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Research Article Open Access
Thuangtong et al., J Clin Exp Dermatol Res 2013, 4:3 DOI:
10.4172/2155-9554.1000182
Volume 4 • Issue 3 • 1000182J Clin Exp Dermatol ResISSN:
2155-9554 JCEDR, an open access journal
*Corresponding author: Pitchaya Maneeprasopchoke, Department
ofDermatology, 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.
In vitro Culture and Histological Characterization of Extracted
Human Hair FolliclesRattapon Thuangtong1, Pitchaya
Maneeprasopchoke1*, Chatchawan Srisawat2, Chinnavuth
Vatanashevanopakorn2, Suchanan Hanamornroongruang3, Sarawut Junnu2
and Saroj Suvanasuthi1 1Department of Dermatology, Faculty of
Medicine Siriraj hospital, Mahidol University, Thailand2Department
of Biochemistry, Faculty of Medicine Siriraj hospital, Mahidol
University, Thailand3Department of Pathology, Faculty of Medicine
Siriraj hospital, Mahidol University, Thailand
Keywords: Human hair follicle; Hair culture; histological
change;Hair measurement
IntroductionHair 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
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 MethodsHuman 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 folliclesIsolated hair follicles were
cultured using a method modified
AbstractBackground: 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.
Journal of Clinical & ExperimentalDermatology
ResearchJournal o
f Clin
ical
&Exp
erimental Dermatology Research
ISSN: 2155-9554
-
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
Volume 4 • Issue 3 • 1000182J Clin Exp Dermatol ResISSN:
2155-9554 JCEDR, an open access journal
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.
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.
Histological changes of in vitro hair folliclesOne 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 analysisThe 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.
ResultsStudy 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 vitroCultured 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
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).
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
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.
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.
-
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
Volume 4 • Issue 3 • 1000182J Clin Exp Dermatol ResISSN:
2155-9554 JCEDR, an open access journal
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
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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
Page 4 of 4
Volume 4 • Issue 3 • 1000182J Clin Exp Dermatol ResISSN:
2155-9554 JCEDR, an open access journal
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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TitleCorresponding authorAbstractKeywordsIntroductionMaterials
and Methods Human hair follicle collection In vitro culture of hair
follicles Examination of hair follicles using a digital microscope
and image analysis Histological changes of in vitro hair follicles
Statistical analysis
ResultsStudy of whole human hair follicle in-vitro Growth of
human hair follicles in vitro Histological change Correlation
between hair diameter, follicle length and growth rate
Discussion AcknowledgementFigure 1Figure 2Figure 3Figure 4Figure
5Table 1References
