./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.

0913-181

I63

1/92’SO5.00

DESC 00141

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

Ke> words:

Organ

culture;

26 October

Human

1991; acccptcd

hair follicles;

Culture

Jindo, Kenji Takamori

and

ofMedicine. To~~YI.Jqxrr?

18 February

conditions:

1992)

Hair growth:

DNA

synthesis

Abstract

L:\perimental normal

results

culture

rcvcaled

conditions

~~natratcd h! histological

findings.

XI-\ ,~tions from both histology \Ve l;wnd I;lr cultures

that culture of human

,~nd the germinative

that although

(‘$5’” air-5”,,

[‘Hlthymidine CO,

As such. organ

and DNA

conditions

uptake

at 37 ‘C). striking culture

conditions

applicable

changes to human

time dependently

occurred

for 12 days under

in the hair bulb cells as dem-

hair follicles

were studied

utilizing

ob-

synthesis.

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

cells successfully

maintained

Zut~~radl~)gr~~p~~~of [“Hlthymldine-labeled

their normal

follicles

showed

morphology localization

bulb DN.A synthesis I :nder condition\ culture

in the hair bulb increased morphological

throughout

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.

Introduction

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,

changed

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).

hair follicles

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.

cells

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

content

in 95 y0 02-5%

condition.

CO, at 37 ‘C. The hair bulb maintained

The degeneration

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

time dependently

its normat

structure

cells lying far from dermal

papillae

(24 h, 48 h, occurred

(72 h, 96 h).

Autoradiographs

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.

167

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.

168

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

169

Fig. 9. :\utoradiographs 95’J,, O,-5”,, primarily Labclling

CO,

of hair follicles at 31 ‘C.

cultured

[ ‘H]Thymidine

for 48 h in

was localized

in the germinative cells below Auber‘s critical line. of nuclei in a few cells of the outer root sheath and

the fibrous

sheath was observed. while no labelling in the cells of dermal papillae.

was seen

Fig. 10. Time course in 95”,, O-5 dependently triplicate

Discussion

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

synthesis

rn hair bulb cultured increased

for 96 h. Each

carried

experiment

using five hair bulbs per culture

means

i SD. Parentheses

midine

( ‘H-TdR)

uously

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.

of DNA

“” CO, at 3 I ‘C. DNA synthesis

by adding

represent

labelling. 2 uCi:ml

was

time out in

dish. Bars represent

the pertods

after [3H]thy-

The follicles were labelled of [jH]thymidinc

contin-

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

170

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 [20], 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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