Thuangtong et al., J Clin Exp Dermatol Res 2013, 4:3
http://dx.doi.org/10.4172/2155-9554.1000182
Clinical & Experimental
Dermatology Research
Open Access
Research Article
In vitro Culture and Histological Characterization of Extracted Human
Hair Follicles
Rattapon Thuangtong1, Pitchaya Maneeprasopchoke1*, Chatchawan Srisawat2, Chinnavuth Vatanashevanopakorn2, Suchanan
Hanamornroongruang3, Sarawut Junnu2 and Saroj Suvanasuthi1
1
2
3
Department of Dermatology, Faculty of Medicine Siriraj hospital, Mahidol University, Thailand
Department of Biochemistry, Faculty of Medicine Siriraj hospital, Mahidol University, Thailand
Department of Pathology, Faculty of Medicine Siriraj hospital, Mahidol University, Thailand
Abstract
Background: In vitro-cultured hair, follicles can serve as an excellent model system for exploring the hair follicle
biology as well as drug delivery targets. In this study, we aimed to investigate how to maintain whole human hair follicles
in vitro and histologically characterize the hair follicles thereof.
Methods: Donated hair follicle specimens from hair transplant operation were cultured in vitro using a medium
consisting of Williams` medium E, L-glutamine, insulin, hydrocortisone, penicillin, streptomycin, and amphotericin B. The
growth of hair follicles was examined daily using a digital microscope. Their histological features were studied at day 0,
7, and 14 to characterize the changes of hair follicles maintained in culture.
Results: Cultured hair follicles continued to grow at a rate of about 200 μm/day during the first four days. Then the
growth rate declined and stopped after an average (range) of 8.4 (7-11) days in culture. Histological study showed an
upward regression of the hair follicles with scattered apoptosis of outer root sheath cells, resembling the follicles in a
catagen stage.
Conclusion: Human hair follicles can be cultured in vitro for at least 7 days before progressing to catagen.
Keywords: Human hair follicle; Hair culture; histological change;
Hair measurement
Introduction
Hair transplantation surgery is indicated for the patients with
male-pattern baldness that is unresponsive to medical treatments.
This procedure can be performed in two different approaches. The first
one is the strip harvesting technique; a strip of scalp is removed under
local anesthesia and then cut into single follicular units, which are then
transplanted back into the balding area in order to have re-growth
hairs. The other approach is Follicular Unit Extraction (FUE), which
is a novel, convenient and effective technique, Individual Hair Follicles
(HF) are extracted and from the occipital scalp and then reinserted
back into the recipient area [1,2].
A follicular unit is a skin appendage with complex structures
containing many cell types resulting from epidermal-dermal
interactions. It consists of epidermis as a matrix, inner root sheath,
bulge, outer root sheath, and dermis such as connective tissues and
dermal papilla. In addition to these components, follicular stem
cells with highly pluripotent properties have been reported to reside
in many parts of the follicle. The stem cells are not only limited to
epidermal stem cells at the bulge but also include melanocyte stem
cells and mesenchymal (nestin-expressing) stem cells [3-8]. These hair
follicles stem cells could potentially serve as gene therapy targets for
regenerative medicine [9-12].
To study the hair follicles biology or use hair follicles cells as drug
delivery or gene therapy targets, in vitro culture of HFs would be of
great use. There are several reports about in vitro culture of hair follicles
published to date [13]. However, most of the studies focused only at
the inferior portion of hair follicles because the length of the whole
hair follicles usually exceeds the low power field of most microscopes.
Moreover, the histological studies of cultured hair follicles are also
limited. In this study, we aimed to culture whole human hair follicles in
vitro and studied their morphology and growth using a novel technique
J Clin Exp Dermatol Res
ISSN: 2155-9554 JCEDR, an open access journal
based on a digital microscope, which has a wider filed, enabling a study
of long hair follicles. The hair follicles were also histologically examined
for any changes after cultured in media up to 14 days.
Materials and Methods
Human hair follicle collection
This study was approved by the institutional review board at
Siriraj Hospital. Human HFs (77 HFs from 10 donors) were obtained
with informed consents from the patients undergoing elective hair
transplantation surgery for androgenic alopecia at the Department
of Dermatology, Siriraj hospital, Mahidol University. All donors
were more than 18 years of age. Single follicular units were obtained
through either the strip harvesting or FUE techniques. Hair follicles
were selected and transported to the laboratory in sterile culture
plates containing normal saline solution, which were placed in an ice
container.
In vitro culture of hair follicles
Isolated hair follicles were cultured using a method modified
*Corresponding author: Pitchaya Maneeprasopchoke, Department of
Dermatology, Siriraj Hospital, 2 Prannok Rd, Bangkoknoi, Bangkok, Thailand, Tel:
+662-419-4333; Email: [email protected]
Received July 25, 2013; Accepted August 30, 2013; Published September 10,
2013
Citation: Thuangtong R, Maneeprasopchoke P, Srisawat C, Vatanashevanopakorn
C, Hanamornroongruang S, et al. (2013) In vitro Culture and Histological
Characterization of Extracted Human Hair Follicles. J Clin Exp Dermatol Res 4:
182. doi:10.4172/2155-9554.1000182
Copyright: © 2013 Thuangtong R, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Volume 4 • Issue 3 • 1000182
Citation: Thuangtong R, Maneeprasopchoke P, Srisawat C, Vatanashevanopakorn C, Hanamornroongruang S, et al. (2013) In vitro Culture and
Histological Characterization of Extracted Human Hair Follicles. J Clin Exp Dermatol Res 4: 182. doi:10.4172/2155-9554.1000182
Page 2 of 4
from Philpott et al. [13]. Each isolated follicle was immediately placed
into a well of a 24-well plate filled with 500 mL of culture medium.
The medium consists of Williams` medium E (Gibco™), which was
developed during the establishment of an enrichment technique for
adult rat hepatocytes based on sequential reduction of the amount
of contaminating fibroblasts during culture. Moreover, It contains
higher amounts of trace elements and exhibits small variations in the
supplementation. In addition, the medium was supplemented with 2
mmol/L L-glutamine (Glutamax®), hydrocortisone 10ng/ml, insulin 10
μg/ml, penicillin 100 U/ml, streptomycin 100 µg/ml, and amphotericin
B 25 µg/ml. In the negative control group, 0.9% NaCl supplemented
with antibiotics was added instead of the medium. All cultures were
incubated at 37°C in an atmosphere of 5% CO2 and 95% air. The
medium was replaced every other day.
an average growth rate of 0.24mm per day, then slowly declined and
eventually stopped growing after an average of 8.4 days after culture
(Figure 3). Accumulative measurement data from 10 subjects, 77
hairs were maintained in modified Williams`medium E and 42 hairs
were maintained in NSS which is the standard solution using in
transplantation operation (Table 1). The hair follicles in NSS were not
growing at all because there are separation of outer root sheath and
basement membrane in histological section (data not shown). The
hair follicle in modified Williams`medium E could be growth average
1.86mm (0.98 – 2.52) with mean growing period of 8.4 days (7-11).
Examination of hair follicles using a digital microscope and
image analysis
A handheld digital microscope (Dino-Lite™ Polarized microscope)
which has a field of view of 9 × 7 mm was used to examine and
photograph the hair follicles instead of the inverted microscope due
to its wider study field. The images of each follicle were taken daily for
2 weeks. DinoXcope program (Dino-Lite™) was used to analyze the
length of hair follicles from digital images. The length measured from
the top end of the cutted follicle to the bottom end of the bulb is defined
as Total Hair Length (THL) and the length of hair shaft above the
epidermis is defined as External Hair Length (EHL). The diameter (D)
of hair shaft was measured at the infundibular opening. The endpoint
of the follicle growth is defined as the time when there is no increase in
total hair length for 3 days.
Figure 1: Human hair follicle isolated for hair transplantation procedure day
0. Intact hair bulb, dermal papila (DP), outer root sheath (ORS), hair shaft (H),
Sebaceous (S) and epidermis (E) are showing.
1 mm
Histological changes of in vitro hair follicles
One to two longitudinal sections of hair follicles at day 0, 7, 14
were bisected and processed in a routine manner that included 10%
neutral-buffered formalin fixation, embedding in paraffin, sectioning
at 4 μ staining with hematoxylin and eosin and examined under a
microscope.
Statistical analysis
The data were shown as means and standard deviation. Statistical
analysis using one way repeated ANOVA was used to show significant
increase of follicle length during in vitro culture. Pearson’s correlation
coefficient was used for correlation analysis. The difference was
considered significant when P value is less than 0.05.
Figure 2: Single follicular unit hair follicle from transplantation operation
showing intact hair bulb, hair shaft, outer root sheath and epidermis (day 0).
Sequential increase in length of hair follicle at day 1, 3, 5, 7 and 10 were
observed.
Results
Study of whole human hair follicle in-vitro
The whole hair follicles including the epidermal part, the isthmus
part and the lower portion could be isolated using FUE techniques. The
images of a whole hair follicles could be clearly and conveniently taken
by a handheld digital microscope (Dino-Lite™ Polarized microscope),
which allows a wider field of study up to 9 × 7 mm; an example is
shown in Figure 1.
Growth of human hair follicles in vitro
Cultured hair follicles continued to grow in vitro, particularly
during the first few days; sequential images of a follicle at various
days after culture clearly demonstrate an elongation of hair shaft
and follicle as shown in Figure 2. When the lengths of hair follicles
were measured and plotted against the time after culture, an increase
of hair length seems to be in linear fashion in the first four days with
J Clin Exp Dermatol Res
ISSN: 2155-9554 JCEDR, an open access journal
Figure 3: Mean of incensement in length of hair from 1 – 14 day in culture
media (77 hairs) and NSS (42hairs) were plot in linear graph.
Volume 4 • Issue 3 • 1000182
Citation: Thuangtong R, Maneeprasopchoke P, Srisawat C, Vatanashevanopakorn C, Hanamornroongruang S, et al. (2013) In vitro Culture and
Histological Characterization of Extracted Human Hair Follicles. J Clin Exp Dermatol Res 4: 182. doi:10.4172/2155-9554.1000182
Page 3 of 4
The average growth rate of all hairs was 0.21mm per day (1.4-2.7). The
average hair diameter of occipital terminal hair of this study was 75.7
μm.
Histological change
The histological character of in vitro hair at day 0, 7 and 14 shows
normal hair follicle at the beginning (Figure 4). At day 7 in culture
medium, bulb remains intact, but decrease of matrix cell and 1-2
necrotic cells in outer root sheath can be observed. At day 14, changing
of bulb shape, separations of matrix cell and scatter apoptosis of outer
root sheath cell can be seen. In some instances upward regression of
and hair follicle may also be observed (data not shown), which resemble
catagen stage.
Correlation between hair diameter, follicle length and growth
rate
Statistical analysis of growing data showing both growth rate and
total growth are synthesized with Pearson’s correlation coefficient
0.836 (p<0.001) (Figure 5). Sizes of hair (all terminal anagen hair)
are not correlated with hair growth while follicle length and hair
growth showed significant negative relation with Pearson’s correlation
coefficient, 0.017 and -0.358 (p<0.05), respectively.
Discussion
Previous studies, focused most on the inferior portion of hair
follicles [13]. A few studies showed growth of whole hair follicles,
Case1
Case2
Case3
Case4
Case5
Case6
Case7
Case8
Case9
Case10
No.of follicles (Media/NSS)
9/5
8/5
9/5
7/3
8/4
8/3
9/4
10/4
10/5
11/4
Diameter (µ)
74.5
75.0
77.7
78.5
76.5
72.8
79.9
69.4
78.8
773.9
Growth period (days)
6.8
7.5
7.8
8.2
8.2
8.3
10.9
9.4
9.5
7.3
8.4/1.2
Growth rate (µ/day) (Media/NSS)
140/0
140/0
216/0
187/0
199/0
252/0
229/0
270/0
260/0
216/0
210/45 and 0/0
Growth (µ)
980/0
922/0
1649/0
1973/0
2081/0
2518/0
2518/0
2365/0
2173/0
1419/0
1862.7/917 and 0/0
2
1
0
1
1
1
2
2
0
1
No growth hair in Williums’ media E
x/S.D.
75.7/8.5
Table 1: Measurement data from 10 cases.
Figure 4: Histology (H&E, 100x) of hair follicle at day 0 (A), Day7 (B), Day14(C). Magnifying 400x picture showing necrotic cell in outer root sheath (D) and Changing
of bulb shape (E) after culture in media for 14 days.
5.A
5.B
Figure 5: A: Show positive coleration between average growth rate and total growth (Pearson’s correlation coefficient 0.836**). B: Show no coleration between hair
shaft diameter and average growth rate (Pearson’s correlation coefficient 0.017 ).
J Clin Exp Dermatol Res
ISSN: 2155-9554 JCEDR, an open access journal
Volume 4 • Issue 3 • 1000182
Citation: Thuangtong R, Maneeprasopchoke P, Srisawat C, Vatanashevanopakorn C, Hanamornroongruang S, et al. (2013) In vitro Culture and
Histological Characterization of Extracted Human Hair Follicles. J Clin Exp Dermatol Res 4: 182. doi:10.4172/2155-9554.1000182
Page 4 of 4
including up to the epidermis, but no histological profile. One of the
obstacles is that the field of study of the inverted light microscope is
limited, at 5mm (4x lens), while average length of human scalp terminal
anagen hair follicle is 5-6 mm. One study used a digital camera and a
measurement program to study whole hair follicle, but it must take take
the follicle out of the medium [14]. We developed a study system using
Dino-Lite™ Polarized microscope, which has the measurable study
field of 9 × 6.7 millimeter. This camera has also been used in dentistry
research. The digital microscope comes with DinoXcope program
(Dino-Lite™), the digital measurement program. This microscope
could be used under the culture plate to observe hair growth without
removing the medium. We checked digital measurement vs. length
reliability by using the standard scale in chamber under culture media
which indicated that length from digital program and true length are
reliable with standard error less than 20 µm per 1000 µm. Our system
shows the clear image of a hair follicle and can be used to observe a
follicle in a culture plate without taking it out of media.
The human hair follicle is source of stem cell reserve [3-8]. Gene
therapy targeted hair follicle might be a novel technique to treat difficult
dermatologic and non-dermatologic diseases [15]. Gene transduction
targeted hair follicular cell can be done by using both viral vector and
non-viral vector [16,17]. Transduction of Green Fluorescent Protein
(GFP) gene to hair follicle can be detected as visible green fluorescence
by fluorescent microscope within 48 hours after transduction. We
show that a whole hair follicle, including sebaceous and epidermis,
could maintain growth in fetal cow serum free media formula for 7-10
day. This 1-week period is enough as a model to study in-vitro hair
follicular gene therapy.
In-vitro maintenance of hair follicles has shown that hairs stop
growing after about 1 week. Philpott et al. shows the histology of in vitro
hair culture at day 9, in the same medium, shows intact morphology
and is seen to remain in the anagen phase [13]. This work was done
with the lower portion of the hair follicle without epidermis. In our
work, histological study at day 7 shows intact morphology. But a few
necrotic cells at the outer root sheath and decreased cell number of the
hair matrix were observed. At day 14, disintegration of hair matrix and
epithelium column was seen; there were also groups of necrotic cells
at outer root sheath, which resemble turning from anagen to telogen
hair [18-20].
In-vivo human scalp anagen hairs grow 0.3-0.4mm per day and the
growth rate increasing in the larger diameter [21]. In-vitro correlation
between the grow rate and the hair diameter had not been defined. In
our work, statistical analysis shows total growth of hair was dependent
on the growth rate, but we could find the relation between hair size and
growth rate.
In summary, we developed a system that can observe in vitro hair
follicle growth using digital dermoscope. The hair follicle can maintain
growth in modified Williams`medium E about 7-10 days, before
turning to catagen and stop growing. The total growth of hair was
dependent on the growth rate but has no relation to the diameter size.
Acknowledgement
Ms. Pimrapat Tengtrakulcharoen for helping in Statistical analysis. Hair
transplantation team for isolation of hair follicle.
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Citation:
Thuangtong
R,
Maneeprasopchoke
P,
Srisawat
C,
Vatanashevanopakorn C, Hanamornroongruang S, et al. (2013) In vitro Culture
and Histological Characterization of Extracted Human Hair Follicles. J Clin Exp
Dermatol Res 4: 182. doi:10.4172/2155-9554.1000182
J Clin Exp Dermatol Res
ISSN: 2155-9554 JCEDR, an open access journal
Volume 4 • Issue 3 • 1000182