Journal of Derrnatological Science, 2 (199 1) 66-70
Culture of human outer root sheath cells from plucked hair follicles in serum-free conditions Seiji Arase, Shoji Katoh, Yasushi Sadamoto, Hideki Nakanishi, Kenji Fujie en Katsuyuki Takeda Department
of Dermatology School of Medicine,
The University of Tokushima, Tokuchima City, Japan
31 March 1990; accepted
Key words: Outer root sheath cells; Serum free conditions;
Abstract We succeeded in culturing human outer root sheath cells (ORSC) in serum-free conditions without a biological feeder layer. The combination of collagen type IV substrate and modified MCDB 153 medium supplemented with bovine pituitary gland extract (K-GM medium) could support the growth of ORSC. These cells can be serially cultivated for at least 4 passages and stored in liquid nitrogen with good recovery. Thus, a large series of experiments using ORSC may be run simultaneously.
Materials and Methods
We previously reported that human outer root sheath cells (ORSC) from plucked hair follicles grew well on collagen type IV substrate in lowcalcium medium [ 1,2]. However, the cells obtained had many disadvantages for experimental use, such as a requirement for fetal bovine serum and an inability of passage. In this communication we report on the culture of human ORSC in serum-free conditions, producing cells which can be passaged more than 4 times.
Culture conditions ORSC were cultured in K-GM medium (modified MCDB 153 medium supplemented with 10 ng/ml epidermal growth factor. 5 pg/ml insulin, 0.5 pg/ml hydrocortisone and 0.5 % of bovine pituitary gland extract [ 31: the complete medium was purchased from Kurabo Co., Osaka, Japan) in 35-mm plastic dishes coated with collagen type IV as previously described [ 1,2] at 37 “C in a humidified 5% CO,/95% air atmosphere. The medium was changed every two days.
to: Seiji Arase, Department of Dermatology School of Medicine, The University of Tokushima, 3 Kuramoto-Cho, Tokuchima City, 770 Japan.
0 1991 Elsevier Science Publishers
Preparation of ORSC and primary culture ORSC were obtained according to the procedure described by Limat and Noser . Briefly, hairs were plucked from the scalp of two healthy subjects (Table I). Follicles with an intact sheath
Variation of the average plating efficiency and colony growth with cell age in the presence Strain
Plating Efficiency (% )*
Collagen type IV
Collagen type IV
2 3 4 5
2.2 15.2 16.5 13.8 5.2 1.6
0.4 9.0 6.2 0.9
10.32 13.45 14.22 13.10 6.03
3.12 4.56 4.90 3.33 1.02
7.10 f 1.48 10.92 f 3.17 6.22 f 1.34
Primary 1 3
2.3 14.7 14.0
0.5 7.1 0.7
9.08 & 1.88 12.46 k 3.73 14.11 k 5.22
6.55 f 1.28 9.03 f 2.12
* the mean of triplicate determinations;
or absence of collagen substrate
** cell number/colony:
were collected and then treated with 0.25% trypsin and 0.02% EDTA in PBS (trypsinEDTA) for 15 min at 37 “C. The ORSC around the follicles were dispersed by gentle pipetting, suspended in the trypsin-EDTA inactivation medium (Eagle’s MEM with 15% fetal bovine serum) and centrifuged at 1000 rpm for 5 min. After removing the medium, the cell pellet was resuspended in K-GM medium and the number of translucent and opaque cells was counted in a hemocytometer. The cells were plated at a density of 8 x lo4 per dish.
f + f + +
the mean k SD of 100 colonies.
per dish. In the 1st to the 5th passage cells, 1 x lo3 cells were plated. After 3 days the cultures were fixed with methanol and stained with Giemsa, and colonies of 4 or more contiguous cells were counted in triplicate. The number of cells per colony was counted in 100 colonies. Cell morphology In all experiments, cultures were studied by a phase contrast microscope, and photographs were taken. Results
Serial subculture When the primary cultures grew to an 80-90 % confluent monolayer, they were subcultured by a 3-min exposure to trypsin-EDTA. First passage cells were plated at a density of 1 x lo5 per dish. Thereafter, the cells were repeatedly subcultured with 1 : 4 splits when the cultures were just prior to confluence. When cell growth slowed cultures were passaged every 4 or 5 days. Colony assa_vfor the determination of plating ef$ciency and celhdar growth The cells were examined at each passage. The primary cells were plated at a density of 5 x lo3
Serial subculture Although we have not confirmed the identity of the cells cultured here either immunohistochemitally or biochemically, the cells obtained from plucked follicles by trypsinization could be outer root sheath cells (ORSC) as discussed by Limat and Noser [ 41. Cultures on collagen type IV multiplied rapidly for a maximum of 4 passages. The growth curves of ORSC from both subjects showed a linear increase in cell number during the initial 8 to 9 generations (Fig. l), following which cellular growth ceased. Even in uncoated dishes, cultures multiplied for 2 passages.
Morphology of ORSC (Fig. 2)
The ORSC showed good growth until passage 4. The cells were monomorphically small and round (Fig. 2A,B,C), however they tended to grow separately through all passages on collagen type IV. Thus cell-to-cell attachment was not clear. Then, at 5th passage, cell growth ceased and individual cells became enlarged, flattened, elongated, and finally made aggregates of cells (Fig. 2D). Even in the absence of collagen type IV substrate, the cells showed good growth until passage 2. The cells were small and polygonal and, interestingly, cell-to-cell attachment was seen from the beginning (Fig. 2E,F). Discussion
( Days 1
Fig. 1. Growth of ORSC in the presence or absence of collagen substrate, beginning with the secondary culture (first passage cells). Each point represents one transfer: (0) ORSClAW and (0) ORSCZAW in the presence of collagen type IV: (0) ORSClAW in its absence.
Plating efficiency (Table I)
and colony growth of ORSC
The plating efficiency of the primary ORSC was about 2% to 2.5% on collagen type IV, which was 4-5 times higher than that in the control dishes. The plating efficiencies of the 1st to 3rd passage cells were about 15% on collagen type IV, comparable to about 8 % in the control dishes. Comparison of the colony size of the cells from various passages indicated that not only the plating efficiency but also the colony growth of the lst, 2nd and 3rd passage ORSC was nearly the same on collagen type IV and slightly higher than that of the primary cells. Colonies were bigger in the presence of collagen type IV than in the absence.
Weterings et al. [ 51 first succeeded in culturing human outer root sheath cells (ORSC) by explanting hair follicles on bovine eye lens capusules. Subsequently several methods for cultivation of human ORSC from plucked hair follicles either as explant outgrowth [ 51 or as dispersed cells [4,6] have been developed. However, in all cases, the special organ (eye lens capusule) [ 51 or the living cells such as 3T3 cells [4,6] have been required to support the growth of ORSC. Recently, in an attempt to simplify culture conditions, we cultured ORSC on different biologic substrates and found that collagen type IV could support the growth of ORSC in low-calcium medium without a living cell feeder layer [ 1,2]. However, the cells obtained there had many disadvantages for experimental use, such as a requirement for fetal bovine serum and an inability of passage. K-GM medium was originally developed for the culture of epidermal keratinocytes [ 3,7,8] and recently the complete medium has become commercially available. Since ORSC and epidermal keratinocytes have the same origin, we presumed that this medium might also support the growth of ORSC. All results indicate that the combination of this medium and collagen type IV substrate is suitable for the culture of ORSC. The cells showed rapid growth until passage 4 under these culture conditions. The colony forming ability of
Fig. 2. Primary culture, 3 days after plating (A). Third passage ORSC, 2 days (B) and 6 days (C) after incubation on collagen type IV. The cells are monomorphically round, but cell-to-cell attachment is unclear. Finally the cells became enlarged and flattened, and formed cellular aggregates at passage 5 (D). In plastic dishes, cell-to-cell attachment is clearly seen in the primary (E) and the second passage cultures (F).
the cells was similar from the 1st to the 3rd passage, after which cellular growth slowed and finally ceased at the 5th passage. Drastic morphologic changes also occurred in the cells in the manner of other types of human epithelial cells at the limits of their proliferative capacity [lo]. The plating efficiency in the primary culture was about 2-2.5 %, 4-5 times higher than that in previous studies [ 11. The plating efficiency in the lst, 2nd and 3rd passage cells was about 15%, 7-8 times higher than that of the primary cells. The primary ORSC were treated by trypsin-EDTA longer, agitated harder and damaged more than the subcultured cells. In addition, the subcultured cells were better adapted to the culture conditions. Because of the very low calcium concentrations of only 0.15 mM in K-GM medium, most of the
subcultured cells may be in the active stage of proliferation. Under these low calcium concentrations, epitherial cells proliferate well but differentiate little [ 1,9,10]. On collagen type IV, cell-to-cell attachment was not clear, whereas ORSC formed colonies with cell-to-cell attachment in uncoated dishes. The reasons for these findings are still unknown. The most important point in this study is that, in K-GM medium, ORSC can be passaged at least 4 times on collagen type IV and 2 times in uncoated dishes when the cells of subconfluence are subcultured with 1 : 4 splits. The ORSC up to 3rd passage showed similar good growth, and they could be stored in liquid nitrogen with a good recovery (data not shown). We can obtain hairs without causing any permanent damage to the
subject. A large numbers of ORSC are easily harvested from a few hairs by the methods described here. Thus, a large series of experiments, including genetic experiments and testing pharmacologic substances for their effects on ORSC could be run simultaneously. References 1 Kuwana R, Arase S, Sadamoto Y, Nakanishi H, Takeda K: A method for culturing human hair follicle cells (II). Nishinihon J Dermatol 50: 271-276, 1988 (in Japanese). 2 Kuwana R, Arase S, Sadamoto Y, Takeda K: A new method for culturing human hair follicle on floating mixed collagen membranes. J Dermatol 17: 11-15, 1990. 3 Boyce ST, Ham RG: Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81 (suppl): 33s-4Os, 1983. 4 Limat A, Noser FK: Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles. J Invest Dermatol 87: 485-488, 1986.
5 Weterings PJJM, Vermorken AJM, Bloemendal H: A method for culturing hair follicle cells. Br J Dermatol 104: 1-5, 1981. 6 Noser FK, Limat A: Organotypic culture of outer root sheath cells from human hair follicles using a new culture device. In Vito Cell Dev Biol 23: 541-545, 1987. 7 Wille JJ, Pittelkow MR, Shipley GD, Scott RE: Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analysis, growth kinetics, and cell cycle studies. J Cell Physiol 121: 31-44, 1984. 8 Shipley GD, Pittelkow MR: Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium. Arch Dermatol 123: 1541a-1544a, 1987. 9 Howley-Nelson P, Sullivan JE, Kung M, Henning H, Yuspa SH: Optimized condition for the growth of human epidermal cells in culture. J Invest Dermato175: 176-182, 1980. 10 Ham RG, Hammond SL: Normal human mammary epitherial cells in serum-free media. In Growth and Differentiation of Mammary Epithelial Cells in Culture. Edited by J Enami and RG Ham, Japan Scientific Societies Press, Tokyo, 1987, pp 59-108.