCGCT TCTGTGACTGACTGCCCATT
3.3D co-culture of hair follicle stem cells and damaged hair follicles in vitro
1) In vitro 3D co-cultivation of soft agar:
a) Preparing agarose liquid with low melting point of 1.2% and 0.7% with tri-distilled
water. After sterilization under high pressure, and keeping it dissolved at 40°C.
b) Preparing 2x hair follicle stem cell culture medium (2xDMEM/F12 culture
medium containing 20% FBS, 200U/mL streptomycin double antibody and 4ng/ml bFGF).
After filtering with 0.1 m disposable sterile filter, it is stored at 37°C.
c) After mixing 1.2% agarose and 2xDMEM/F12 culture medium at the ratio of 1:1,
taking 2ml of the mixed solution, which is injected into a 35cm 2 culture dish, and putting
it into an incubator for later use after cooling and solidification.
d) Digesting the hair follicle stem cells, preparing a cell suspension of1.Ox105 cells
/mL with the hair follicle stem cell culture solution, and counting.
e) After mixing 0.7% agarose and 2x hair follicle stem cell culture medium in a sterile
test tube according to the ratio of 1: 1, adding 1/10 volume of hair follicle stem cell
suspension into the tube. Mixing them well, and inject them into a petri dish paved with
1.2% agarose with the same amount as above.
f) Recruiting 10 volunteers with pathological alopecia, taking 30 hair follicles from the hair loss area of each person. They are randomly divided into control group, hair follicle stem cell group and hair follicle stem cell+ drug group, with 10 in each group; The control group is directly prepared into frozen sections of 5 m thick and frozen at -20 °C. The remaining two groups are repeatedly washed with PBS solution containing high concentration of cyanine and streptomycin for 3 times. When the upper layer of agarose is in semi coagulation state, inserting the upper layer of agarose and putting it into the incubator for 3D co-culture.
g) During the culture period, adding 10 UL PBS or drugs into each hair follicle root
in the hair follicle stem cell group, the hair follicle stem cell + drug group, once every 2
days, for a total of 10 days. Observing the hair growth of the damaged hair follicles.
h) After the culture, the hair follicle samples of hair follicle stem cell group and hair
follicle stem cell+drug group are made into frozen sections with a thickness of 5[m for
subsequent detection.
2) HE staining of hair follicles: frozen sections are solidified with 4%
paraformaldehyde for 10mmin. After washing with tap water for 3 times, soaking the sections
with Harris hematoxylin staining solution for 3-5min, then washing with tap water;
Differentiating with 1% hydrochloric acid alcohol for 3-5s, and washing with tap water;
Soaking the sections in 0.2% ammonia solution for 1 min until it returns to blue, and rinsing
with tap water; Dehydrating the sections with 95% alcohol and anhydrous ethanol, sealing
with xylene transparent and neutral gum; Observing and taking pictures under the
microscope. Making a contrast of the pathological differences between damaged hair
follicles and co-cultured hair follicles.
3) Immunofluorescence staining of hair follicles: frozen sections are solidified with
4% paraformaldehyde for 10min, which is washed with PBS for 3 times, treated with 10%
goat serum blocking solution containing 0.25% Triton-100 for 20min, and incubated
overnight at 4°C with appropriately diluted primary antibodies (K19 and integrin P1). The
next day, after washing with PBS for three times, the fluorescent secondary antibody
corresponding to the species of the primary antibody is added to react at 37C for 30min;
Wshing the sections with PBS, re-staining with DAPI and sealing. Observing under
fluorescence microscope and taking pictures. Compare the difference between damaged
hair follicles and co-cultured hair follicle stem cells.
1. Clinical study on the treatment of pathological alopecia with allogeneic hair follicle
stem cells
Example 1 Patients' condition record of hair follicle regeneration technology
The patient, male, 31 years old, was admitted to hospital for "alopecia for many years",
with alopecia grade 7. Specialist condition: the front hairline and bilateral frontal eminence
moved up obviously, only the core area, the back center area and the top rotation area had
fluffy hair growth, and the scalp was visible, while the hair in the back occipital area was
sparse and soft, and the scalp was visible. Diagnosis: Androgen alopecia.
Treatment course:
On November 25, 2019, the patient was admitted to the hospital at 08:00. Routine
examination of blood routine and infectious diseases were performed before the operation,
and the contraindications of operation were excluded. The scalp was cleaned by routine
haircut. At 08:20, he entered the operating room and underwent autologous hair follicle extraction (FUE) under local anesthesia. At 08:50, 52 single hair follicles and 33 double hair follicles were extracted and stored in nutrient solution containing reagent bottle for low temperature preservation and laboratory culture.
On December 27th, 2019, the patient was instructed to abstain from drinking and fast
for 8 hours before operation one day in advance. Carrying out 08:30 preoperative design,
that is, the height of anterior hairline is 7.5cm, and the height of bilateral frontal eminence
is 7.0cm. The planting area is about 4.0cm under the hairline, which is about 334 square
centimeters. Cleaning the scalp with regular haircut. At 9: 30, the hair follicle cells were
taken from the laboratory and stored at 4°C in the hospital. At 09:55, they entered the
operating room and were implanted with autologous hair follicle cells under basic
anesthesia and local anesthesia. The planting instrument is a hair follicle cell planting gun
designed and produced independently, and the output volume is about 10 microliters each
time, with a total of 4 files, and each file is increased by one file, that is, 10 microliters;
The number of hair follicle primordia injected once is about 10-15 (there is extravasation
when injecting needles every time. It is estimated that 3-5 hair follicle cells are planted at
each point, and the normal hair density is about 50-70 hairs per square centimeter, so the
amount of cells planted this time is more suitable). Anesthetic ratio: 0.9% sodium chloride
injection 30 ml, 2% lidocaine hydrochloride injection 5 ml, ropivacaine injection 5 ml. The
hair follicle cell planting gun is set to a depth of about 4 mm, the planting density is about
needles per square centimeter, and the needle insertion angle is vertical. During the
planting process, there was liquid extravasation. The operation ended at 18:25, and the
planting area was about 334 square centimeters. After operation, amoxicillin capsule was
taken orally for three days. The patient is told to keep the wound clean and avoid infection.
On December 28, 2019, on the first day after surgery, the patient's vital signs were
stable and the wound was slightly painful and tolerable. Physical examination: there was a
little plasma exudation and reddish on the wound surface of the implant area, so no special
treatment was needed. The patient was told to keep the wound dry and clean, avoid
scratching and collision.
On December 29th, 2019, the second day after the operation, the patient's vital signs
were stable and no special discomfort was mentioned. Physical examination: the wound in
the planting area was dry and clean, the skin is reddish, and a little scab was formed. The
patient was told to keep the wound dry and clean, avoid scratching and collision.
On December 30, 2019, on the third day after operation, the patient's vital signs were
stable and no special discomfort was found. Physical examination: the wound in the
planting area was dry and clean, without obvious redness and swelling, with good wound
healing and scab formation. The patient was told to keep the wound dry and clean, avoid
scratching and collision, and the oral antibiotic treatment could be stopped. Keep observing.
On January 4th, 2020, on the 9th day after operation, the patient's vital signs were
stable, and the wound itching was unbearable. Physical examination: The wound in the
planting area was covered with old scab and necrotic epidermis, and the wound healed well
without swelling and exudation. Itching was caused by stimulating nerve endings during
the growth of hair follicle cells. Patients were instructed to spray normal saline to relieve
itching and avoid scratching. Keep observing.
On January 7th, 2020, on the 12th day after operation, the patient's vital signs were
stable, and the itching and discomfort in the planting area were not relieved, which was caused by the stimulation of sensory nerve endings by hair follicle cells in the growth process. Description: Recently, when washing scalp with shampoo, there is a tingling sensation. Considering that shampoo stimulates skin and nerve sensitivity occurs in the repair process of skin sensory nerve in planting area, patients were instructed to change baby shampoo with less irritation and gently wash scalp for symptomatic treatment.
Physical examination: The wound in the planting area was clean and dry, and the wound
has healed well. Observation under the hair follicle detector: there were signs of hair
growth at the primordium of the regrown hair follicle, with 1-3 new hairs in each pore,
which were brown and gray in color and soft in texture. It was also observed that some hair
follicles remained subcutaneous and did not break through the epidermis. Therefore, it was
impossible to count the survival rate and density, so patient was instructed to have regular
outpatient reexamination to understand the growth status of hair follicle cells. The
observation continues.
On January 11, 2020, on the 16th day after operation, the patient's vital signs were
stable, the itching symptoms in the planting area basically disappeared, and there was no
pain when cleaning the scalp. Physical examination: the skin in the planting area was dry
and clean, without redness and swelling, with soft villi growing, sparse hair density, ideal
angle and uniformity, and no obvious improvement in appearance compared with that
before operation. Observation under the hair follicle detector: there are soft hairs growing
in the hair follicle cells, with 1-3 new hairs in each pore, with no obvious increase in the
number. The patient is told to have a weekly outpatient review.
On January 19, 2020, on the 24th day after operation, the patient's vital signs were
stable and no special discomfort was mentioned. Physical examination: the skin in the planting area was dry and clean, without redness and swelling, with a small amount of healthy hair growth and soft villi growth, which was denser than last week. Hair density, angle and uniformity are ideal. Compared with other planting areas, the bilateral frontal area grew slowly, and only sparse and light-colored, soft fluffy hair grew. Considering that male hair loss mostly starts from bilateral frontal areas, whether the slow growth of hair follicle cells in this area was related to its internal environment remains to be confirmed by clinical observation and laboratory test results. Observation by hair follicle detector: healthy hair grew in the hair follicle cells of the returning species, and the growth of soft hair was more than before, with 1-3 new hairs in each pore. Instructing the patient to review the clinic once a week. Keeping observing.
On April 9th, 2020, in the 15th week after operation, the patient was unable to come
to the hospital for regular reexamination due to the epidemic situation in Wuhan, and
recovered well after operation. About 6 weeks after operation, hair loss occurred in the
planting area. Considering the normal physiological phenomenon that hair follicle cells
could not develop into healthy hair at one time in the process of intradermal growth and
development, it is expected that it will take 2-3 times to develop into healthy hair. Physical
examination: only sparse and short villi grow in bilateral frontal eminence; Soft, slightly
sparse and dark hair grows in the core area, central area and apical gyrus, with a length of
about 1.0cm. The appearance of planting area improved significantly compared with that
before operation. During the growth of hair follicle cells, there was no concentrated growth
of one pore, which resulted in excessive hair in one pore, and there was no unsatisfactory
hair uniformity, angle and density. However, the growth time of hair follicle cells was
inconsistent, and the texture was inconsistent during hair growth. It was considered that the development of hair follicle cells was influenced by internal environment and operation.
Observation by hair follicle detector: hair follicle cells in bilateral frontal area grow slower
than those in other areas, and soft hair continues to grow in other hair follicle cells, and the
density is close to normal. There are 1-3 new hairs in each pore, and the growth rate of new
hairs is inconsistent. Instructing the patient to have a monthly outpatient review. The
observation continues.
On May 17, 2020, the patient recovered well in the 20th week after operation, without
any discomfort such as itchy head and abnormal sensation, and had regular diet and sleep.
Physical examination: only sparse and short villi grew in bilateral frontal eminence, which
was not obviously improved compared with 5 weeks ago and needs to be observed later;
Hair in the regeneration planting area in the core area and the front area of the center
became thicker than that in the front part, and there were a lot of villi growing, and the
appearance was obviously improved compared with that in 5 weeks. Observation under the
hair follicle detector: the regenerated hair follicles in bilateral frontal area had growth
phenomenon, but there was no growth and thickening change compared with before, and
they grew in fluffy shape with low density. There were still soft hairs growing in the hair
follicle cells of the remaining species, and the density was close to normal. There were 1
3 new hairs in each pore, and the growth rate of new hairs was inconsistent. Instructing the
patient to review the clinic once a month. Keeping the observation.
June 17, 2020, 24 weeks after operation, the patient recovered well after operation.
The hair loss symptoms occurred in the recent period, the diet and sleep were regular, and
there were no bad habits of the patient. Physical examination: there were only sparse and
short villi growing in bilateral frontal horns, which was not significantly improved compared with that before 4 weeks. The hair in the regeneration planting area of core area and central anterior area became thinner and softer than that of 4 weeks ago, only a few scattered healthy hair grew, and the appearance was worse than 4 weeks ago. Observation under the hair follicle detector: the regenerated hair follicles in bilateral frontal areas grew in a villous shape, the density was fair, and there was no obvious shedding phenomenon.
There were 1-3 new hairs in each pore. At 24 weeks after operation, hair loss and softening
occurred in the implant area. The factors considered were 1: the growth period of hair
follicle cells was too short in the process of growth and development. 2: It is caused by
malnutrition during growth.
At present, there is no obvious healthy hair growth in bilateral frontal horn planting
area, and the density is lower than other planting areas. At present, the high concentration
of dihydrotestosterone distribution and malnutrition in hair follicle growth need clinical
observation and verification.
Example 2
Li Fan, a 28 year old male, was admitted to the hospital for "alopecia for many years".
The grade of alopecia was grade 7. The special condition: the anterior hairline and bilateral
frontal homs moved up obviously, only villous hair growth was found in the central
posterior area and the apical rotation area, the scalp was visible, and the posterior occipital
hair was thin and soft, and the scalp was visible. Diagnosis: androgen alopecia.
Treatment course:
On October 17, 2019, he was admitted to the hospital at 13:30. Before surgery, he
routinely checked blood routine and infectious diseases to rule out surgical contraindications; Cleaning the scalp with regular haircut. At 14:00, he entered the operating room, and at 14:00, he underwent autologous hair follicle extraction (FUE) under local anesthesia. At 14:30, 91 single hair follicles were extracted and 23 double hair follicles were stored in reagent bottle containing nutrient solution for laboratory culture.
On November 11th, 2019, at 10: 00, the hair follicle primordium was taken from the
laboratory and stored at 4°C in the hospital. At 13:40, the hair follicle primordium was put
into the operating room for "autologous hair follicle cell implantation" under local
anesthesia. The planting instrument is a hair follicle cell planting gun designed and
produced independently, and the output volume is about 5 microliters each time, a total of
9 files, and each file is increased by one file, that is, 5 microliters; The number of hair
follicle cells injected once is about 10-15 (there is extravasation when injecting needles
every time. It is estimated that 3-5 hair follicle cells are planted at each point, and the
normal hair density is about 50-70 hairs per square centimeter, so the amount of cells
planted this time is more suitable). Anesthetic ratio: 30 ml of 0.9% sodium chloride
injection, 5 ml of 2% lidocaine hydrochloride injection, 5 ml of ropivacaine injection. The
anesthetic effect was not good during operation, so the anesthetic with the above ratio was
added once. The hair follicle cell planting gun is set to a depth of about 4 mm, the planting
density is about 50 needles per square centimeter, and the needle insertion angle is vertical.
During the planting process, the extravasation of liquid is serious, and the extravasation
still exists when the needle depth is readjusted. It is considered that the concentration of
base liquid is too high, which can not be absorbed and diffused rapidly in tissues. There is
a lot of bleeding in the book, and the rubber band around the head can be considered to
stop bleeding after reoperation. The operation ended at 16:30, and it was unbearable to end the operation ahead of time because of pain, so basic anesthesia and local anesthesia should be considered for re-operation. About 2/3 bags of hair follicle cells were planted, and the planting area was about 65 square centimeters. After operation, amoxicillin capsule was taken orally for three days. Instructing the patient to keep the wound clean and avoid infection.
On November 12th, 2019, the first day after operation, the patient's vital signs were
stable, and the wound was slightly painful and tolerable. Physical examination: There was
a little plasma exudation in the wound surface of the planting area, which was reddish and
didi not need special treatment. Instructing the patient to keep the wound clean and avoid
scratching and collision.
On November 13, 2019, the second day after the operation, the patient's vital signs
were stable and no special discomfort was found. Physical examination: The wound in the
planting area is dry and clean, with reddish skin and a little scab formation. The patient
was instructed to keep the wound dry and clean to avoid scratching and collision.
On November 14th, 2019, the third day after operation, the patient's vital signs were
stable and no special discomfort was mentioned. Physical examination: the wound in the
planting area was dry and clean, without obvious redness and swelling, with good wound
healing and scab formation. Instructing the patient to keep the wound dry and clean, he
may wash the scalp with warm water and stop the oral antibiotic treatment, and avoid
scratching and collision.
On November 18th, 2019, on the 7th day after operation, the patient's vital signs were
stable and the wound was itchy and uncomfortable. Physical examination: The wound surface in the planting area was clean and dry, and the wound surface heals well without swelling and exudation. Itching was caused by stimulating nerve endings during the growth of hair follicle cells. The patient was instructed to spray normal saline to relieve itching and avoid scratching.
On November 19th, 2019, on the 8th day after operation, the patient's vital signs were
stable and the planting area was itchy and uncomfortable, so he was informed to come to
the hospital for reexamination and arrived at the hospital at 18:30. Physical examination:
the wound surface in the planting area was clean and dry, and it had healed, covered with
scab and necrotic epithelial tissue, and cleaned with alcohol. Observation under the hair
follicle detector: there were signs of hair growth at the primordium of the regrown hair
follicle, with 2-5 new hairs in each pore, which were black in color and good in texture. It
was also observed that some hair follicles remained subcutaneous and did not break
through the epidermis. Therefore, it was impossible to count the survival rate and density,
so the patients should be informed to have an outpatient reexamination every three days to
understand the growth status of hair follicle cells.
On November 26th, 2019, the 15th day after operation, the patient's vital signs were
stable and no special discomfort was mentioned. Physical examination: the skin in the
planting area was dry and clean, without redness and swelling, with soft villi growing, ideal
hair density, angle and uniformity, and no obvious improvement in appearance compared
with that before operation. Observation under the hair follicle detector: there were soft hairs
growing at the hair follicle cells of the returning species, with 2-5 new hairs in each pore.
Instructing the patient to have a weekly outpatient review.
On December 3rd, 2019, on the 22nd day after operation, the patient's vital signs were
stable and no special discomfort was found. Physical examination: the skin in the planting
area was dry and clean, without redness and swelling, with a small amount of healthy hair
growth and soft villi growth, which was denser than last week, and the hair density, angle
and uniformity were ideal. Compared with last week, the appearance of hair growth at the
right frontal angle was obviously improved. Observation under the hair follicle detector:
healthy hair grew in the hair follicle cells of the returning species, and the growth of soft
hair was more than before, with 2-5 new hairs in each pore. Asking the patient to have a
weekly outpatient review.
On December 9th, 2019, on the 28th day after operation, the patient's vital signs were
stable and no special discomfort was found. Physical examination: the skin in the planting
area was dry and clean, the position of the front hairline moved down obviously compared
with that before the operation, and the hair texture and density in the core area and the left
frontal eminence also increased obviously compared with last week. The appearance of
planting area improved significantly compared with that before operation. During the
growth process of hair follicle cells four weeks after operation, there was no concentrated
growth of one pore resulting in excessive hair in a single pore, and no unsatisfactory hair
uniformity, angle and density. However, the growth time of hair follicle cells was
inconsistent, and the texture was inconsistent during hair growth. Considering that the
development of hair follicle cells was influenced by internal environment and operation.
Observation under the hair follicle detector: there were still soft hairs growing at the hair
follicle cells of the returning species, which were denser than before. There were 2-5 new
hairs in each pore, and the growth speed of new hairs was inconsistent, and the length of 5 hairs in a single pore was uneven. Informing the patient to have a weekly outpatient review
On March 22nd, 2020, in the 16th week after operation, the patient was unable to
come to the hospital for regular reexamination due to the epidemic situation in Wuhan. The
patient recovered well after operation, and the hair in the planting area was thicker than
before. The patient is instructed to have a monthly outpatient review.
On April 21st, 2020, in the 20th week after operation, the patient recovered well
without any special discomfort. Physical examination: the hair in the planting area was
sparse and soft, with the length of about 3.0cm, in which the hair growth in the core area
and the front center area was ideal, and the villi in the back center area grow more and were
sparse, which was related to the relatively small number of hair follicle cells planted here
during the operation. The texture and density of the planting area were significantly
improved compared with those before operation. Observation under hair follicle detector:
there were still soft hairs growing at the hair follicle cells of the returning species, and
healthy hairs grew more than before. Instructing the patient to have a weekly outpatient
review.
On May 17, 2020, 24 weeks after operation, the patient recovered well without any
special discomfort. Physical examination: the density and texture of hair in the planting
area improved significantly compared with that before 4 weeks, and the number of healthy
hair increased, with the length of about 4.0cm. There was no obvious difference between
the hair in the back center and the surrounding area. Observation under the hair follicle
detector: there were still soft hairs growing at the hair follicle cells, the density was close
to normal 90%, healthy hairs grew more than before, and the length of double hairs with more than one pore was different. Instructing the patient to review the clinic once a week.
On June 30th, 2020, 32nd week after operation, the patient recovered well without
special discomfort. Physical examination: the density and texture of hair in the planting
area improved obviously compared with that before 6 weeks, and the number of healthy
hair increased, but more soft hair grew. It may be caused by malnutrition during hair follicle
development and growth. Observation under the hair follicle detector: there were still soft
hairs growing at the hair follicle cells, the density was close to normal 90%, healthy hairs
grew more than before, and the length of double hairs with more than one pore was different.
Asking the patient to have a weekly outpatient review.
Example 3
A 51-year-old patient was admitted to hospital because of "alopecia for many years".
The grade of alopecia was sparse and moderate in female. Specialist condition: bilateral
frontal eminence moved up obviously, with a little villus growing and scalp visible. Hair
in core area, central area and apical rotation area was thin and soft, and scalp is visible. The
hair in the occipital region was sparse and soft, and the scalp was visible. Diagnosis:
Androgen alopecia.
Treatment course:
On December 4, 2019, the patient was admitted to the hospital at 09:30. Before the
operation, the blood routine and infectious diseases were routinely examined to rule out
the contraindications of surgery; Regular small-area haircut on the occipital region to clean
the scalp. The patient entered the operating room at 10: 00, and underwent autologous hair
follicle extraction (FUE) under local anesthesia. At 10:30, 61 single hair follicles were extracted and 32 double hair follicles were stored in reagent bottle containing nutrient solution for laboratory culture.
On January 4th, 2020, the patient was instructed to abstain from drinking and fasting
for 8 hours before operation one day in advance. Performing preoperative design at 08:30:
the height of the front hairline be unchanged, and the height of bilateral frontal eminence
also be unchanged, so it was only encrypted. The planting area was the whole core area,
central area and apical rotation area, which was about 262 square centimeters. Cleaning the
scalp regularly. At 9: 30, the hair follicle cells were taken from the laboratory and stored
at 4°C in the hospital. At 09:55, they entered the operating room and were implanted with
autologous hair follicle cells under basic anesthesia and local anesthesia. The planting
instrument was a hair follicle cell planting gun designed and produced independently, and
the output volume was about 10 microliters each time, with a total of 4 files, and each file
is increased by one file, that is, 10 microliters; The number of hair follicle primordia
injected once was about 10-15 (there is extravasation when injecting needles every time. It
is estimated that 3-5 hair follicle cells were planted at each point, and the normal hair
density was about 50-70 hairs per square centimeter, so the amount of cells planted this
time was more suitable). Anesthetic ratio: 30 ml of 0.9% sodium chloride injection, 5 ml
of 2% lidocaine hydrochloride injection, 5 ml of ropivacaine injection. The hair follicle
cell planting gun was set to the depth of about 4 mm, the planting density was about 50
needles per square centimeter, and the needle insertion angle was vertical. Liquid
extravasation occurred during the implantation, and the operation ended at 13:50. After
operation, amoxicillin capsule was taken orally for three days. Instructing the patient to
keep the wound dry and clean, avoid infection, scratching and collision.
On January 5, 2020, the first day after operation, the patient's vital signs were stable,
and the wound was slightly painful and tolerable. Physical examination: There was a little
blood exudation in the wound surface of the planting area, which was reddish and did not
need special treatment. Instructing the patient to keep the wound clean and avoid scratching
and collision.
On January 7th, 2020, on the third day after operation, the patient's vital signs were
stable and no special discomfort was founded. Physical examination: the wound in the
planting area was dry and clean, without obvious redness and swelling, with good wound
healing and scab formation. Informing the patient to keep the wound dry and clean, she
may wash the scalp gently with warm water and stop the oral antibiotic treatment, and
avoid scratching and collision.
On January 9, 2020, the fifth day after operation, the patient's vital signs were stable,
and the wound was itchy and uncomfortable, which was tolerable. Physical examination:
the wound in the planting area was covered with a little old scab and necrotic epidermis,
and the wound healed well without swelling and exudation. Itching may be caused by
stimulating nerve endings during the growth of hair follicle cells. Patients are advised to
wash their scalp with warm water and avoid scratching.
On January 19th, 2020, on the 9th day after operation, the patient's vital signs were
stable, and the itching and discomfort in the planting area were not relieved, which was
caused by the stimulation of sensory nerve endings by hair follicle cells in the growth
process. Physical examination: the wound in the planting area is clean and dry, and the
wound has healed well. It can be seen by naked eyes that there were a few healthy and black hair growing in the planting area, which is about 0.2cm long. Whether it was formed by the development and growth of hair follicle cells needs further clinical observation and confirmation. Observation under the hair follicle detector: there were signs of hair growth at the primordium of the regrown hair follicle, with 1-3 new hairs in each pore, which were brown and gray in color and soft in texture. Occasionally, healthy hair follicles grew. It was also observed that some hair follicles remained subcutaneous and did not break through the epidermis. Advising the patient to have regular outpatient reexamination to attain the growth status of hair follicle cells.
On April 1, 2020, in the 14th week after operation, the patient was unable to come to
the hospital for regular reexamination due to the epidemic situation in Wuhan, which
indicated that the postoperative recovery was good and the hair growth was slow. Through
the observation of patients' photos, it could be seen that the hair grows well in the top
planting area of the head and can cover the scalp normally. Instructing the patient to have
a monthly outpatient review. And the observation continues.
The above is the basic principles, main features and advantages of the present
invention. It should be understood by those skilled in the art that the present invention is
not limited by the above embodiments. The above embodiments and descriptions only
illustrate the principles of the present invention. Without departing from the spirit and
scope of the present invention, there will be various changes and improvements in the
present invention, all of which fall within the scope of the claimed invention. The claim
scope of that present invention is defined by the append claims and their equivalents.
Claims (10)
1. A hair follicle microarray co-isolation system is characterized by it comprises a hair
follicle stem cell suspension and damaged hair follicles to be repaired.
2. The hair follicle microarray co-isolation system according to claim 1, wherein the
method for isolating the hair follicle stem cell comprises the following steps:
(1) Taking healthy hair follicles and washing them repeatedly with penicillin
streptomycin PBS solution;
(2) Under the dissecting microscope, cutting off the bulge of the outer root sheath of
the hair follicle by microsurgery, which is placed in a petri dish; Adding hair follicle stem
cell isolation medium, which is incubated in the incubator of 5%CO2 at 37°C;
(3) Supplementing the same amount of medium 1.5mL the next day, observing under
an inverted microscope the outer root sheath of the hair follicle, and carrying out isolating
after the stem cells have reached 80% fusion;
(4) Digesting the hair follicle stem cells, preparing a hair follicle stem cell suspension
of 1.Ox105 cells /mL with the hair follicle stem cell isolation solution, and counting;
(5) After mixing 1.2% agarose and DMEM/F12 isolation medium according to the
mass ratio of 1:1, taking 2ml of the mixed solution which is injected into a 35cm 2 isolation
dish, and putting it into an incubator for later use after cooling and solidification;
(6) After mixing 0.7% agarose and hair follicle stem cell isolation medium in the ratio
of 1:1 in a sterile test tube, adding 1/10 volume of hair follicle stem cell suspension to the
tube, which are mixed well. Then injecting medium I into the petri dish, the amount added is equal to medium I;
(7) Replacing the isolation medium once every 2 days for a total of 10 days.
3. The hair follicle microarray co-isolation system according to claim 2, wherein the
hair follicle stem cell isolation medium contains 10% FBS, 100U/mL penicillin double
antibody, 2ng/ml bFGF DMEM/F12.
4. The hair follicle microarray co-isolation system according to claim 2, characterized
in that the culture medium I is mixed with 1.2% agarose and DMEM/F12 culture medium
according to the ratio of 1:1, then 2ml of the mixed solution is injected into a 35cm 2 culture
dish, which is put into an incubator for later use after being cooled and solidified.
5. The hair follicle microarray co-isolation system according to claim 2, wherein the
digestion solution of the hair follicle stem cell digestion contains a phosphate buffer
solution containing 0.1% pancreatin and 0.008% EDTA by mass.
6. The hair follicle microarray co-isolation system according to claim 2, wherein the
healthy hair follicle is taken from the occipital part of the head.
7. The hair follicle microarray co-isolation system according to claim 5, which is
characterized in that the digestion condition is 37 °C for 3-5 minutes.
8. The hair follicle microarray co-isolation system according to claims 1-7, wherein
the damaged hair follicles to be repaired are repeatedly washed with penicillin
streptomycin PBS solution, and the follicles are inserted into the upper agar of the solidified
hair follicle stem cell suspension. Then they are put into an incubator for 3D co-isolation
to form a hair follicle microarray co-isolation system.
9. The application of a hair follicle microarray co-isolation system in drugs for
treating pathological alopecia is characterized in that the hair follicle microarray co-culture
system according to any one of claims 1-6 is prepared into drugs for treating pathological
alopecia, damaged hair follicles in hair loss areas are implanted into the drugs for repair
culture, and repaired hair follicles are implanted into hair loss areas.
10. The application of the hair follicle microarray co-isolation system in treating
pathological alopecia according to claim 9, it is characterized by implanting successfully
repaired hair follicles in the hair loss area, and an oral drug is offered, and the oral drug is
thymosin.
-1/2- 2020103546
Figure 1.
The skin HE of nude mice in each group after injection for 5 weeks
-2/2- 2020103546
Figure 2.
The hair growth status after subcutaneous injection of HFSC
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