./rw~itr/ 1~1nr,71iurr~/(,fiic,~1/ Sc~wwr. 3 (1992) 163-17 1 1’3 1992 Else\,ier Science Publishers B.V. All rights reserved.
Organ culture conditions of human hair follicles Ryusuke Imai, Yuko Miura, Kouki Mochida, Toshimasa Hideoki Ogawa Dcprtvlem
(!/Dermutolog~~.Jwrendo L’t~iuer.vir~~ School (Received
Jindo, Kenji Takamori
~~natratcd h! histological
XI-\ ,~tions from both histology \Ve l;wnd I;lr cultures
that culture of human
,~nd the germinative
As such. organ
at 37 ‘C). striking culture
changes to human
for 12 days under
in the hair bulb cells as dem-
of 95”” O,-5”,, CO, a~ 3 1 ’ C were superior when compared to normal culture conditions attempting to maintain the normal morphology of hair germinative cells. The hair bulb
hair f~>llicles when
bulb DN.A synthesis I :nder condition\ culture
in the hair bulb increased morphological
the 96 and 48 h culture
in the germinative
increased time dependently for 96 h after culture initiation. of 95”,, O,-5”” CO, at 31 ‘C. the synthesis of DNA in hair germinative tnetlx)d rnd! prolc LISC~LIIfor studies on the human hair growth mechanism.
The biology of hair growth is an area in which many basic questions remain unanswered. Critical studies designed to answer these questions require development of a cell culture and an organ culture system suitable for hair follicles. Weterring et al. [ 1] described a method for culturing epithelial cells derived from the outer root sheath of the human hair follicle. Several similar methC’~J~~~,(/)OIII~~,(‘I~L.L’ to: Ryusuke Imai, <)g!. Juntcnd~, Ilnivrrsit) School Bunl\\o-ku. Tokyo 113.Japan.
Department of Medicine,
cells was observed.
Such an organ
ods have been reported [X3]. Jahoda and Oliver  have reported the establishment of in vitro cultures of dermal papilla cells from rat vibrissa follicles. The culture of human dermal papilla cells has also been reported by Messenger et al.  and Katsuoka et al. . Recently, Tanigaki et al.  reported on the culture of murine hair cells, including hair bulb germinative cells. Although many investigators have reported on cell culture systems, an efficient culturing technique for the hair germinative cells of hair bulbs is presently unavailable. While it is apparent that cultured hair cells, being only one aspect of the complex hair struc-
Fig. 3. Schema of the hair organ culture method. The culture dish contained approximately 0.75 ml of media in the central hole, over which a stainless Five hair follicles
mesh and lens paper
on the lens paper
in each of
Fig. 1. Freshly isolated dium under
normal anagen hair follicles an inverted microscope.
culture system. Some reports on organ cultured mouse hair follicle have been published [8-141. Recently, Philpott et al [ 151 reported the maintenance and growth of human hair follicles in vitro. In that investigation, they cultured follicles under normal culture conditions (95% air-5% CO, at 37 “C). On the other hand, Kondo et al. [ 161 reported on the organ culture of human hair follicles under high oxygen content conditions (95% 02-5% CO, at 36 “C).
n 7 (6
Fig. 4. Time course Fig. 2. Section
ture, present some problems with regard to culturing, these problems may be resolved by organ culture of hair follicles. The biology of hair growth can be observed in total by utilizing such an organ
in the hair bulb cul-
turedin 957; air-5”/, CO, at 37 “C. DNA synthesis increased time dependently for 12 days. Each experiment was carried out in triplicate using tive hair bulbs per culture dish. Bars represent means + SD. Parentheses represent the time after [3H]thymidine (3H-TdR) labeling. The follicles were labelled continuously
Fig. 5. Morphology culture
of hair bulb cultured The changes
in 95”,, air-5”,
The purpose of the herein reported study was to establish an appropriate organ culture condition (02 concentration and temperature) for studying the biology of human hair growth. In this paper we report on culture conditions for human hair follicles that may be more suitable than those normally utilized. Materials and Methods lsolrttior~qf‘h~?laFl
C. The structure
of hair bulb had alread>
2-l h after
gradually progressed during the culture periods (48 h. 72 h. 96 h). The hair bulb’s germinative also degenerated in all samples (14 h. 48 h, 77 h, 96 h).
Utilizing a stereo microscope, normal anagen hair follicles of the human scalp were obtained by use of a knife and tweezers from the operative skin of four patients with benign scalp tumor. Figures 1 and 2 show the microscopic features of the isolated normal anagen hair follicles.
Isolated hair follicles were placed in Ham F12 (GIBCO) containing X0”;, fetal bovine serum (GIBCO), 500 unit/ml of penicillin (GIBCO) and 500 u&/ml streptomycin (GIBCO) for 20 min. After washing in Ham F 12, the hair follicles were cultured in Ham Fl2 containing 30”(, fetal bovine serum under various conditions. As shown in Fig. 3, culture dishes (Falcon 3037) contained the media in the central hole, over which a stainless mesh (Falcon 3037) and lens paper were placed. Five hair follicles were placed on the lens paper in each dish and were sunk into the medium so as to be surrounded by a film of medium. Cultures were carried out in either 95 “(, air-5 o. CO, or 95”,, 02-5(Jl, CO, at various temperatures (37, 3 1 and 25 ‘C) using a
Fig. 6. Morphology
of hair bulb cultured
72 h, 96 h) in a high oxygen
in 95 y0 02-5%
CO, at 37 ‘C. The hair bulb maintained
at 48 h and developed
temperature gradient incubator: TN-2 12 (Toyo Kagakusangyo Co., Ltd., Tokyo). The media was not changed during the culture period when morphological observations were carried out. On the other hand, during the observation of DNA synthesis, the media was changed to Ham F12 containing 20% fetal bovine serum and 2 uCi/ml of t3H]thymidine after 24 h, and was then changed thereafter every 3 days. Therefore, the follicles were labelled continuously by [ 3H]thymidine. Morphology
Cultured follicles were fixed overnight in 10% buffered formalin and processed for paraffin embedding. Sections were cut into 4 urn thicknesses and stained with hematoxylin-eosin. Slides were examined using light microscopy.
of hair germinative
cells lying far from dermal
(24 h, 48 h, occurred
(72 h, 96 h).
Localization of [‘Hlthymidine in cultured follicles was determined with autoradiographs. Follicles were cultured for 24 h in Ham F 12 containing 20 y0 fetal bovine serum, and were then labeled for 24 h by adding 2 @i/ml of [3H]thymidine to each of the media in 95 y0 O,-5 y0 CO* at 3 1 oC. The follicles were then fixed overnight in 10% buffered formal and processed for paraffin embedding. Sections were cut into 4 urn thicknesses, dipped in NR-M2 nuclear track emulsion (Konica), and stored in a dark container containing desiccant for 14 days at -20 “C. The emulsion was developed at room temperature for 10 min in Konicadol (Konica), rinsed in water, then fixed in Konifix (Konica) for 10 min. Slides were counterstained with hematoxylin-eosin and examined using light microscopy.
FIN. 7. Morphology 71 h. 96 h). The
its normal atructurc (24 h, 4X h. of hair bulb cultured in 95 O0 O,-5 “” CO, at 3 1 ‘C. The hair bulb maintained hair bulb’s germinative cells maintained their normal morphology (24 h, 48 h). A slight degeneration of the germinative cells lying far from dermal papillae occurred at 72 and 96 h.
Measuren~ent qf DNA in hair bulb Cultured follicles were rinsed in lo”, TCA. The hair bulbs only were cut from the follicles with a knife, placed in a microhomogenizer (Wheaton 200 # 357848), and then homogenized in 200 1.11 of lo’,, TCA. Measurement of DNA in the hair bulb homogenate was achieved by the methods described by Kissane and Robins [ 171. The measurement of fluorescence intensity was carried out using a Hitachi Fluorescence Spectrophotometer. Meusurernent qf DNA synthesis in hair bulb DNA synthesis in cultured hair follicles was investigated by measuring the incorporation of [‘Hlthymidine by DNA in the hair bulb consti-
tuting cells. Briefly, hair follicles were cultured in Ham F12 containing 204;, fetal bovine serum. After 24 h, L3H]thymidine was added (2 uCi/ml), and the samples were then subjected to further culturing under various conditions. The cultured follicles were subsequently rinsed in lo”, TCA. The hair bulbs only were cut from the follicles with a knife, placed in microhomogenizer, and then homogenized in 200 ul of IO”, TCA. The [ ‘Hlthymidine content in the homogenates of the hair bulb was measured by a scintillation counter (Beckman LS-3801). The [3H]thymidine count was divided by the content of DNA in hair bulb homogenate, the result of which indicated the amount of DNA synthesis in the cultured hair bulb.
Fig,. 8. Morphology of hair bulb cultured in 95”,/, O,-5% CO, al: 25 “C. The hair bulb and the germinative cells maintained their normal morphology (2 4 h , 48 h, 12 h, 96 h).
Results Effect of O2 concentration on organ culture Initially, the effect of O2 concentration on the organ culture was observed. The [3H] thymidine uptake of hair bulb DNA cultured in Ham F12 containing 20% fetal bovine serum in 95% air5% CO, at 37 “C increased linearly up to 7 days in conjunction with the increasing culture period, eventually plateauing at 12 days (Fig. 4). Figure 5 illustrates the micrographs of follicles cultured for 24, 48, 72 and 96 h under the same conditions. The hair bulb’s structure commenced changing within 24 h of culture initiation, and continued to do so time dependently. Germinative cells of the hair bulb also demonstrated degeneration after 24 h culture. In 95% 0,-5x CO, at 37 “C the hair bulb maintained its normal structure through-
out the 96 h culture, although degeneration of the hair germinative cells occurred at 48 h culture and continued to do so time dependently (Fig. 6). EfSect of temperature on organ culture We then investigated the effect of temperature on hair follicle culture in 95% O,-5 y0 CO1. At 3 1 ‘C, the hair bulb maintained its normal structure through 96 h culture (Fig. 7). The hair bulb’s germinative cells also maintained their normal morphology during 48 h culture, although a slight degeneration of the germinative cells lying far from the dermal papillae occurred at 72 and 96 h culture. At 25 “C, the hair bulb and germinative cells maintained their normal structure throughout the 96 h culture period (Fig. 8), although the [ ‘HIthymidine uptake was remarkably suppressed (data not shown). Accordingly, the autoradiographic
Fig. 9. :\utoradiographs 95’J,, O,-5”,, primarily Labclling
of hair follicles at 31 ‘C.
for 48 h in
in the germinative cells below Auber‘s critical line. of nuclei in a few cells of the outer root sheath and
sheath was observed. while no labelling in the cells of dermal papillae.
Fig. 10. Time course in 95”,, O-5 dependently triplicate
Under normal culture conditions (95”,, air5”,, CO, at 37 “C). the [3H]thymidine uptake in the hair bulb increased time dependently for 12 days (Fig. 4). Although the data appears to indicate that the hair bulb cells maintained their cell viability for 12 days, the histological findings revealed striking morphological changes of the hair bulb cells (Fig. 5). As such, these findings
rn hair bulb cultured increased
for 96 h. Each
using five hair bulbs per culture
i SD. Parentheses
localization of [“Hlthymidine and DNA synthesis in hair bulb in 95”, O,-5”; CO, at 3 1 ‘C was studied. Figure 9 shows the autoradiographs of hair follicles cultured in 95”, 02-59, CO, at 3 1 ‘C. Autoradiographs of [ 3H]thymidinelabelled follicles cultured for 48 h showed that [ “Hlthymidine was localized primarily in the germinative cells below Auber’s critical line. Labelling of the cells of the outer root sheath and the fibrous sheath was rarely observed. Labelling of the dermal papilla cells was not observed. Figure 10 shows the time course of [“Hlthymidine uptake by DNA of follicles cultured in 95”, O,5”,, CO, at 3 1 ‘C. The above-mentioned [ 3H]thymidine uptake increased linearly and time dependently throughout the culture period.
“” CO, at 3 I ‘C. DNA synthesis
labelling. 2 uCi:ml
time out in
dish. Bars represent
The follicles were labelled of [jH]thymidinc
to the media.
revealed that our organ culture of hair follicles carried out under the stated conditions was, in fact, unsuccessful and, furthermore, that histological findings are absolutely necessary when estimating the success of an organ culture. We therefore continued to study the culture conditions of hair follicles utilizing observations from histology and DNA synthesis. The hair bulb’s germinative cells probably obtain nutrition and oxygen from vascular tissue which is rich in dermal papillae. The cell cycle of hair germinative cells is very short when compared to that of the epidermal basal cells [ 181. Such findings suggest that hair germinative cells require richer oxygen than that of the epidermal basal cells. We therefore attempted to culture follicles under a high oxygen content condition at 37 ‘C. Under such conditions. although the degeneration of hair germinative cells could not be suppressed after 48 h culture, the hair bulb maintained its normal structure (Fig. 6). thereby suggesting that a high oxygen content condition is more suitable than normal culture conditions. Ikeda et al. [ 191 reported on the influence of culturing temperature with respect to changes induced in the organ culture of psoriatic skin. Although skin explant degeneration occurred at
37 ‘C, degeneration was almost completely inhibited when explants were cultured at 3 1 and 24 oC. Accordingly, we cultured hair follicles at reduced temperature (3 1 “C, 25 “C). The degeneration of hair germinative cells, which occurred when follicles were cultured at 37 “C (Fig. 6) was almost inhibited when follicles were cultured at 3 1 a and 25 “C (Figs. 7,8). The temperature of normai skin has been reported as being approximately 33 “C , thereby suggesting that it may be more appropriate to culture follicles at 3 1 oC rather than 25 “C. We therefore continued further investigation with culture conditions that included a high oxygen content (95% 02-5 % CO1) and a reduced temperature (3 1 “C). Autoradiographs of [3H]thymidine in Fig. 9 showed localization primarily in the germinative cells below Auber’s critical line. This finding indicates that the synthesis of DNA occurs in the hair germinative cells and not in the dermal papilla cells. As shown in Fig. 10, [3H]thymidine incorporation of the hair bulb increased time dependently until 96 h culture. This finding indicates that the germinative cells of the hair bulb synthesize DNA for 96 h in vitro. The findings in Figs. 9 and 10 indicate that human hair follicles in vitro also maintained cell viability from the standpoint of DNA synthesis. The results shown in Figs. 7, 9 and 10 indicate that culture conditions of 95 y0 O,-5% CO, at 3 1 ‘C are suitable for culturing human hair follicles, at least from the standpoint of both histology and DNA synthesis. Philpott et al. [ 161 reported that hair follicles grow in vitro under conditions of 95% air-5 y0 CO? at 37 “C. They observed that hair follicle length, increased time dependently and that [ 3H]thymidine uptake, [ 14C]leucine uptake and hair keratin synthesis occurred in the cultured hair follicle. We are presently examining the hair follicle length [ 14C]leucine uptake and hair keratin synthesis under the culture conditions that we have proposed herein, and will report the results of such examination in a subsequent paper. We reported herein that culture conditions of 95 p0 02-5% CO, at 3 1 PC are suitable for cultures of human hair follicles when attempting to
maintain the normal morphology of hair germinative cells and that, utilizing said conditions, the synthesis of DNA in hair germinative cells can be investigated. Such an organ culture method may prove useful for studies on the human hair growth mechanism.
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