Arch Dermatol Res (1990) 282:442-445
Organ culture of human scalp hair follicles: effect of testosterone and oestrogen on hair growth S. Kondo, Y. Hozumi, and K. Aso Department of Dermatology, Yamagata University School of Medicine, Yamagata City, Yamagata 990-23, Japan Received March 10, 1990
Summary. Whole human scalp hair follicles were cultured. The follicles were dissected from skin pieces of normal scalp and put into 1.5 ml of incubation medium in a closed 5 ml glass tube under an atmosphere of 95% 0 2 and 5% CO2. The tube was rolled at 15 rpm at 36~ Remarkable hair growth was noticed for 7 to 8 days. Hair root sheaths also grew with the hair shafts. The structure of the hair bulbs was well maintained for at least 6 days, and then the hair matrix ceils started to degenerate. Fetal calf serum was not essential for hair growth in vitro, but increased the growth rate slowly. Testosterone and oestrogen inhibited hair growth in vitro to a similar extent. The minimum effective doses of both hormones to suppress hair growth were around 5 ng]ml, which corresponds well to the normal plasma level of testosterone in adult males in vivo, suggesting that scalp hair growth may be critically controlled by testosterone in adult males. Key words: O r g a n c u l t u r e - H u m a n hair follicle - H a i r growth -
Recently, cell cultures o f h u m a n or a n i m a l hair follicles i n c l u d i n g the o u t e r r o o t sheath a n d the d e r m a l p a p i l l a have b e e n e m p l o y e d for the s t u d y o f hair g r o w t h m e c h a n isms [8, 9, 1 1 - 1 3 ] . However, these studies r e p o r t o n l y d a t a for i n d i v i d u a l p a r t s o f the whole follicle. It is n o w necessary to observe the whole follicle in vitro. I n o r g a n culture, successful hair g r o w t h for 7 to 8 days in 3 to 5-day-old mice has b e e n r e p o r t e d [1, 2]. H a t t o r i  also r e p o r t e d o r g a n c u l t u r e o f m o u s e hair follicles a n d the effect of s e r u m c o m p o n e n t s . We r e p o r t the o r g a n c u l t u r e o f h u m a n scalp hair follicles to observe hair g r o w t h a n d some effects o f b o t h sex h o r m o n e s o n hair g r o w t h in vitro.
Offprint requests to: S. Kondo
Materials and methods Dulbecco's modified minimumessential medium (DMEM) and fetal calf serum (FCS) were purchased from Dainihon Pharmaceutical Co. (Osaka, Japan). Testosterone enanthate (Testinon Depot, 125mg/ml in sesame oil) was purchased from Mochida Pharmaceutical Co. (Tokyo). Oestradiol dipropionate (Ovahormon Depot, 5mg/ml in oil) was purchased from Teikokuzoki Pharmaceutical Co. (Tokyo). Thymidine-3H (TdR-3H) was obtained from Amersham Japan.
Organ culture The culture methods have previously been reported . Scalp skin specimens with hair follicles were obtained. Whole hair follicles were dissected out of the skin specimens under a dissecting microscope. Each hair follicle was put into 1.5 ml of incubation medium in a 5 ml siliconized glass tube (culture tube), into which a mixture of 95% 02 and 5% CO2 was blown through a tube from a gas bomb. The culture tube was closed with a rubber plug, and rolled at 15 rpm at 36~ in a modified roller tube culture incubator (Type SB-2, Ikemoto Rikakogyo Co. Ltd, Tokyo). The lengths of the hair follicles in the culture tubes were measured every 24 h through the tube wall under a dissecting microscope at x 20. The incubation medium was renewed every 3 days. At the end of culture, the hair follicles were fixed in 10% formalin and stained with haematoxylin-eosin (HE).
Incubation media DMEM was supplemented with 0 to 20% FCS, plus 0.4 gg/ml hydrocortisone (HC), 50 gg/ml streptomycin and 50 U/ml penicillin.
Addition of hormones Both testosterone and oestrogen in oil were dissolved in 99.5% ethanol and added to the culture giving a final concentration of alcohol in the medium of 0.5%. This resulted in the sesame oil in Testinon Depot being diluted 106 or more times and that in Ovahormon Depot being diluted 4 x l0 s or more times in the culture medium.
S. Kondo et al. : Organ culture of human hair follicle
Fig. 1. Growth curve of human scalp hair follicles in organ culture.
Scalp hair follicles in the growing stage from a normal 17-yearold male cultured in DMEM + 20% FCS. Q, no addition of sex hormones; O, addition of 125 ng/ml testosterone; x, addition of 125 ng/ml oestrogen
Fig. 2. The effect of FCS concentrations on human scalp hair growth
in organ culture. Scalp hair follicles in the growing stage from a normal 6-year-old female were cultured. Each point represents the mean value of 3 - 5 hair follicles per concentration. 0, 1 day of culture; A, 2 days of culture; I , 3 days of culture; • 5 days of culture
Autoradiography The effect of hormones Some follicles were incubated with 2 gCi/ml TdR-3H for 14 h (overnight), fixed in 10% formalin, paraffin-sectioned and submitted to autoradiography.
Organ culture of scalp hair follicles The hair shafts grew steadily for 7 - 8 days at a rate of 0 . 1 5 - 0 . 4 ram/day. They grew linearly for about 6 days and then the growth rate decreased (Fig. 1). The hair shaft grew well in D M E M without FCS, but the addition of 1 0 - 5 0 % FCS increased the growth rate by up to 100% (Fig. 2).
Histological analysis of cultured follicles Microscopic observations revealed rapid growth of the hair shaft (Fig. 3 A - C ) as well as maintenance of intact hair bulb structures for about 6 days in culture (Fig. 3 D, E). Hair root sheaths also grew with the hair shafts (Fig. 3B, C), Degeneration of the hair matrix started at about day 7 or 8 of culture (Fig. 3F). Histological observations revealed that most hair bulb cells and the interface between the dermal papilla and the matrix were well maintained at day 3 (Fig. 3 G) as well as at 5 days (Fig. 3 I) of culture. The degenerating hair bulb showed disintegration of the interface between the dermal papilla and the matrix (Fig. 3 H).
Autoradiography For at least 5 days of culture many 3H-TdR-labelled matrix cells were seen in the lower part of the hair bulb (Fig. 4).
The addition o f either hormone suppressed the hair growth rate (Figs. i and 5). Both hormones suppressed the growth rate in a similar manner during culture (Fig. 1). The minimum suppressive concentration of both hormones was about 5 ng/ml, ranging from 3 to 8 ng/ml, and the maximum suppressive rate was about 70% for both hormones (Fig. 5), although the suppressive rate was around 50% in the case of the experiment in Fig. 1.
Recent studies o f dermal papillae in cell culture have revealed some important relationships between the hair matrix and the dermal papilla. Dermal papilla cells cultured in vitro and implanted into an animal induced the hair growth activity of the hair matrix in vivo . However, there have been no reports about the success in cell culture of hair matrix cells, indicating the difficulty of keeping them in vitro. In our organ culture system, hair matrix cells actively propagated for at least 5 days, proved by the presence of TdR-3H-tabelled cells in the hair matrix. The hair root sheath grew with the hair shaft in vitro at the same rate, probably because root sheath cells were growing at the hair matrix as well as at their cut ends. Degeneration of the hair matrix cells was recognized at about day 7 of culture. The reason for the hair matrix cell degeneration may be lack of oxygen and nutrients in the centre of the hair bulb, inadequate incubation medium, or lack o f growth factors. The first reason is unlikely because o f the high 02 pressure in the gas phase and the high rotation rate of the culture tubes in the culture system, as in a previous report of psoriatic skin culture . The second and third reasons may be more probable, although the effect of concentration of FCS was slight.
S. Kondo et al. : Organ culture of human hair folIicle
Fig. 3 A - H . Histological observation of hair follicles in organ culture. Scalp hair follicles from a 35-year-old male were cultured in DMEM + 20% FCS. A hair follicle in organ culture was observed each day under a dissecting microscope: A, day 0; B, day 2; and C, day 5 at • and D, day 1; E, day 6; and F, day 8 at x60. A - C show the fast growth of a hair shaft. The root sheath is also growing together with the hair shaft (arrows). In C the hair shaft looks narrow because of flattening of the shaft by chance. D and E show well maintained hair bulb structures, but F shows a disappearing matrix structure. G shows the well-maintained structure of a hair bulb including the hair matrix and dermal papilla of day 3 of culture, while H shows the degenerating hair matrix of day 7 of culture (arrowheads) ( • 150, HE staining). I shows a cross section of the lower part of a hair bulb cultured for 5 days, demonstrating the well-maintained hair bulb structure, including matrix and dermal papilla cells, and the interface between the matrix and dermal papilla (small arrows). Some cells with clear cytoplasm corresponding to melanocytes can be seen (large arrows) ( x 350, HE staining)
Fig. 4 A - C . Autoradiography of the hair matrix in organ culture. There are many 3H-TdRlabelled cells (arrows) observed in the lower hair bulb corresponding to the hair matrix at (A) day 1, (B) day 3 and (C) day 5 of organ culture in DMEM § 20% FCS. Pa, dermal papilla, x 750
C o n c e n t r a t i o n of
60 Hormones [ng/ml~
Fig. 5. The effect of sex hormone concentration on human hair growth in organ culture. Scalp hair follicles from a 35-year-old male cultured for 3 days in DMEM + 10% FCS. Both sex hormones remarkably inhibited hair growth in organ culture, x, testosterone; O, oestrogen
B o t h sex h o r m o n e s , testosterone and oestrogen, lowered the g r o w t h rate o f the hair follicle but did n o t delay the degeneration in vitro. It is interesting that the patterns o f the inhibitory effects o f b o t h h o r m o n e s were similar: the m i n i m u m inhibitory concentrations o f b o t h h o r m o n e s were a r o u n d 5 ng/ml. The n o r m a l testosterone levels in plasma range f r o m 3 to 10 ng/ml in adult males, and f r o m 0.2 to 0.8 ng/ml in adult females, while n o r m a l oestrogen (as oestradiol in plasma) concentrations are f r o m 0.05 to 0.186 ng/ml in adult females and m u c h less in adult males. J u d g i n g f r o m these data, scalp hair g r o w t h m a y be critically controlled by testosterone in adult males in vivo. The action o f oestrogen in reducing the rate o f hair g r o w t h has also been shown experimentally in the guinea pig, a l t h o u g h in this instance, the d u r a t i o n o f a n a g e n was prolonged, n o t shortened . K a t s u o k a et al.  d e m o n s t r a t e d a n d r o g e n - b i n d i n g protein in the nuclei o f the papillary cells o f scalp hair using a cytochemical staining m e t h o d . T a k a y a s u and A d a c h i  d e m o n s t r a t e d 5c~-reductase activity in h u m a n
S. Kondo et al. : Organ culture of human hair follicle hair follicles, indicating that testosterone is converted to the active form, 5c~-dihydrotestosterone, and utilized in h u m a n hair follicles. However, the existence o f a n d r o g e n receptors has n o t been confirmed yet in the hair follicle except in the dermal papilla, a n d the m e c h a n i s m o f the a n d r o g e n action is n o t k n o w n . The relationship between the dermal papilla a n d the hair matrix cells m u s t be i m p o r t a n t and should be studied, especially with regard to sex h o r m o n e s . F r a t e r et al.  stated that, in the presence o f F C S or 1 2 - 1 4 d a y rat serum, rat hair g r o w t h was well maintained for 2 days in o r g a n culture, b u t in the presence o f 1 8 - 2 0 day rat serum or adult rat serum, the rat hair bulb degenerated markedly, suggesting the presence o f g r o w t h factor(s) in fetal or y o u n g animal serum. In our experiment with h u m a n scalp hair follicles, the fact that hair shafts grew in media w i t h o u t F C S m i g h t indicate that hair g r o w t h requires no g r o w t h factors, or that the autocrine or paracrine f o r m s o f g r o w t h factor(s) exist in hair matrix cells or in dermal papilla cells. F u t u r e studies will d e m o n s t r a t e any conditions or factors which p r o l o n g the m a i n t e n a n c e o f hair bulb structures in o r g a n culture.
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