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1 Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006, pp 인체두피모낭의장기간배양을위한기관배양배지의개발 l içl Ç i Ç i Çm Ç k*ç q**ç o ne t 3 26 *tk p e ne q 221 ** t ne e o 4 ( o 10p r, o 20p }ˆ) Development of Organ Culture Medium for Long Term Culture of Human Hair Follicle Bo-Young Yoo,GHee-Hoon Yoon, Yeon-Ho Shin, Young-Kwon Seo, Doo-Hoon Lee, Kye-Yong Song*, Sung-Joo Hwang** and Jung-Keug Park Department of Chemical and Biochemical Engineering, Dongguk University, 3-26, Pil-dong, Chung-gu, Seoul , Korea *Department of Pathology, Chung-Ang University, 221, Heukseok-dong, Dongjak-gu, Seoul , Korea **SJ Hair Hair Clinic, Dongwon Btd, 513-1, Sinsa-dong, Kangnam-gu, Seoul , Korea (Received 1 December 2005; accepted 20 January 2006) k l l p~ svl p pn l p rp mp k s p k kp m. nˆk ~~ e p p qp r p p l ~ v s p e m. ~ v kl pn Williams E mediump p Philpott medium q ~ k ˆ (B ) s p DHGM(Dongguk hair growth medium)p pn m. IMDM p DHGMp s p pn m. l Philpott medium IMDMp k p s p p qp 9p 12p r l, p k p dž pv, CK19 p p l p k l p (regression) pl p Ž l., DHGMp k p rp k q p s p 25p v v rp p v 3 p k p dž pv, r rp CK19 p ˆ l. p k ˆ v s p ~ nl p q k tn l p p Ž. p k p q n l r l k n ˆ rp kl pn pp p. h Abstract We successfully isolated human anagen hair follicles from human scalp skin by microdissection and tried to culture them under various conditions. First we confirmed negative effect of serum on human hair follicle organ culture. As a next step serum-free medium compositions, Philpott medium, IMDM, and DHGM (Dongguk hair growth medium) were tried. Philpott medium is a general medium for hair organ culture based on Williams E medium and DHGM is a special self-developed medium containing high amino acids and vitamins (B group) composition. As results, hair follicle in Philpott medium and IMDM showed anagen phase morphological structure, but rapid loss of hair elongation, low alkaline phosphatase expression, and very low expression of CK19. It is thought these hair follicles rapidly regressed from apoptosis. However, hair follicles in DHGM showed long term anagen phase morphological structure, continuous hair elongation, high alkaline phosphatase, and CK19 expression. These results demonstrate that high amino acids and vitamins (B group) composition are essential to in vitro long term human hair follicle organ culture and this culture medium will be useful in basic study of hair biology or application study to the development of alopecia treatment drugs. Key words: Hair, Organ Culture, Medium, Hair Length, CK19, Alkaline Phosphatase To whom correspondence should be addressed. jkpark@dongguk.edu 58

2 1. p qv n m(ectoderm) t m(mesoderm) o p k s p q p (organ)p., p t p rq p p ve, p, l q p }p ol l [1]. o (dermal papilla)m v(matrix) pp -v qn (epidermal-dermal interaction)l p q (anagen), (catagen), v (telogen) p lv p t (life cycle) p ~ pl. pm p p q ov sr o ˆ l l 15~20 p t r p k r [2]. p q,, v t l r q r qp kv k r pv k. ˆ m qp ˆ l r l p dšd (testosterone)p 5α- o (5α-reductase)l p p d Šd (dihydortestosterone, DHT)p r, p p k r n~(androgen receptor)m o p ˆ p r p [3]. o r p ˆ rp p pl rkp, q rp p pn ~ e p t p. p p sq ( )p pn n s(species) p e [5, 20], p o qlrp (stumptailed macaque)p nl np p l e dv k [19]. k ol e p rp Žp p erp. p o l p~ p ~ nl k (organ culture) p p e m. rp p r o l k v s p l m, ~ (serum)p rrp o p k r p l q ~ v s p e p v p. ~l p qp r transforming growth factor-β(tgf-β) l pl ~ nl p s o p k r. 1~20Í kv ~p ~ k n ~ v k n p q p l v p p plp ~ p nl ~ pq p ke p k n l l n l sp p rm l kl ~ v rp [5, 6, 7] p rp n v Philpott [6]p p vp Williams E mediuml 10 ug/ml insulin, 10 ug/ml transferrin, 10 ng/ml sodium selenite 10 ng/ml hydrocortisone p ~ s p. p l Kealey [5]p Philpott mediuml ˆ (L-glutamine)p 2 mm p 4 mm ~ l k p p ATP p v p m. pm p l qp k q kp l o, m kp plk nl pl rkp. p r o n v rp m mk p r p l rp l. p q p~ p q kp o k vp 59 v p q p l n m o p n (outer root sheath), (inner root sheath), v (matrix), (melanocyte) p, t m o p o (dermal papilla), v(dermal sheath) p sq. epš p(cytokine)p qpq(growth factor) r e r (paracrine signaling) k vrrp - qn(direct cell-cell interaction)p ~ nl l r o v p r m ~ n m v p p rp mk p v l p k rp mk p p l q kp l n p r m. v, sp l p p s p k v vl l qpq p ~ l p s p lq l mp l l p q kn v q~p s p m. p rp t p e pk kr~(usa FDA)p p qrp e (Minoxidil)p, l p p oveˆ p k r [8]. e p ~ nl o p ke qp v vascular endothelial growth factor(vegf)p p q l p v ep v p k r [4]. svl v m, q (mesenteric ischemia)p nl k p p pe(glycine) ~ pro-apoptotic gene productp baxm caspase-3 p t anti-apoptotic gene productp bcl2 p p p l [18]. Tanaka [24] p L- (L-serine)p e p sp oveˆ, L- pep anti-apoptotic gene productp Bcl-w v e apoptosis r eˆ p p. p l p ke k ˆ p bcl-2 p v e apoptosis r p [23]. nrp s p pv kv, ~ ~p kp tp r (s ) ~ s l k o rp k lr v p k ˆ p o p l v p. p ˆp n k k p p IMDM (Iscove s Modified Dulbecco s medium) v s p ˆp k ˆ (B )p 2 o vp DHGM (Dongguk hair growth medium)p m. p rp ~ nl p l vop (glucose) ˆ (L-glutamine)p pn p 2,000 mg/l ˆ p 4mM v e [11]. ~ p o t svl r Cu 2+m ˆp l p p p qp p p, ~ nl t svp q p Cu p m o 10-4 µg/mlp ~ m [10, 11, 12]. n ~k (bovine serum albumin, BSA)p Cum l peroxyl radical trap qnp p k r pl peroxidel p apoptosis r eˆ p Ž [11]. p v q ve t p o t l q svp DNA kr ol tn qnp Znp rp [13, 14, 15], endonucleasesp r r l pl v Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

3 60 o mëo Ëel Ë m Ëp Ë në të r (keratinocyte)p apoptosis qnp lr qnp p v p pp p Ž l vl ~ m [13, 16]. rp kl pn insulin, transferrin, sodium selenite hydrocortisone p Philpott medium ~ m. p v pn l kp mp, p ˆ, p q, k p dž pv r, sv r p m. 2. m 2-1. m p~ p pe e p p wk e q p l n p n e e r s vl e qp p ll n m. svp kn vl k e e p Philpott [5]p p pn l p m. Scalpel blade No. 10p pn l (epidermis)m o (papillary dermis)p r okp v (reticular dermis)m v (hypodermis) l p m. p svp p~ p kp tosvp r m. kl p mp o m v p p l q p n mp p qp n l rn m h h l mk mz m h 24-well plate(spl, Korea)l 1 jp v 500 µl ~ l k kp ee mp 2p p v m. p n v Philpott medium, IMDM, DHGMp ~ p p lp n Sigma r p n m. Philpott mediump Williams E medium(gibco BRL, USA)l 10 µg/ml insulin, 10 ng/ml hydrocortisone, 10 µg/ml transferrin, 10 ng/ml sodium selenite 50 U/ml gentamicin(welgene, Korea)p l rs m. r ~p p o l Philpott mediuml 5Í nˆk ~(fetal bovine serum, FBS) ~ l k m. IMDM(Welgene, Korea) DHGMl kp ~ nl 4 mm( s ) L-glutamine, trace elements(10 4 µg/ml cupric sulfate, 10 4 µg/ml zinc sulfate, 10 4 µg/ml manganese sulfate, µg/ml iron sulfate, 20 mg/l bovine serum albumin) l tl. p v vp s p Table 1. v s vmp v ~ el mp p ˆ m p v n ql m z l m on j m m ~o p p qp p~ p p p m. 2 p rk vm 10 p v n p~ (stereoscope, KSZ, Korea) pn l o lk o v r mp e eqpp 0p l 25p v vp l m. p p l r v p r mp p s o v r e v o44 o k Table 1. Composition of Philpott medium, IMDM, and DHGM Philpott medium IMDM DHGM INORGANIC SALTS CaCl CuSO 4 ²5H 2 O Fe(NO 3 )²9H 2 O KCl KNO MgSO MnCl 2 4H 2 O NaCl NaHCO Na 2 HPO ZnSO 4 7H 2 O OTHER COMPONENTS D-Glucose Glutathione Methyl Linoleate Sodium Pyruvate HEPES Phenol Red AMINO ACIDS L-Alanine L-Arginine HCl L-Asparagine(anhydrous) L-Aspartic acid L-Cystine L-Cystine 2HCl L-Glutamic acid L-Glutamine L-Glycine L-Histidine HCl 2H 2 O L-Isoleucine L-Leucine L-Lysine HCl L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophane L-Tyrosine 2Na 2H 2 O L-Tyrosine L-Valine VITAMINS Ascorbic acid Biotin D-Ca Pantothenate Choline Chloride Ergocalcipherol Folic acid i-inositol Menadione Sodium Bisulfate Niacinamide Pyridoxal HCl Riboflavin α-tochopherol Phosphate, Disodium Thiamine HCl Vitamin A Acetate Vitamin B

20 o C l 20 pe o l sq k p dž pv m. k p dž pv 96-well plate(bottom black, SPL, Korea)l well 20 µlp k p dž pv nkp ~ 20 µlp MgCl 2m 160 µlp fluorescence assay buffer ~ m.

4 v m pr p l m o qp q pn l p r m (m o±5 µm). v s l p r p o v ~ e l v m p ˆ okp m. v rp qp, o m d svp q l l p q o m d sv p p eq o m d svp l t epithelial strand, o m d p l p v k v p l k rpp t p pp p ˆ l. p~ p q kp o k vp m g m mr h ~o o p p o l ˆrp k p dž pv(alkaline phosphatase) kp o kp l (microdissection)p pn l o p m. o v nkp PRO- PREP(Intron, Korea) 100 µll o 10 p sv pn l o m. 20 o C l 20 pe o l sq k p dž pv m. k p dž pv 96-well plate(bottom black, SPL, Korea)l well 20 µlp k p dž pv nkp ~ 20 µlp MgCl 2m 160 µlp fluorescence assay buffer ~ m. pr l 4-methylumbelliferyl phosphate vp well 1µl ~ q p l t fluorometer(victor 3, Perkin Elmer, USA) pn l excitation Ž qp 340 nm, emission Žqp 440 nm r l p p r m [17] os i n k kp ee p 10Í l r Ž p rs sv r m p r p r l d p l e H/E m (hematoxylin/eosin staining)p e e l p m. ve p v pp sq t r r l m p vp CK19p p m [21, 22]. r svr d p 0.3Í H 2 O 2 nkl l endogeneous peroxidase p lr, 2Í k ~ 0.5Í ~p n l p ~ p ktl. p t svr d p 1 ~p RCK108(DAKO, USA) ml 2e k peˆ, 2 ~ biotinylated horse anti-mouse IgG(1:200 dilution, Vector, USA) n l k 30 pe. p Tris m nkp 5 3 } m. avidin-biotin(zymed Lab, USA) eˆ daiaminobenzidine (DAB)p m. Hematoxylinp sm p, 3Í glutaraldehyde nkp r l gel-mount(biomedia, USA) p l m. 3. y 3-1. m op p svp vr v kn v Fig. 1. Schematic diagram of procedure to isolate hair follicle from scalp dermis. Fig. 2. Microscopy of isolated human hair follicle (a: anagen phase, b: catagen phase, c: telogen phase). l k q mp 4e p l p m (Fig. 1). q p 80Í p p p plp p ~ p p sp Ž p lpp p pl. p n s p l r p k pl. p p~ p l o m v l rp l l p s v p p l p q p (80Í p, Fig. 2(a)), l rp l p o m v ˆ l p p v p p l (Fig. 2(b), (c)) m hk s mk mz m h n 5Í ~p p, l q p l m v o e p d svp vep lp k 4pwl dm o l k p d o ˆ o s p p l (Fig. 3). Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

p sr p r (1Í)p ~l k 4pl 7pw pl p ˆ p k ~ l l p v p Ž l. ~p p q kl r p Ž l p e l r m. q kn v s p p~ p ql p o s l l q n Philpott medium p s p n m.

2 BSA, and trace elements (Arrows indicate starting points). Fig. 6.

5 62 o mëo Ëel Ë m Ëp Ë në të r Fig. 3. Regression of hair bulb structure caused by fetal bovine serum after 4 days of organ culture (a: Philpott medium only b: Philpott medium with 5 FBS). p sr p r (1Í)p ~l k 4pl 7pw pl p ˆ p k ~ l l p v p Ž l. ~p p q kl r p Ž l p e l r m. q kn v s p p~ p ql p o s l l q n Philpott medium p s p n m. k kp ˆ p o p ~ (X30)l l v v mp m, p ~ p p p l m ne p 5 r s l e p o m. p m o v ~ l mp mp, v p kp p k yp p o yp l o p rl m l. sp v r n v kn p l r v r p k(10 µl/cm ) p ~ l m 2 p f r m. k 2pw v vl n m p qp el p plp kr Fig. 5. Morphology of organ cultured hair follicle in IMDM supplemented with 10 ng/ml hydrocortisone, 10 µg/ml insulin, 10 µg/ml transferrin, and 10 ng/ml sodium selenite, 0.2 BSA, and trace elements (Arrows indicate starting points). Fig. 6. Morphology of organ cultured hair follicles in DHGM supplemented with 10 ng/ml hydrocortisone, 10 µg/ml insulin, 10 µg/ml transferrin, and 10 ng/ml sodium selenite, 0.2 BSA, and trace elements (Arrows indicate starting points). Fig. 4. Morphology of organ cultured hair follicles in Philpott medium, which is Williams E medium supplemented with 10 ng/ml hydrocortisone, 10 µg/ml insulin, 10 µg/ml, transferrin, 10 ng/ml sodium selenite, and 0.2 BSA (Arrows indicate starting points). o44 o k perp Žo p p pl (Fig. 4, 5, 6). ~ l m k kl to svp q p rp l l perp v v v p p qp k p v k p ˆ p kp r r l p l p ˆp 3 o sv p pn l to svp p rp l p Ž. p v p Philpott mediump n k 9pw

6 p~ p q kp o k vp 63 Fig. 7. Increase of hair length of organ cultured hair follicles in Philpott medium, IMDM and DHGM (*p<0.01). Fig. 8. Alkaline phosphatase expression of organ cultured hair follicles in different medium (*p<0.01). p v r ˆ v kk. k (9p) kp p v pp mm/day p l. IMDMp n k 12pw v v rp v mp (Fig. 7), 12p kp p v pp mm/day r l. DHGM p n k 20pw p p v v k 10pw vp p v pp mm/day ~ p v p 0.4 mm/daym p pv kkp 25pw v p v pl. Philpott mediump k l srp k 2 p p p v ˆ l (Fig. 7). e l n p p p vq r p n mp ~ pl llp p Ž. k t l p n pp p v ˆ e p q p pp Table 2l ˆ. s l k p lvl q p pp p plp IMDM DHGMp n q p p Philpott mediuml ˆ p p pl. Philpott mediump n k 2pw q p pp k 12pw 29.2Í IMDMp n 54.2Í, DHGMp n 66.7Íp l p p p pl (Table 2). n p o p o l p k v k p dž pvp r p k 4pw Philpott mediuml DHGMp n p 3 p v k p ˆ p p p k 12pw v ov l (Fig. 8). k k p s p o H/E m t l prp ˆ CK19l lsv mep ee m [11, 12]. p rp l sq r t CK19, q l, Ž o, l k r, v l Žo p n p r p yp p n p n l r~rp l ˆ p k r. Philpott mediump 10p k l p v k k CK19 DHGMp k l n p n l p p pl (Fig. 9(b), (d)). p r rp CK19 p k p (early catagen phase) l p p Ž l rp pl p p m. 4. s rp p v s p p Philpott mediump IMDMp k p s p q } pv p qp lp, p k p dž pv, CK19 p p l p k p l p (regression) pl p Ž DHGMp k p rp k q p s, v rp p v p k p dž pv r rp CK19 p ˆ l. p k ˆ v s p ~ nl p q k t n l p p Ž, p k p q Table 2. Number of hair follicles at growth phase Day of culture Philpott medium 19/24(79.2Í) 14/24(58.3Í) 12/24(50.0Í) 7/24(29.2Í) Media IMDM 20/24(83.3Í) 18/24(75.0Í) 13/24(54.2Í) 13/24(54.2Í) DHGM 24/24(100Í) 20/24(83.3Í) 19/24(79.2Í) 16/24(66.7Í) Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

64 o mëo Ëel Ë m Ëp Ë në të r Fig. 9. Histological analysis of organ cultured hair follicles at 10th day (a, b: in Philpott medium; c, d: in DHGM) (Brown color indicates CK19-positive cells).

7 64 o mëo Ëel Ë m Ëp Ë në të r Fig. 9. Histological analysis of organ cultured hair follicles at 10th day (a, b: in Philpott medium; c, d: in DHGM) (Brown color indicates CK19-positive cells). n l l k n ˆ rp kl pn pp p. l lqo l l l( r )p vol p lp pl. y 1. Maria, I. M. and Marjana, T. C., Epidermal Stem Cells: the Cradle of Epidermal Determination, Differentiation and Wound Healing, Biol. Cell., 97(3), (2005). 2. Jahoda, C. A. and Reynolds, A. J., Dermal-Epidermal Interactions: Adult Follicle-derived Cell Populations and Hair Growth, Dermatol. Clin., 14(4), (1996). 3. Eicheler, W., Happle, R. and Hoffmann, R., 5α-Reductase Activity in the Human Hair Follicle Concentrates in Dermal Papilla, Arch. Dermatol. Res., 290, (1998). 4. Messenger, A. G. and Rundegren, J., Minoxidil: Mechanism of Action on Hair Follicle, Br. J. Dermatol., 150, (2004). 5. Philpott, M. P., Green, M. R. and Kealey, T., Human Hair Growth in vitro, J. Cell. Sci., 97, (1990). 6. Westgate, G. E., Gibson, W. T., Kealey, T. and Philpott, M. P., Prolonged Maintenance of Human Hair Follicles in vitro in a Serum-free Medium, Br. J. Dermatol., 129, (1993). 7. Childs, C. B., Proper, J. A., Tucker, R. F. and Moses, H. L., Serum Contains a Platelet-derived Transforming Growth Factor, Proc. Natl. Acad. Sci. USA., 79, (1982). 8. Sica, D. A., Minoxidil: an Underused Vasodilator for Resistant or Severe Hypertension, J. Clin. Hypertens., 6(5), (2004). 9. Vjayalakshhmi, B., Sesikeran, B., Udaykumar, P., Kalyanasundaram, S. and Raghunath, M., Effects of Vitamin Restriction and Supplementation on Rat Intestinal Epithelial Cell Apoptosis, Free. Radic. Biol. Med., 38(12), (2005). 10. Uno, H. and Kurata, S., Chemical Agents and Peptides Affect Hair Growth, J. Invest. Dermatol., 101(1 Suppl), (1993). 11. Thomas, C. E., The Influence of Medium Components on Cu(2+)-Dependent Oxidation of Low-density Lipoproteins and its Sensitivity to Superoxide Dismutase, Biochim. Biophys. Acta., 1128(1), 50-57(1992). 12. Trachy, R. E., Fors, T. D., Pickart, L. and Uno, H., The Hair Follicle-stimulating Properties of Peptide Copper Complexes. Results in C 3 H Mice., Ann. N. Y. Acad. Sci., 26(642), (1991). 13. Lewin, B. and Genes, V., Oxford University Press Inc., New York(1994). 14. Paus, R. and Pecker, S., Biology of Hair and Nail, In Bolognia, J. L., Jorizzo, J. L., Rapini, R. P., eds Dermatology. Mosby-Wolfe, London, (2003). 15. Sheng, Y., Pero, R. W., Olsson, A. R., Bryngelsson, C. and Hua, J., DNA Repair Enhancement by a Combined Supplement of Carotenoids, Nicotinamide, and Zinc, Cancer. Detect. Prev., 22, (1998). 16. Marini, M. and Musiani, D., Micromolar Zinc Affects Endonucleolytic Activity in Hydrogen Peroxide-mediated Apoptosis, Exp. Cell. Res., 239(2), (1998). 17. Handjiski, B. K., Eichmuller, S., Hofmann, U., Czarnetzki, B. M. and Paus, R., Alkaline Phosphatase Activity and Localization During the Murine Hair Cycle, Br. J. Dermatol., 131(3), (1994). 18. Theresa, J., Enrico, A., Anil, H. and Sreedhar, K., Glycine Prevents the Induction of Apoptosis Attributed to Mesenteric Ischemia/ reperfusion Injury in a Rat Model, Surgery, 134, (2003). 19. Uno, H., Cappas, A. and Schlagel, C., Cyclic Dynamics of Hair Follicles and the Effect of Minoxidil on the Bald Scalps of Stumptailed Macaques, Am. J. Dermatopathol., 7(3), (1985). 20. Philpott, M. P., Green, M. R. and Kealey, T., Rat Hair Follicle Growth in vitro., Br. J. Dermatol., 127(6), (1992). 21. Commo, S., Gaillard, O. and Bernard, B. A., The Human Hair Follicle Contains two Distinct K19 Positive Compartments in the Outer Root Sheath: a Unifying Hypothesis for Stem Cell Reservoir?, Differentiation, 66(4-5), (2000). 22. Michel, M., Torok, N., Godbout, M. J., Lussier, M., Gaudreau, P., Royal, A. and Germain, L., Keratin 19 as a Biochemical Marker of Skin Stem Cells in vivo and in vitro: Keratin 19 Expressing Cells are Differentially Localized in Function of Anatomic Sites, and Their Number Varies with Donor Age and Culture Stage., J. Cell. Sci., 109, (1996). 23. Yang, L., Zhang, B., Toku, K., Maeda, N., Sakanaka, M. and Tanaka, J., Improvement of the Viability of Cultured Rat Neurons by the Non-essential Amino Acids L-serine and Glycine that Upregulates Expression of the Anti-apoptotic Gene Product Bcl-w., Neurosci. Lett., 295(3), (2000). o44 o k

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<30372E31362D323028BDC5C7F6C5C32DB9CCB1B970626D292E666D> k m v r1 r1 16~20 (2009) J. Korean Academy of Managed Care Pharmacy Vol. 1, No. 1 Pharmacy Benefit Management iç 1# x sƒ, 1 w w w Overview on Pharmacy Benefit Management in USA Kyung Eob ChoiGand Hyun